Anti-mesothelin antigen-binding molecule and uses thereof

Abstract

The present disclosure provides antibodies and polypeptides that specifically bind to mesothelin (MSLN), including bispecific antibodies that bind to both MSLN and T cell antigens (e.g., CD3), as well as pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects using these antibodies.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 262,156, filed October 6, 2021, and U.S. Provisional Application No. 63 / 369,989, filed August 1, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing The electronically submitted Sequence Listing XML (Name: 193452_SL; Size: 95,711 bytes; Creation Date: September 8, 2022) is incorporated by reference in its entirety herein.

[0003] The present disclosure relates to antibodies specific for human mesothelin (MSLN), including bispecific antibodies that bind to human mesothelin (MSLN) and T cell antigens (e.g., CD3), and methods of use thereof. [Background technology]

[0004] Mesothelin (MSLN) is a cell surface glycoprotein with normal expression restricted to mesothelial cells lining the pleura, pericardium, and peritoneum. The mesothelin gene encodes a 71 kDa precursor protein that is cleaved by the endoprotease furin into a 40 kDa membrane-associated protein called mesothelin and a 31 kDa shed fragment called megakaryocyte potentiating factor (MPF) that is released from the cell. The soluble form of the mesothelin protein is likely the result of an aberrant splicing event that results in a frameshift mutation and premature termination, which deletes amino acids at the C-terminus that are responsible for the association of the protein with the cell membrane (Hassan et al., Clinical Cancer Research, 10:3937-3942, 2004).

[0005] Mesothelin is highly expressed in various tumor types, including mesothelioma, pancreatic cancer, ovarian cancer, and gastric and lung adenocarcinoma. The limited distribution of mesothelin on normal tissues makes it a promising target for tumor-specific therapy. Evidence suggests that the extracellular domain of membrane-bound mesothelin is shed from tumor cells, but the exact mechanisms associated with this shedding remain to be elucidated (Ho and Lively, Cancer Epidemiol Biomarkers Prev., 15(9):1751, 2006). These soluble forms of mesothelin may hinder tumor cell-directed immunotherapy by producing an antigen sink that binds to anti-MSLN antibodies.

[0006] CD3 is a homodimeric or heterodimeric 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 a dimeric arrangement of four different chains, including gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby triggering T cell activation in a manner similar to engaging the TCR with peptide-loaded MHC molecules. Bispecific antibodies capable of binding to CD3 and a target antigen have been proposed for therapeutic uses, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0007] Thus, there is a need for therapeutic approaches for targeting and T cell-mediated killing of tumor cells expressing MSLN, including bispecific antibodies that bind to MSLN and T cell antigens (e.g., CD3). Summary of the Invention

[0008] The present disclosure provides antibodies and polypeptides that specifically bind to MSLN (e.g., human MSLN), including bispecific antibodies that bind to both MSLN and T cell antigens (e.g., CD3). Also provided are pharmaceutical compositions that include these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating subjects using these antibodies. The antibodies disclosed herein are particularly advantageous in that they have a high isoelectric point and unexpectedly fast serum clearance in human FcRn knock-in mice. This type of pharmacokinetic profile has been shown to result in increased biodistribution and tissue retention in subjects compared to antibodies with low isoelectric points and slow serum clearance (see, e.g., Li et al., MAbs., 6(5):1255-64, 2014; Boswell et al., Bioconjug Chem., 21(12):2153-63, 2010). The applicant anticipates that this increased biodistribution will be advantageous for the treatment of tumors in human subjects.

[0009] In one aspect, the disclosure provides an antibody that specifically binds to human MSLN, the antibody comprising a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence represented by SEQ ID NO: 11, and a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence represented by SEQ ID NO: 12, wherein the antibody does not comprise SEQ ID NO: 4 and / or 8.

[0010] In one embodiment, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 3, 13, and 5, respectively.

[0011] In one embodiment, the antibody comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 6, 7, and 14, respectively.

[0012] In one embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 3, 13, 5, 6, 7, and 14, respectively.

[0013] In one embodiment, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences represented by SEQ ID NOs: 3, 30, and 5; 3, 29, and 5; 3, 31, and 5; 3, 32, and 5; 3, 33, and 5; or 3, 34, and 5, respectively.

[0014] In one embodiment, the antibody comprises CDRL1, CDRL2, and CDRL3 amino acid sequences represented by SEQ ID NOs: 6, 7, and 35; 6, 7, and 36; 6, 7, and 37; 6, 7, and 38; 6, 7, and 39; or 6, 7, and 40, respectively.

[0015] In one embodiment, the antibodies are selected from the group consisting of SEQ ID NOs: 3, 30, 5, 6, 7, and 35; 3, 29, 5, 6, 7, and 35; 3, 29, 5, 6, 7, and 36; 3, 29, 5, 6, 7, and 37; 3, 29, 5, 6, 7, and 38; 3, 29, 5, 6, 7, and 39; 3, 29, 5, 6, 7, and 40; 3, 30, 5, 6, 7, and 36; 3, 30, 5, 6, 7, and 3 7;3, 30, 5, 6, 7, and 38;3, 30, 5, 6, 7, and 39;3, 30, 5, 6, 7, and 40;3, 31, 5, 6, 7, and 35;3, 31, 5, 6, 7, and 36;3, 31, 5, 6, 7, and 37;3, 31, 5, 6, 7, and 38;3, 31, 5, 6, 7, and 39;3, 31, 5, 6, 7, and 40;3, 32, 5, 6, 7, and 35;3 , 32, 5, 6, 7, and 36;3, 32, 5, 6, 7, and 37;3, 32, 5, 6, 7, and 38;3, 32, 5, 6, 7, and 39;3, 32, 5, 6, 7, and 40;3, 33, 5, 6, 7, and 35;3, 33, 5, 6, 7, and 36;3, 33, 5, 6, 7, and 37;3, 33, 5, 6, 7, and 38;3, 33, 5, 6, 7, and 39;3, 3 3, 34, 5, 6, 7, and 38; 3, 34, 5, 6, 7, and 39; or 3, 34, 5, 6, 7, and 40.

[0016] In one embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO:11.

[0017] In one embodiment, the antibody comprises a VH amino acid sequence of SEQ ID NO: 18, 17, 19, 20, 21, or 22.

[0018] In one embodiment, the antibody optionally comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0019] In one embodiment, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, where the variant heavy chain constant region binds to an FcγR with a lower affinity than the wild-type heavy chain constant region binds to the FcγR.

[0020] In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:53, 54, 55, 56, or 72.

[0021] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 83, 84, 41, 42, 43, 44, 45, 46, 66, 67, 68, 69, 70, or 71.

[0022] In one embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO:12.

[0023] In one embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO: 24, 23, 25, 26, 27, or 28.

[0024] In one embodiment, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 48, 47, 49, 50, 51, or 52.

[0025] In one embodiment, the VH and VL comprise the amino acid sequences set forth in SEQ ID NOs: 18 and 24, 17 and 23, 17 and 24, 17 and 25, 17 and 26, 17 and 27, 17 and 28, 18 and 23, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, 19 and 25, 19 and 26, 19 and 27, 19 and 28, 20 and 23, 20 and 24, 20 and 25, 20 and 26, 20 and 27, 20 and 28, 21 and 23, 21 and 24, 21 and 25, 21 and 26, 21 and 27, 21 and 28, 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, or 22 and 28, respectively.

[0026] In one embodiment, the heavy and light chains are selected from the group consisting of SEQ ID NOs: 83 and 48, 84 and 48, 83 and 49, 84 and 49, 83 and 50, 84 and 50, 83 and 51, 84 and 51, 83 and 52, 84 and 52, 83 and 47, 84 and 47, 41 and 47, 41 and 48, 41 and 49, 41 and 50, 41 and 51, 41 and 52, 42 and 47, respectively. , 42 and 48, 42 and 49, 42 and 50, 42 and 51, 42 and 52, 43 and 47, 43 and 48, 43 and 49, 43 and 50, 43 and 51, 43 and 52, 44 and 47, 44 and 48, 44 and 49, 44 and 50, 44 and 51, 44 and 52, 45 and 47, 45 and 48, 45 and 49, 45 and 50, 45 and 51, 45 and and 52, 46 and 47, 46 and 48, 46 and 49, 46 and 50, 46 and 51, 46 and 52, 66 and 47, 66 and 48, 66 and 49, 66 and 50, 66 and 51, 66 and 52, 67 and 47, 67 and 48, 67 and 49, 67 and 50, 67 and 51, 67 and 52, 68 and 47, 68 and 48, 68 and 49, 68 and 50 , 68 and 51, 68 and 52, 69 and 47, 69 and 48, 69 and 49, 69 and 50, 69 and 51, 69 and 52, 70 and 47, 70 and 48, 70 and 49, 70 and 50, 70 and 51, 70 and 52, 71 and 47, 71 and 48, 71 and 49, 71 and 50, 71 and 51, or 71 and 52.

[0027] In one embodiment, the antibody further comprises a CD3 binding portion.

[0028] In one embodiment, the CD3 binding moiety is a polypeptide. In one embodiment, the CD3 binding moiety is an antibody. In one embodiment, the CD3 binding moiety is a single chain fragment variable (scFv). In one embodiment, the scFv comprises the amino acid sequence represented by SEQ ID NO:76.

[0029] In one embodiment, the CD3 binding moiety is covalently linked to the light chain. In one embodiment, the CD3 binding moiety is covalently linked to the C-terminus of the light chain. In one embodiment, the CD3 binding moiety is covalently linked to the C-terminus of the light chain via a peptide linker, optionally comprising the amino acid sequence set forth in SEQ ID NO: 63, 64, 73, 74, or 75.

[0030] In one embodiment, the light chain comprises an amino acid sequence set forth in SEQ ID NO:77, 78, 79, 80, 81, or 82.

[0031] In one aspect, the disclosure provides an antibody that specifically binds to human MSLN, the antibody being selected from the group consisting of SEQ ID NOs: 83 and 77, 84 and 77, 41 and 77, 42 and 77, 43 and 77, 44 and 77, 45 and 77, 46 and 77, 66 and 77, 67 and 77, 68 and 77, 69 and 77, 70 and 77, 71 and 77, 83 and 78, 84 and 78, 41 and 78, 42 and 78, 43 and 78, 44 and 78, 45 and 78, 46 and 78, 66 and 78, 67 and 78, 68 and 78, 69 and 78, 70 and 78, 71 and 78, 83 and 79, 84 and 79, 41 and 79, 42 and 79, 43 and 79, 44 and 79, 45 and 79, 46 and 79, 66 and 79, 67 and 79, 68 and 79, 69 and 79, 70 and 79, 71 and 79, 83 and 80, 84 and 80, 41 and 80, 42 and 80, 43 and 80, 44 and 80, 45 and 80, 46 and 80, 66 and 80, 67 and 80, 68 and 80, 69 and 80, 70 and 80, 71 and 80, 83 and 81, 84 and 81, 41 and 81, 42 and 81, 43 and 81, 44 and 81, 45 and 81, 46 and 81, 6 and light chains comprising the amino acid sequences represented by: 6 and 81, 67 and 81, 68 and 81, 69 and 81, 70 and 81, 71 and 81, 83 and 82, 84 and 82, 41 and 82, 42 and 82, 43 and 82, 44 and 82, 45 and 82, 46 and 82, 66 and 82, 67 and 82, 68 and 82, 69 and 82, 70 and 82, or 71 and 82.

[0032] In one embodiment, the heavy and light chains comprise the amino acid sequences set forth in SEQ ID NOs: 83 and 77, respectively.

[0033] In one aspect, the disclosure provides a polypeptide comprising a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence represented by SEQ ID NO: 11, the polypeptide not comprising SEQ ID NO: 4. In one embodiment, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences represented by SEQ ID NOs: 3, 13, and 5, respectively. In one embodiment, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences represented by SEQ ID NOs: 3, 30, and 5; 3, 29, and 5; 3, 31, and 5; 3, 32, and 5; 3, 33, and 5; or 3, 34, and 5, respectively. In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 18, 17, 19, 20, 21, or 22.

[0034] In one embodiment, the polypeptide comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 83, 84, 41, 42, 43, 44, 45, 46, 66, 67, 68, 69, 70, or 71.

[0035] In one aspect, the disclosure provides a polypeptide comprising a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 12, the polypeptide not including SEQ ID NO: 8. In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 6, 7, and 14, respectively. In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 6, 7, and 35; 6, 7, and 36; 6, 7, and 37; 6, 7, and 38; 6, 7, and 39; or 6, 7, and 40, respectively. In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 24, 23, 25, 26, 27, or 28.

[0036] In one embodiment, the polypeptide comprises a light chain comprising the amino acid sequence of SEQ ID NO: 77, 47, 48, 49, 50, 51, 52, 78, 79, 80, 81, or 82.

[0037] In one aspect, the disclosure provides a polypeptide comprising an amino acid sequence represented by SEQ ID NO:73, 74, or 75.

[0038] In one embodiment, an antibody or polypeptide disclosed herein is conjugated to a cytotoxic agent, cytostatic agent, toxin, radionuclide, or detectable label.

[0039] In one aspect, the disclosure provides a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of an antibody disclosed herein; or a polypeptide disclosed herein.

[0040] In one aspect, the disclosure provides a vector comprising a polynucleotide disclosed herein.

[0041] In one aspect, the disclosure provides a recombinant host cell comprising: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a first polynucleotide encoding a heavy chain variable region, or heavy chain, of an antibody disclosed herein, and a second polynucleotide encoding a light chain variable region, or light chain, of an antibody disclosed herein; (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region or heavy chain of an antibody disclosed herein, and a second vector comprising a second polynucleotide encoding a light chain variable region or light chain of an antibody disclosed herein.

[0042] In one aspect, the disclosure provides a pharmaceutical composition comprising an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, and a pharma- ceutically acceptable carrier or excipient.

[0043] In one aspect, the disclosure provides a method of producing an antibody, the method comprising culturing a host cell disclosed herein under suitable conditions such that the polynucleotide is expressed and the antibody is produced.

[0044] In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0045] In one aspect, the present disclosure provides for the use of an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein for the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[0046] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein for use in a medicament.

[0047] In one aspect, the present disclosure provides an antibody disclosed herein, a polypeptide disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein for use in treating cancer in a subject in need thereof. [Brief description of the drawings]

[0048] [Figure 1A] Graphs showing the Ka and Kd (left and right, respectively) of light chain variants of anti-MSLN 1A12 antibody. Control MAb represents anti-MSLN 1A12 antibody alone. [Figure 1B] Graph showing the effect of the same light chain mutations on antibody expression. In FIG. 1B, the y-axis is a measurement of absorbance intensity in milliabsorbance units (mAU) and the x-axis is in minutes, used to determine the retention time of each peak. [Diagram 2] Graphs showing the Ka and Kd (left and right, respectively) of heavy chain variants of anti-MSLN 1A12 antibody. Control MAb represents anti-MSLN 1A12 antibody alone. [Diagram 3] 1 is a graph showing the affinity of various pairs of light and heavy chain variants of the anti-MSLN 1A12 antibody to MSLN. The control MAb represents the anti-MSLN 1A12 antibody alone. [Figure 4] Schematic diagram of a bispecific IgG-scFv antibody comprising a Fab domain (e.g., an anti-cancer Fab domain) and an immunoglobulin Fc domain linked to a scFv domain (e.g., an anti-T cell scFv domain). The x's shown in the CH2 domain represent optional mutations that block the interaction of the antibody with the Fc receptor. [Diagram 5]A and B are size-exclusion chromatograms showing protein concentration before (FIG. 5A) and after (FIG. 5B) Ni-NTA purification of a bispecific molecule comprising an anti-MSLN 1A12 binding domain and a humanized UCHT1 anti-CD3 scFv portion, with the structure illustrated in FIG. 4. FIG. 5A shows a size-exclusion chromatogram obtained from running a crude protein extract containing the bispecific molecule through a Superdex 200 10 / 300 GL pre-packed gel filtration column. FIG. 5B shows a size-exclusion chromatogram obtained from running a Ni-NTA purified protein extract containing the bispecific molecule through a Superdex200 10 / 300 GL pre-packed gel filtration column, resulting in a homogenous single peak of protein centered at about 11 mL. In FIGS. 5A and 5B, the y-axis is a measurement of absorbance intensity in milliabsorbance units (mAU) and the x-axis is in milliliters (mL), used to determine the retention volume of each peak. [Figure 6] A and B are size-exclusion chromatograms showing protein concentration before (FIG. 6A) and after (FIG. 6B) Ni-NTA purification of a bispecific molecule comprising an anti-MSLN 1A12 binding domain and a Micro194 anti-CD3 scFv portion, with the structure illustrated in FIG. 4. FIG. 6A shows a size-exclusion chromatogram obtained from running a crude protein extract containing the bispecific molecule through a Superdex 200 10 / 300 GL pre-packed gel filtration column. FIG. 6B shows a size-exclusion chromatogram obtained from running a Ni-NTA purified protein extract containing the bispecific molecule through a Superdex200 10 / 300 GL pre-packed gel filtration column, resulting in a homogenous single peak of protein centered at about 13 mL. In FIGS. 6A and 6B, the y-axis is a measurement of absorbance intensity in milliabsorbance units (mAU) and the x-axis is in milliliters (mL), used to determine the retention volume of each peak. [Figure 7]5 is a graph showing the affinity for MSLN of bispecific molecules containing the indicated anti-MSLN 1A12 binding domain and an anti-CD3 binding portion, each having the structure shown in Figure 4. The control MAb represents the anti-MSLN 1A12 antibody alone. [Figure 8A] 5 is a graph showing the results of the ability of bispecific molecules comprising an anti-MSLN 1A12 binding domain and an anti-CD3 binding moiety, each having the structure shown in Figure 4, to induce cytotoxicity in SKOV3 human ovarian cancer cells. Cytotoxicity is reported as the percentage of dead cells versus the concentration of bispecific molecule, determined for SKOV3 cells on day 5 after contacting the cells with the bispecific molecule. [Figure 8B] 5 is a graph showing the results of the ability of bispecific molecules comprising an anti-MSLN 1A12 binding domain and an anti-CD3 binding moiety, each having the structure shown in Figure 4, to induce cytotoxicity in OVCAR3 human ovarian cancer cells. Cytotoxicity is reported as the percentage of dead cells relative to the concentration of bispecific molecule, determined for OVCAR3 cells on day 2 after contacting the cells with the bispecific molecule. [Figure 9] 9A and 9B are graphs showing the results of the ability of a bispecific molecule comprising an anti-MSLN 1A12 binding domain and an anti-CD3 binding portion, having the structure shown in FIG. 4, to induce cytotoxicity in SKOV3 cells (FIG. 9A), and the ability of a TriTAC molecule to bind MSLN, CD3, and serum albumin (FIG. 9B). The ability of the bispecific and TriTAC molecules to induce cytotoxicity is challenged by soluble MSLN (TriTAC+sMSLN and Ig-scFv+sMSLN, respectively). [Figure 10]FIG. 5 is a graph showing tumor growth over time in NOD / SCID / IL-2Rγcnull (NSG) mice engrafted intraperitoneally with luciferase expressing SKOV3 cells treated with bispecific molecules comprising an anti-MSLN 1A12 binding domain and an anti-CD3 OKT3 binding moiety having the structure illustrated in FIG. 4. Untreated mice and mice treated with activated T cells (ATC) alone were used as controls. The y-axis is the amount of light emitted in photons / second (p / s) detected by bioluminescence imaging. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present disclosure provides anti-MSLN antibodies and polypeptides, including bispecific antibodies that bind to both MSLN and T cell antigens (e.g., CD3). Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of using these antibodies to treat a subject. The antibodies disclosed herein are particularly useful for treating cancer in a subject.

[0050] definition The term "MSLN" as used herein refers to mesothelin. The amino acid sequence of human mesothelin can be found in Accession No. Q13421 (UniProtKB). MSLN is a cell surface glycoprotein that is highly expressed in pancreatic cancer, ovarian cancer, mesothelioma, and some other cancer types. Mesothelin is expressed in normal mesothelial cells that cover the pleura, pericardium, and peritoneum, but has a restricted distribution on normal tissues. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless expressly indicated to be derived from a non-human species. Thus, the term "MSLN" refers to human MSLN, unless otherwise specified to be derived from a non-human species (e.g., "mouse MSLN", "monkey MSLN", etc.).

[0051] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma.

[0052] As used herein, the term "antibody(s)" includes full length antibodies, antigen-binding fragments of full length antibodies, and molecules comprising the CDRs, VH regions, and / or VL regions of an antibody. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, signal domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypes (anti-Id antibodies (e.g., anti-anti-Id antibodies), and any of the above). In certain embodiments, the antibody described herein refers to a polyclonal antibody population. The antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibody described herein is an IgG antibody, or a class (e.g., human IgG1 or IgG4), or subclass thereof. In certain embodiments, the antibody is a humanized monoclonal antibody. In another particular embodiment, the antibody is a human monoclonal antibody.

[0053] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens or two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (excluding the Fc region). Multispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single chain antibodies or linked single chain Fvs (scFvs), camelized antibodies, affibodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide linked Fvs (sdFvs). Multispecific antibodies can be antibodies of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2 a or IgG2 b In certain embodiments, the multispecific antibodies described herein are IgG antibodies, or classes (e.g., human IgG1, IgG2, or IgG4), or subclasses thereof.

[0054] As used herein, the term "CDR" or "complementarity determining region" refers to the non-adjacent antigen combining sites found within the variable regions of heavy and light chain polypeptides. These particular regions are described, for example, in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, and the definitions include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" refers to the CDRs defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDRs defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy and light chain CDRs of an antibody are defined using different conventions. In certain embodiments, the heavy and / or light chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human MSLN).CDRH1, CDRH2, and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent the light chain CDRs.

[0055] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino terminal 110-120 or about 110-125 amino acids in a mature heavy chain and about the amino terminal 90-115 amino acids in a mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable region are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0056] As used herein, the terms "VH" and "VL" refer to the heavy and light chain variable regions, respectively, of an antibody as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0057] As used herein, the term "constant region" is as is common in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors).

[0058] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, e.g., subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0059] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0060] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are analogous terms for antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex). Such binding is as would be understood by one of skill in the art. For example, a molecule that specifically binds to an antigen can generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, BIAcore®, KinExA3000 instruments (Sapidyne Instruments, Boise, ID), or assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K that is at least 2 logs (e.g., a factor of 10), 2.5 logs, 3 logs, 4 logs lower than the K when the molecule nonspecifically binds to another antigen. A, or larger K A binds to the antigen.

[0061] As used herein, "affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0062] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as set forth in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al, Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of the above is incorporated herein by reference in its entirety.

[0063] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or prophylactic measures as described herein. "Treatment" methods employ administration of an antibody to a subject having a disease or disorder, or predisposed to having such a disease or disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disease or disorder or a recurrent disease or disorder, or to prolong the survival of the subject beyond that expected in the absence of such treatment.

[0064] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of the therapy that achieves a desired prophylactic or therapeutic effect.

[0065] As used herein, the term "subject" includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.

[0066] The term "isolated" as used herein with respect to an antibody or polynucleotide refers to an antibody or polynucleotide that has been separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) that are present in the natural source of the antibody or polynucleotide. All examples of "isolated antibodies" described herein are further contemplated as antibodies that may, but do not have to be isolated. All examples of "isolated polynucleotides" described herein are further contemplated as polynucleotides that may, but do not have to be isolated. All examples of "antibodies" described herein are further contemplated as antibodies that may, but do not have to be isolated. All examples of "polynucleotides" described herein are further contemplated as polynucleotides that may, but do not have to be isolated.

[0067] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be obtained using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of KarlinS&Altschul SF(1990)PNAS87:2264-2268, modified KarlinS&Altschul SF(1993)PNAS90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al.,(1990)J Mol Biol 215:403, the contents of which are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed to obtain nucleotide sequences that are homologous to the nucleic acid molecules described herein using the NBLAST nucleotide program parameters set, for example, score=100, word length=12. BLAST protein searches can be performed to obtain amino acid sequences homologous to the protein molecules described herein using the XBLAST program parameters set, for example, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, the contents of which are incorporated herein by reference in their entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0068] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0069] Anti-MSLN antibody In one aspect, the disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN). The amino acid sequences of exemplary antibodies are presented in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

[0070] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a VH domain that comprises one, two, or all three of the CDRs of the VL domain disclosed in Table 1. In certain embodiments, the antibody comprises a CDRH1 of a VH domain listed in Table 1. In certain embodiments, the antibody comprises a CDRH2 of a VH domain listed in Table 1. In certain embodiments, the antibody comprises a CDRH3 of a VH domain listed in Table 1.

[0071] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a VL domain that comprises one, two, or all three of the CDRs of the VL domain disclosed in Table 1. In certain embodiments, the antibody comprises a CDRL1 of the VL domain listed in Table 1. In certain embodiments, the antibody comprises a CDRL2 of the VH domain listed in Table 1. In certain embodiments, the antibody comprises a CDRL3 of the VL domain listed in Table 1.

[0072] The individual CDRs of the antibodies disclosed herein may be determined according to any CDR numbering scheme known in the art.

[0073] In certain embodiments, one or more of the CDRs of an antibody disclosed herein may be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0074] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the Kabat numbering scheme.

[0075] In certain embodiments, one or more of the CDRs of an antibody disclosed herein can be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entireties).

[0076] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the Chothia numbering system.

[0077] In certain embodiments, one or more of the CDRs of an antibody disclosed herein may be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety. See also, for example, "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0078] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the MacCallum numbering system.

[0079] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7:132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212, both of which are incorporated by reference in their entireties; and Lefranc MP et al., (2009) Nucleic Acids Res 37:D1006-D1012.

[0080] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the IMGT numbering system.

[0081] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0082] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the AbM numbering scheme.

[0083] In certain embodiments, the CDRs of an antibody disclosed herein can be determined according to the AHo numbering system as described in Honegger and Pluckthun, A., J. Mol. Biol. 309:657-670 (2001), which is incorporated by reference in its entirety.

[0084] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and comprise the CDRs of the antibodies disclosed in Table 1 herein, as determined by the AHo numbering system.

[0085] In certain embodiments, the individual CDRs of the antibodies disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, where the structural analysis identifies residues within the variable region(s) that are predicted to contact the epitope region of MSLN.

[0086] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence represented by SEQ ID NO: 11, and a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence represented by SEQ ID NO: 12, where each CDR is independently determined according to the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, or by structural analysis of the multispecific molecule, and the structural analysis identifies residues within the variable region(s) that are predicted to contact an epitope region of MSLN (e.g., human MSLN). In some embodiments, the antibody does not include SEQ ID NO: 4 and / or SEQ ID NO: 8.

[0087] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences represented by SEQ ID NOs: 3, 13, and 5, respectively.

[0088] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences represented by SEQ ID NOs: 6, 7, and 14, respectively.

[0089] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences represented by SEQ ID NOs: 3, 13, 5, 6, 7, and 14, respectively.

[0090] In certain embodiments, the disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN), wherein the antibodies comprise CDRH1, CDRH2, and CDRH3 amino acid sequences represented by SEQ ID NOs: 3, 29, and 5; 3, 30, and 5; 3, 31, and 5; 3, 32, and 5; 3, 33, and 5; or 3, 34, and 5, respectively.

[0091] In certain embodiments, the disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN), wherein the antibodies comprise CDRL1, CDRL2, and CDRL3 amino acid sequences represented by SEQ ID NOs: 6, 7, and 35; 6, 7, and 36; 6, 7, and 37; 6, 7, and 38; 6, 7, and 39; or 6, 7, and 40, respectively.

[0092] In certain embodiments, the disclosure provides antibodies that specifically bind to MSLN (such as human MSLN), the antibodies being selected from SEQ ID NOs: 3, 29, 5, 6, 7, and 35; 3, 29, 5, 6, 7, and 36; 3, 29, 5, 6, 7, and 37; 3, 29, 5, 6, 7, and 38; 3, 29, 5, 6, 7, and 39; 3, 29, 5, 6, 7, and 40; 3, 30, 5, 6, 7, and 35; , 30, 5, 6, 7, and 36;3, 30, 5, 6, 7, and 37;3, 30, 5, 6, 7, and 38;3, 30, 5, 6, 7, and 39;3, 30, 5, 6, 7, and 40;3, 31, 5, 6, 7, and 35;3, 31, 5, 6, 7, and 36;3, 31, 5, 6, 7, and 37;3, 31, 5, 6, 7, and 38;3, 31, 5, 6, 7, and 39;3, 31, 5, 6, 7, and 40;3, 32, 5, 6, 7, and 35;3, 32, 5, 6, 7, and 36;3, 32, 5, 6, 7, and 37;3, 32, 5, 6, 7, and 38;3, 32, 5, 6, 7, and 39;3, 32, 5, 6, 7, and 40;3, 33, 5, 6, 7, and 35;3, 33, 5, 6, 7, and 36;3, 33, 5, 6, 7, and 37;3, 33, 5, 6, 7, and 38;3, 33, 5, 6, and 3, 34, 5, 6, 7, and 39; 3, 33, 5, 6, 7, and 40; 3, 34, 5, 6, 7, and 35; 3, 34, 5, 6, 7, and 36; 3, 34, 5, 6, 7, and 37; 3, 34, 5, 6, 7, and 38; 3, 34, 5, 6, 7, and 39; or 3, 34, 5, 6, 7, and 40.

[0093] In certain embodiments, an antibody is provided that specifically binds to MSLN (e.g., human MSLN) comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence represented by SEQ ID NO: 11. In certain embodiments, the present disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising a VH comprising an amino acid sequence represented by SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence represented by SEQ ID NO: 11.

[0094] In certain embodiments, the present disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence represented by SEQ ID NO: 12. In certain embodiments, the present disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising a VL comprising an amino acid sequence represented by SEQ ID NO: 12. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence represented by SEQ ID NO: 12.

[0095] In certain embodiments, the present disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:11, and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), comprising a VH comprising the amino acid sequence of SEQ ID NO: 11 and a VL comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence represented by SEQ ID NO: 11, and the amino acid sequence of the VL consists of the amino acid sequence represented by SEQ ID NO: 12.

[0096] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 11 and 12, respectively. In certain embodiments, the VH and VL amino acid sequences consist of the amino acid sequences set forth in SEQ ID NOs: 11 and 12, respectively.

[0097] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) comprising a VH amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21, or 22. In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) consisting of a VH amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21, or 22.

[0098] In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) comprising the VL amino acid sequence of SEQ ID NO: 23, 24, 25, 26, 27, or 28. In certain embodiments, the present disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) consisting of the VL amino acid sequence of SEQ ID NO: 23, 24, 25, 26, 27, or 28.

[0099] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the VH and VL are set forth in SEQ ID NOs: 17 and 23, 17 and 24, 17 and 25, 17 and 26, 17 and 27, 17 and 28, 18 and 23, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, 19 and 25, 19 and 26, 19 and 27, 19 and 28, 20 and 23, 20 and 24, 20 and 25, 20 and 26, 20 and 27, 20 and 28, 21 and 23, 21 and 24, 21 and 25, 21 and 26, 21 and 27, 21 and 28, 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, or 22 and 28. In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the VH and VL are set forth in SEQ ID NOs: 17 and 23, 17 and 24, 17 and 25, 17 and 26, 17 and 27, 17 and 28, 18 and 23, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, 19 and 25, 19 and 26, 19 and 27, 19 and 28, 20 and 23, 20 and 24, 20 and 25, 20 and 26, 20 and 27, 20 and 28, 21 and 23, 21 and 24, 21 and 25, 21 and 26, 21 and 27, 21 and 28, 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, or 22 and 28.

[0100] In certain embodiments, the disclosure provides antibodies that cross-compete with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 11 and 12, respectively, for binding to MSLN (e.g., human MSLN). In certain embodiments, the disclosure provides antibodies that cross-compete with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 17 and 23, 17 and 24, 17 and 25, 17 and 26, 17 and 27, 17 and 28, 18 and 23, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, 19 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, 19 and 28, 19 and 29, 19 and 30, 19 and 31, 19 and 32, 19 and 33, 19 and 34, 19 and 35, 19 and 36, 19 and 37, 19 and 38, 19 and 39, 19 and 40, 19 and 41, 19 and 42, 19 and 43, 19 and 44, 19 and 45, 19 and 46, 19 and 47, 19 and 48 ...3, 19 and 44, 19 and 45, 19 and and 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, or 22 and 28.

[0101] In certain embodiments, the disclosure provides antibodies that bind to the same or overlapping epitopes of MSLN (e.g., epitopes of human MSLN) as the antibodies described herein, e.g., antibodies comprising VH and VL amino acid sequences set forth in SEQ ID NOs: 11 and 12, respectively. ...7 and 23, 17 and 24, 17 and 25, 17 and 26, 17 and 27, 17 and 28, 18 and 23, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and 28, 19 and 23, 19 and 24, respectively. , 19 and 25, 19 and 26, 19 and 27, 19 and 28, 20 and 23, 20 and 24, 20 and 25, 20 and 26, 20 and 27, 20 and 28, 21 and 23, 21 and 24, 21 and 25, 21 and 26, 21 and 27, 21 and 28, 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, or 22 and 28.

[0102] In certain embodiments, the epitope of the antibody may be determined by, but is not limited to, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange combined mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry, array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping), etc. In the case of X-ray crystallography, crystallization may be achieved using any of the methods known in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350; McPherson A(1990) Eur J Biochem 189:1-23; Chayen NE(1997) Structure 5:1269-1274; McPherson A(1976) J Biol Chem 251:6300-6303, all of which are incorporated herein by reference in their entireties. Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, Molecular Simulations, Inc.; see e.g., Meth Enzymol (1985) volumes 114&115, eds Wyckoff HW et al.; U.S. Patent Application 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, all of which are incorporated herein by reference in their entireties. Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art.For example, see Champe M et al., (1995) (supra) and Cunningham BC & wells JA (1989) (supra) for a description of mutagenesis techniques, such as alanine scanning mutagenesis techniques. In certain embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis testing. Furthermore, antibodies that recognize and bind to the same or overlapping epitopes of MSLN (e.g., human MSLN) can be identified using routine methods, such as immunoassays, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as MSLN (e.g., human MSLN). Numerous types of competitive binding assays are known: for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15), solid-phase direct biotin-avidin EIA (Cheung RC et al., (1988) Mol Immunol 25(1):7-15), al., (1990) Virology 176:546-52), and direct labeling RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entireties.Typically, such assays involve the use of purified antigen (e.g., MSLN, e.g., human MSLN) bound to a solid surface or cells bearing either an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, the test immunoglobulin inhibits specific binding of the reference or antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more when the competing antibody is present in excess. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibodies. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibody to the antigen is then measured using a radioactive or enzymatic label. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp. 386-389, all of which are incorporated herein by reference in their entireties.

[0103] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 83, 84, 41, 42, 43, 44, 45, 46, 66, 67, 68, 69, 70, or 71. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence represented by SEQ ID NO: 83, 84, 41, 42, 43, 44, 45, 46, 66, 67, 68, 69, 70, or 71.

[0104] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a light chain comprising an amino acid sequence represented by SEQ ID NO: 47, 48, 49, 50, 51, or 52. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 48, 49, 50, 51, or 52.

[0105] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), comprising a heavy chain and a light chain, the heavy chain and the light chain being set forth in SEQ ID NOs: 83 and 48, 84 and 48, 83 and 49, 84 and 49, 83 and 50, 84 and 50, 83 and 51, 84 and 51, 83 and 52, 84 and 52, 83 and 47, 84 and 47, 41 and 47, 41 and 48, 41 and 49, 41 and 50, 41 and 51, 41 and 52, 42 and 47, 42 and 48, 42 and 49, 42 and 50, 42 and 51, 42 and 52, 43 and 47, 43 and 48, 43 and 49, 43 and 50, 43 and 51, 43 and 52, 44 and 47, 44 and 48, 44 and 49, 44 and 50, 44 and 51, 44 and 52, 45 and 47, 45 and and 48, 45 and 49, 45 and 50, 45 and 51, 45 and 52, 46 and 47, 46 and 48, 46 and 49, 46 and 50, 46 and 51, 46 and 52, 66 and 47, 66 and 48, 66 and 49, 66 and 50, 66 and 51, 66 and 52, 67 and 47, 67 and 48, 67 and 49, 67 and 50, 67 and 51, 67 and 52, 68 and 47, 68 and and 51, 68 and 52, 69 and 47, 69 and 48, 69 and 49, 69 and 50, 69 and 51, 69 and 52, 70 and 47, 70 and 48, 70 and 49, 70 and 50, 70 and 51, 70 and 52, 71 and 47, 71 and 48, 71 and 49, 71 and 50, 71 and 51, or 71 and 52.

[0106] In certain embodiments, the amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 83 and 48, 84 and 48, 83 and 49, 84 and 49, 83 and 50, 84 and 50, 83 and 51, 84 and 51, 83 and 52, 84 and 52, 83 and 47, 84 and 47, 41 and 47, 41 and 48, 41 and 49, 41 and 50, 41 and 51, 41 and 52, 42 and 47, 42 and 48, 42 and 49, 42 and 50, 42 and 51, 42 and 52, 43 and 47, 43 and 48, 43 and 49, 43 and 50, 43 and 51, 43 and 52, 44 and 47, 44 and 48, 44 and 49, 44 and 50, 44 and 51, 44 and 52, 45 and 47, 45 and 48, 45 and 49, 45 and 50, 45 and 51, 4 5 and 52, 46 and 47, 46 and 48, 46 and 49, 46 and 50, 46 and 51, 46 and 52, 66 and 47, 66 and 48, 66 and 49, 66 and 50, 66 and 51, 66 and 52, 67 and 47, 67 and 48, 67 and 49, 67 and 50, 67 and 51, 67 and 52, 68 and 47, 68 and 48, 68 and 49, 68 and 50, 68 and 51, 68 and 52, 69 and 47, 69 and 48, 69 and 49, 69 and 50, 69 and 51, 69 and 52, 70 and 47, 70 and 48, 70 and 49, 70 and 50, 70 and 51, 70 and 52, 71 and 47, 71 and 48, 71 and 49, 71 and 50, 71 and 51, or 71 and 52.

[0107] In certain embodiments, the disclosure provides antibodies that specifically bind to MSLN (e.g., human MSLN) and a T cell antigen (e.g., CD3).

[0108] In certain embodiments, the present disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding moiety. Any CD3 binding moiety known in the art can be used in the antibodies and polypeptides disclosed herein, including, but not limited to, polypeptides, aptamers, and small molecule CD3 binding moieties. In certain embodiments, the CD3 binding moiety is an antibody. In certain embodiments, the CD3 binding moiety is a single chain fragment variable (scFv). The VH and VL amino acid sequences of exemplary anti-CD3 antibodies (e.g., scFv) are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0109] In certain embodiments, the CD3 binding portion is an anti-CD3 antibody (e.g., an scFv) that comprises the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the amino acid sequence of the CD3 binding portion consists of the amino acid sequence set forth in SEQ ID NO:76.

[0110] In certain embodiments, the CD3 binding moiety is covalently linked to the light chain. In certain embodiments, the CD3 binding moiety is covalently linked to the C-terminus of the light chain. In certain embodiments, the CD3 binding moiety is covalently linked to the C-terminus of the light chain via a peptide linker. In certain embodiments, the peptide linker comprises the amino acid sequence represented by SEQ ID NO: 63, 64, 73, 74, or 75.

[0111] In certain embodiments, an antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding moiety has a light chain comprising an amino acid sequence represented by any one of SEQ ID NOs: 77, 78, 79, 80, 81, or 82. In certain embodiments, an antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding moiety has a light chain consisting of an amino acid sequence represented by any one of SEQ ID NOs: 77, 78, 79, 80, 81, or 82.

[0112] In certain embodiments, antibodies that specifically bind to MSLN (e.g., human MSLN) and further comprise a CD3 binding portion are selected from the group consisting of SEQ ID NOs: 83 and 77, 84 and 77, 41 and 77, 42 and 77, 43 and 77, 44 and 77, 45 and 77, 46 and 77, 66 and 77, 67 and 77, 68 and 77, 69 and 77, 70 and 77, 71 and 77, 83 and 78 ...85 and 77, 86 and 77, 87 and 77, 88 and 77, 89 and 77, 90 and 77, 91 and 77, 92 and 77, 93 and 78, 94 and 77, 95 and 77, 96 and 77, 97 and 77, 98 and 77, 99 and 77, 100 and 101, 102 and 103, 104 and 105, 106 and 107, 108 and 109, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 1 4 and 78, 41 and 78, 42 and 78, 43 and 78, 44 and 78, 45 and 78, 46 and 78, 66 and 78, 67 and 78, 68 and 78, 69 and 78, 70 and 78, 71 and 78, 83 and 79, 84 and 79, 41 and 79, 42 and 79, 43 and 79, 44 and 79, 45 and 79, 46 and 79, 66 and 79, 67 and 79, 68 and 79, 69 and 79, 70 and 79, 71 and 79, 83 and 80, 84 and 80, 41 and 80, 42 and 80, 43 and 80, 44 and 80, 45 and 80, 46 and 80, 66 and 80, 67 and 80, 68 and 80, 69 and 80, 70 and 80, 71 and 80, 83 and 81, 84 and 81, 41 and 81, 42 and 81, 43 and 81, 44 and 81, 45 and 81, 46 and and 81, 66 and 81, 67 and 81, 68 and 81, 69 and 81, 70 and 81, 71 and 81, 83 and 82, 84 and 82, 41 and 82, 42 and 82, 43 and 82, 44 and 82, 45 and 82, 46 and 82, 66 and 82, 67 and 82, 68 and 82, 69 and 82, 70 and 82, or 71 and 82.

[0113] In certain embodiments, antibodies that specifically bind to MSLN (e.g., human MSLN) and further comprise a CD3 binding portion are selected from the group consisting of SEQ ID NOs: 83 and 77, 84 and 77, 41 and 77, 42 and 77, 43 and 77, 44 and 77, 45 and 77, 46 and 77, 66 and 77, 67 and 77, 68 and 77, 69 and 77, 70 and 77, 71 and 77, 83 and 78 ...85 and 77, 86 and 77, 87 and 77, 88 and 77, 89 and 77, 90 and 77, 91 and 77, 92 and 77, 93 and 78, 94 and 77, 95 and 77, 96 and 77, 97 and 77, 98 and 77, 99 and 77, 100 and 101, 102 and 103, 104 and 105, 106 and 107, 108 and 109, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 1 4 and 78, 41 and 78, 42 and 78, 43 and 78, 44 and 78, 45 and 78, 46 and 78, 66 and 78, 67 and 78, 68 and 78, 69 and 78, 70 and 78, 71 and 78, 83 and 79, 84 and 79, 41 and 79, 42 and 79, 43 and 79, 44 and 79, 45 and 79, 46 and 79, 66 and 79, 67 and 79, 68 and 79, 69 and and 79, 70 and 79, 71 and 79, 83 and 80, 84 and 80, 41 and 80, 42 and 80, 43 and 80, 44 and 80, 45 and 80, 46 and 80, 66 and 80, 67 and 80, 68 and 80, 69 and 80, 70 and 80, 71 and 80, 83 and 81, 84 and 81, 41 and 81, 42 and 81, 43 and 81, 44 and 81, 45 and 81, 46 and The heavy chains and light chains include those having the amino acid sequences represented by the following: 81, 66 and 81, 67 and 81, 68 and 81, 69 and 81, 70 and 81, 71 and 81, 83 and 82, 84 and 82, 41 and 82, 42 and 82, 43 and 82, 44 and 82, 45 and 82, 46 and 82, 66 and 82, 67 and 82, 68 and 82, 69 and 82, 70 and 82, or 71 and 82.

[0114] In certain embodiments, an antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding moiety comprises heavy and light chains that comprise the amino acid sequences set forth in SEQ ID NOs: 83 and 77, respectively. In certain embodiments, an antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding moiety comprises heavy and light chains consisting of the amino acid sequences set forth in SEQ ID NOs: 83 and 77, respectively.

[0115] In certain embodiments, the antibody that specifically binds to MSLN (e.g., human MSLN) and further comprises a CD3 binding portion is a bispecific antigen-binding molecule having the structure of FIG. 4. Molecules having the structure of FIG. 4 are bispecific and bivalent for each of the target antigens (e.g., MSLN and CD3) and are also referred to herein as IgG-scFv molecules. These molecules comprise a conventional monospecific antibody structure in which the antigen-binding domain is specific for MSLN, and also comprise two scFv domains, each covalently linked to the C-terminus of two light chain constant regions, where the scFv domains are oriented in a VH-VL orientation (i.e., the VH portion of each of the two scFv domains is covalently linked to the C-terminus of the respective light chain constant region), and specific for a T cell antigen (TCA) (e.g., CD3). In any of the bispecific molecules disclosed herein, including those having the structure of FIG. 4, the anti-cancer or anti-MSLN antigen-binding domain(s) may be derived from any of the anti-MSLN antibodies disclosed herein. For example, the anti-MSLN antigen-binding domain may comprise the CDRs and / or variable regions of the anti-MSLN antibody disclosed herein. The anti-MSLN monospecific antibody or anti-MSLNxanti-TCA (e.g., CD3) bispecific antigen-binding molecule of the present disclosure may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0116] In certain embodiments, the antibodies disclosed herein are conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, a cytotoxic agent is capable of inducing the death or destruction of a cell in contact with the agent. In certain embodiments, a cytostatic agent is capable of preventing or substantially reducing the proliferation of a cell in contact with the agent and / or inhibiting the activity or function of a cell. In certain embodiments, the cytotoxic agent or cytostatic agent is a chemotherapeutic agent. In certain embodiments, a radionuclide is an isotope 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 Re. In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0117] Any immunoglobulin (Ig) constant region may be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). b ).

[0118] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), optionally comprising a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0119] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0120] In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 72, 53, 54, 55, or 56. In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 72, 53, 54, 55, or 56.

[0121] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced in the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or the hinge region (residues 216-230, numbered according to the EU numbering system) of an antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0122] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein, such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), for example, as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered, for example, to facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0123] In certain embodiments, one, two or more amino acid mutations (e.g., substitution, insertion or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to change (e.g., shorten or extend) the half-life of the antibody in vivo.See, for example, International Publication Nos. WO02 / 060919, WO98 / 23289; and WO97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 (all of which are incorporated herein by reference in their entirety).For example, mutations that change (e.g., shorten or extend) the half-life of the antibody in vivo. In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or FcRn-binding fragment thereof (preferably the Fc or hinge-Fc fragment), to shorten the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or FcRn-binding fragment thereof (preferably the Fc or hinge-Fc fragment), to extend the half-life of the antibody in vivo. In certain embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In certain embodiments, the IgG1 constant region of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety.This type of mutant IgG, referred to as "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0124] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1 (numbered according to the EU numbering system) and / or the hinge region (residues 216-230, numbered according to the EU numbering system) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or a fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to change the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., J. Immunol. 1999, 143:1311-1315, and WO 97 / 13311, the disclosure of which is incorporated herein by reference. al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entireties.

[0125] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, where the variant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D, and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (according to the EU numbering system). In certain embodiments, FcγRIIB is expressed in cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0126] In further embodiments, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 (numbered according to the EU numbering system) can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation of the constant region domain (by point mutation or other means) may reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization, each of which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604, incorporated herein by reference in its entirety).In various embodiments, one or more of the following mutations in the constant region of the antibodies described herein may occur: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; S239D substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution, which are numbered according to the EU numbering system.

[0127] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of the antibody described herein.

[0128] In certain embodiments, the antibodies described herein comprise an IgG1 constant region with an N297Q or N297A amino acid substitution, as numbered according to the EU numbering system. In certain embodiments, the antibodies described herein comprise an IgG1 constant region with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, as numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant region with a mutation selected from the group consisting of L234A, L235A, and combinations thereof, as numbered according to the EU numbering system. In another embodiment, the antibodies described herein comprise an IgG1 constant region with a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, as numbered according to the EU numbering system. In certain embodiments, the amino acid residues of the constant region of the antibodies described herein at positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain, as numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, numbered according to the EU numbering system.

[0129] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of the antibodies described herein, numbered according to the EU numbering system, can be replaced with a different amino acid residue, thereby causing the antibody to have altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogene et al.), which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 (numbered according to the EU numbering system) in the N-terminal region of the CH2 domain of the antibodies described herein are modified, thereby altering the ability of the antibody to fix complement. This approach is described further in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 5, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 (numbered according to the EU numbering system). This approach is described in detail in International Publication No. WO00 / 42072, the entire contents of which are incorporated herein by reference.

[0130] In certain embodiments, the antibodies described herein comprise a modified constant region of IgG1, which modification enhances the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 μg / mL of the antibody can induce cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of MSLN-expressing cells within 1, 2, or 3 hours, as assessed by the methods described herein and / or known to those of skill in the art. In certain embodiments, the modified constant region of IgG1 comprises the substitutions S239D and I332E, numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 comprises the substitutions S239D, A330L, and I332E, numbered according to the EU numbering system. In certain embodiments, the modified constant region of IgG1 comprises the following substitutions, numbered according to the EU numbering system: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, and the percentage of Tregs undergoing cell death is at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold higher than the percentage of effector T cells undergoing cell death.

[0131] In certain embodiments, the antibodies described herein comprise an IgG4 antibody constant region, in which a serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with a proline. In certain embodiments, the disclosure provides an antibody that specifically binds to MSLN (e.g., human MSLN), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 16 or 22.

[0132] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein that has two heavy chain constant regions.

[0133] Pharmaceutical Compositions Provided herein are compositions comprising the anti-MSLN antibodies disclosed herein having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, and the like. hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0134] In certain embodiments, the pharmaceutical composition comprises an anti-MSLN antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an anti-MSLN antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In certain embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for increasing or promoting the activity of MSLN (e.g., human MSLN) and treating conditions such as cancer. In certain embodiments, the present disclosure relates to a pharmaceutical composition of the present disclosure comprising an anti-MSLN antibody of the present disclosure for use as a medicament. In another embodiment, the present disclosure relates to a pharmaceutical composition of the present disclosure for use in a method for the treatment of cancer.

[0135] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Sequestering or chelating agents of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0136] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by injection, either subcutaneous, intramuscular, or intravenous, is also contemplated herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, in solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also include one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also include small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0137] Preparations for parenteral administration of antibodies include sterile solutions ready for injection, sterile, dry soluble products that can be combined with a solvent immediately prior to use, such as lyophilized powders, subcutaneous tablets, sterile suspensions ready for injection, sterile, dry insoluble products that can be combined with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0138] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0139] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigate, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0140] The anti-MSLN antibodies disclosed herein may be formulated as aerosols for topical application, such as inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entireties). These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or a microfine powder for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will, in certain embodiments, have a diameter of less than 50 microns, and in certain embodiments, less than 10 microns.

[0141] The anti-MSLN antibodies disclosed herein can be formulated for local or topical application, for example, topical application to the skin and mucous membranes, for example, the eye, in the form of gels, creams, and lotions, and for application to the eye, or for intracapsular or intrathecal application. Topical administration is contemplated for transdermal delivery, administration to the eye or mucous membranes, or inhalation therapy. Nasal solutions of antibodies can also be administered alone or in combination with other pharma- ceutically acceptable excipients.

[0142] Transdermal patches, such as iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference.

[0143] In certain embodiments, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder and can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein, or a pharma- ceutically acceptable derivative thereof, in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients or other pharmacological components of the powder that improve stability, or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as a citrate buffer, a sodium phosphate buffer, or a potassium phosphate buffer, or other such buffer known to those of skill in the art, in certain embodiments, a buffer with a pH of about neutral. The solution is then sterile filtered under standard conditions known to those of skill in the art, followed by lyophilization to provide the desired formulation. In certain embodiments, the resulting solution will be apportioned into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at room temperature at about 4°C. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. When reconstituted, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amount can be empirically determined.

[0144] The anti-MSLN antibodies disclosed herein and other compositions provided herein can also be formulated to be targeted to specific tissues, receptors, or other areas of the body of the subject being treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,071,570 ... See, for example, Nos. 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In certain embodiments, the antibodies described herein are targeted to tumors.

[0145] Compositions to be used for in vivo administration will generally be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0146] Method and use In another aspect, the present disclosure provides a method of treating a subject using the anti-MSLN antibody disclosed herein. Any disease or disorder in a subject that would benefit from reduced MSLN (e.g., human MSLN) function can be treated using the anti-MSLN antibody disclosed herein. In certain embodiments, the disease or disorder is resistant to a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody). In certain embodiments, the disease or disorder recurs after treatment with a checkpoint targeting agent (e.g., an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, or an antagonistic anti-PD-1 antibody).

[0147] The anti-MSLN antibodies disclosed herein are particularly useful for inhibiting immune system tolerance to tumors and can therefore be used as immunotherapy for subjects with cancer. For example, in certain embodiments, the present disclosure provides methods for inhibiting the proliferation of T cells (e.g., CD8 + cytotoxic T cells, CD4 + The present disclosure provides a method for increasing activation of T helper cells, NKT cells, effector T cells, or memory T cells, comprising administering to a subject an effective amount of an anti-MSLN antibody or pharmaceutical composition thereof as disclosed herein. In certain embodiments, the present disclosure provides a method for treating cancer in a subject, comprising administering to a subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0148] Cancers that may be treated with the anti-MSLN antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In certain embodiments, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as lung, breast, ovarian, lymphatic, gastrointestinal (e.g., colon), anal, genital and urogenital tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neuronal or glial cells), head and neck, skin (e.g., melanoma), and pancreatic, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, hepatic cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small intestine cancer, and esophageal cancer. The cancer may be early stage, mid-stage, late stage, or metastatic. In certain embodiments, the cancer is resistant to a checkpoint targeting agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody). In certain embodiments, the cancer recurs after treatment with a checkpoint targeting agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody).

[0149] In certain embodiments, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), renal cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptors, progesterone receptors, or Her2 / neu, e.g., triple-negative breast cancer), ovarian cancer, colon cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, esophageal-gastric cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal carcinoma, thyroid cancer, cervical cancer, epithelial carcinoma, peritoneal cancer, or lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease).

[0150] In certain embodiments, the cancer is a blood cancer, such as leukemia, lymphoma, or myeloma.In certain embodiments, the cancer is a leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CMML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In certain embodiments, the cancer is lymphoma, for example, B cell lymphoma, diffuse large B cell lymphoma (DLBCL), activated B cell-like (ABC) diffuse large B cell lymphoma, germinal center B cell (GCB) diffuse large B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma.In certain embodiments, the cancer is myeloma, for example, multiple myeloma.

[0151] In another embodiment, the cancer is selected from carcinoma (eg, advanced or metastatic carcinoma), melanoma, or lung adenocarcinoma, eg, non-small cell lung carcinoma.

[0152] In certain embodiments, the cancer is lung cancer, e.g., lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0153] In certain embodiments, the cancer is melanoma, for example, progressive melanoma. In certain embodiments, the cancer is progressive or unresectable melanoma that does not respond to other therapies. In other embodiments, the cancer is melanoma with BRAF mutation (e.g., BRAF V600 mutation). In still other embodiments, the anti-MSLN antibody or pharmaceutical composition disclosed herein is administered after treatment with anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0154] In another embodiment, the cancer is hepatocellular carcinoma, e.g., advanced hepatocellular carcinoma, with or without associated viral infection, e.g., chronic viral hepatitis.

[0155] In another embodiment, the cancer is prostate cancer, e.g., aggressive prostate cancer.

[0156] In yet another embodiment, the cancer is a myeloma, e.g., multiple myeloma.

[0157] In yet another embodiment, the cancer is a renal cancer, for example, a renal cell carcinoma (RCC) (eg, metastatic RCC, clear cell renal cell carcinoma (CCRCC), or papillary cell carcinoma of the kidney).

[0158] In yet another embodiment, the cancer is selected from lung cancer, melanoma, renal cancer, breast cancer, colon cancer, leukemia, or a metastatic lesion of cancer.

[0159] In certain embodiments, the method further comprises administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent. In certain embodiments, the chemotherapeutic agent is a demethylating agent (e.g., azacytidine). In certain embodiments, the chemotherapeutic agent is a DNA damage inducing agent (e.g., gemcitabine). In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-PD-1 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD96 antibody, an antagonistic anti-CEACAM1 antibody, an agonist anti-CD137 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, and an antagonist anti-PD-1 antibody, wherein the MSLN (e.g., human MSLN) antibody or pharmaceutical composition disclosed herein synergizes with the checkpoint targeting agent.

[0160] In certain embodiments, the present disclosure relates to an antibody and / or pharmaceutical composition of the present disclosure for use in a method of the present disclosure, the method further comprising administering an additional therapeutic agent to the subject. In certain embodiments, the present disclosure relates to (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent for use as a medicament. In certain embodiments, the present disclosure relates to (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent for use in a method for treating cancer. In further embodiments, the present disclosure relates to a pharmaceutical composition, kit, or kit-of-parts comprising (a) an antibody and / or pharmaceutical composition of the present disclosure, and (b) an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent.

[0161] In certain embodiments, anti-PD-1 antibodies are used in the methods disclosed herein. In certain embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Mediimmune. In certain embodiments, the anti-PD-1 antibody is PDR001, developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0162] Further non-limiting examples of anti-PD-1 antibodies that may be used in the therapeutic methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated by reference in their entirety for all purposes: U.S. Pat. No. 6,808,710; U.S. Pat. No. 7,332,582; U.S. Pat. No. 7,488,802; U.S. Pat. No. 8,008,449; U.S. Pat. No. 8,114,845; U.S. Pat. No. 8,168,757; U.S. Pat. No. 8,354,509; U.S. Pat. No. 8,686,119; U.S. Pat. No. 8,735,553; U.S. Pat. No. 8,747,847; U.S. Pat. No. 8,779,105; U.S. Pat. No. 8,927,697; U.S. Pat. No. 8,993,731; U.S. Pat. No. 9,102,727; U.S. Pat. No. 9,205,148; U.S. Patent Application Publication No. U.S. Pat. No. S2013 / 0202623A1; U.S. Patent Application Publication No. US2013 / 0291136A1; U.S. Patent Application Publication No. US2014 / 0044738A1; U.S. Patent Application Publication No. US2014 / 0356363A1; U.S. Patent Application Publication No. US2016 / 0075783A1; and PCT Publication No. WO2013 / 033091A1; PCT Publication No. WO2015 / 036394A1; PCT Publication No. PCT Publication No. WO2014 / 179664A2; PCT Publication No. WO2014 / 209804A1; PCT Publication No. WO2014 / 206107A1; PCT Publication No. WO2015 / 058573A1; PCT Publication No. WO2015 / 085847A1; PCT Publication No. WO2015 / 200119A1; PCT Publication No. WO2016 / 015685A1; and PCT Publication No. WO2016 / 020856(A1).

[0163] In certain embodiments, anti-PD-L1 antibodies are used in the methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is atezolizumab, developed by Genentech. In certain embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene, and Mediimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

[0164] Non-limiting examples of anti-PD-L1 antibodies that may be used in the therapeutic methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated by reference in their entirety for all purposes: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Patent Application Publication No. US2010 / 0203056(A1); U.S. Patent Application Publication No. US2003 / 0232323(A1); U.S. Patent Application Publication No. US2013 / 0323249(A1); U.S. Patent Application Publication No. US2014 / 0341917(A1); U.S. Patent Application Publication No. US2015 / 0323249(A1); No. US2014 / 0044738(A1); U.S. Patent Application Publication No. US2015 / 0203580(A1); U.S. Patent Application Publication No. US2015 / 0225483(A1); U.S. Patent Application Publication No. US2015 / 0346208(A1); U.S. Patent Application Publication No. US2015 / 0355184(A1); and PCT Publication No. WO2014 / 100079(A1); PCT Publication No. WO2014 / 022758(A1); PCT Publication No. WO2014 / 055897(A2); PCT Publication No. WO2015 / 061668(A1); PCT Publication No. WO2015 / 109124(A1); PCT Publication No. WO2015 / 195163(A1); PCT Publication No. WO2016 / 000619(A1); and PCT Publication No. WO2016 / 030350(A1).

[0165] In certain embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab, developed by Bristol-Myers Squibb.

[0166] In certain embodiments, the anti-MSLN antibodies disclosed herein are administered to a subject in combination with a compound that targets an immunomodulatory enzyme(s), such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Thus, in certain embodiments, the additional therapeutic agent is a compound that targets an immunomodulatory enzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certain embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see, e.g., WO2010 / 005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In certain embodiments, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In certain embodiments, the anti-MSLN antibody disclosed herein is administered to a subject in combination with an IDO inhibitor for treating cancer. The IDO inhibitor described herein for use in treating cancer is present in a solid dosage form of a pharmaceutical composition, such as a tablet, pill, or capsule, the pharmaceutical composition comprising an IDO inhibitor and a pharma- ceutically acceptable excipient. Thus, the antibody described herein and the IDO inhibitor described herein can be administered separately, sequentially, or simultaneously as separate dosage forms. In certain embodiments, the antibody is administered parenterally and the IDO inhibitor is administered orally. In certain embodiments, the inhibitor is selected from the group consisting of epacadostat (Incyte Corporation), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics).Epacadostat is described in PCT Publication No. WO2010 / 005958, which is incorporated herein by reference in its entirety for all purposes.In certain embodiments, the inhibitor is epacadostat.In another embodiment, the inhibitor is F001287.In another embodiment, the inhibitor is indoximod.In another embodiment, the inhibitor is NLG919.

[0167] In certain embodiments, the anti-MSLN antibodies disclosed herein are administered to a subject in combination with a vaccine. The vaccine can be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine.

[0168] In certain embodiments, the anti-MSLN antibodies disclosed herein are administered to a subject in combination with an adjuvant. Depending on the treatment situation, various adjuvants may be used. Non-limiting examples of suitable adjuvants include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Montanide ISA (incomplete Seppic adjuvant), Ribi adjuvant system (RAS), Titer Max, muramyl peptides, Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvant, nitrocellulose-absorbed antigens, encapsulated or entrapped antigens, 3 De-O-acylated monophosphoryl lipid A (3 D-MPL), immunostimulatory oligonucleotides, Toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, saponins, Quil A, QS-21, QS-7, immune stimulatory complexes such as ISCOMATRIX. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules, such as poly(A) and poly(U).Combinations of the above adjuvants may also be used.For example, see U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety.In certain embodiments, the adjuvant used herein is QS-21 STIMULON.

[0169] In certain embodiments, the anti-MSLN antibody disclosed herein is administered to a subject in combination with an additional therapeutic agent comprising a TCR. In certain embodiments, the additional therapeutic agent is a soluble TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a TCR. Thus, in certain embodiments, the present disclosure relates to an antibody and / or pharmaceutical composition of the present disclosure in combination with an additional therapeutic agent comprising a TCR for use as a medicament and / or for use in a method for treating cancer.

[0170] In certain embodiments, the anti-MSLN antibodies disclosed herein are administered to a subject in combination with a cell expressing a chimeric antigen receptor (CAR). In certain embodiments, the cell is a T cell.

[0171] In certain embodiments, the anti-MSLN antibody disclosed herein is administered to a subject in combination with a TCR mimetic antibody.In certain embodiments, the TCR mimetic antibody is an antibody that specifically binds to a peptide-MHC complex.For non-limiting examples of TCR mimetic antibodies, see, for example, U.S. Patent No. 9,074,000 and U.S. Publication No. US2009 / 0304679A1 and US2014 / 0134191(A1).All of these are incorporated herein by reference in their entirety.

[0172] In certain embodiments, the anti-MSLN antibodies disclosed herein are administered to a subject in combination with a bispecific T cell engager (BiTE) (e.g., as described in WO2005061547A2, which is incorporated herein by reference in its entirety) and / or a dual affinity retargeting antibody (DART) (e.g., as described in WO2012162067A2, which is incorporated herein by reference in its entirety). In certain embodiments, the BiTE and / or DART specifically bind to a tumor-associated antigen (e.g., a polypeptide overexpressed in a tumor, a polypeptide derived from an oncovirus, a polypeptide comprising a tumor-specific post-translational modification, a polypeptide specifically mutated in a tumor) and a molecule on an effector cell (e.g., CD3 or CD16). In certain embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), and optionally, the BiTE and / or DART comprises the VH and VL sequences of cetuximab. In certain embodiments, the tumor-associated antigen is Her2 (e.g., human Her2), and optionally, the BiTE and / or DART comprises the VH and VL sequences of trastuzumab. In certain embodiments, the tumor-associated antigen is CD20 (e.g., human CD20).

[0173] The anti-MSLN antibody and additional therapeutic agent (e.g., chemotherapeutic agent, radiotherapeutic agent, checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, soluble TCR, TCR-expressing cell, chimeric antigen receptor expressing cell, and / or TCR mimetic antibody) can be administered separately, sequentially, or simultaneously as separate dosage forms. In certain embodiments, the anti-MSLN antibody is administered parenterally and the IDO inhibitor is administered orally.

[0174] The antibodies or pharmaceutical compositions described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and by formulating with an aerosolizing agent for use as a spray. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intraarterially. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered to a tumor draining lymph node.

[0175] The amount of antibody or composition that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0176] The exact dosage to be used in the composition depends on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the judgment of the physician and the circumstances of each subject. For example, the effective dosage may vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is a human or an animal, other drugs administered, or whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, such as transgenic mammals, may also be treated. Therapeutic dosages may generally be optimally titrated to optimize safety and efficacy.

[0177] The anti-MSLN antibodies described herein can also be used to assay MSLN (e.g., human MSLN) protein levels in biological samples using classical immunohistological methods known to those skilled in the art, such as immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, e.g., glucose oxidase; radioisotopes, e.g., iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody that recognizes the anti-MSLN antibody described herein can be labeled and used in conjunction with the anti-MSLN antibody to detect MSLN (e.g., human MSLN) protein levels. Thus, in certain embodiments, the present disclosure relates to the use of the anti-MSLN antibody of the present disclosure for in vitro detection of MSLN (e.g., human MSLN) protein in a biological sample. In further embodiments, the present disclosure relates to the use of the anti-MSLN antibody of the present disclosure for assaying and / or detecting MSLN (e.g., human MSLN) protein levels in a biological sample in vitro, optionally where the anti-MSLN antibody is conjugated to a radionuclide or detectable label and / or carries a label as described herein, and / or where immunohistological methods are used.

[0178] Assaying the expression level of MSLN (e.g., human MSLN) protein is intended to include measuring or estimating the level of MSLN (e.g., human MSLN) protein in a first biological sample, either qualitatively or quantitatively, directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing with disease-related protein levels in a second biological sample). The MSLN (e.g., human MSLN) polypeptide expression level in a first biological sample can be measured or estimated and compared to a standard MSLN (e.g., human MSLN) protein level, which can be taken, for example, from a second biological sample obtained from an individual without the disorder or determined by averaging levels from a population of individuals without the disorder. As is understood in the art, once a "standard" MSLN (e.g., human MSLN) polypeptide level is known, it can be used repeatedly as a standard for comparison. Thus, in a further embodiment, the present disclosure relates to an in vitro method for assaying and / or detecting MSLN protein levels, e.g., human MSLN protein levels, in a biological sample, including qualitatively or quantitatively measuring or estimating the level of MSLN protein, e.g., human MSLN protein, in a biological sample by immunohistological methods.

[0179] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cell source potentially expressing MSLN (e.g., human MSLN). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs).

[0180] The anti-MSLN antibodies described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications that are well known and standard to those skilled in the art and based on the present description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro determination and evaluation of immune system status and / or immune response may be used to predict, diagnose, and monitor, including those known or suspected to have immune system dysfunction, or to evaluate patient samples for expected or desired immune system response, antigen response, or vaccine response. Determination and evaluation of immune system status and / or immune response is also useful to determine the suitability of a patient for drug clinical trials or for administration of a particular chemotherapeutic agent, radiotherapeutic agent, or antibody (including combinations thereof) compared to a different agent or antibody. This type of prognostic and diagnostic monitoring and evaluation has already been performed using antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), where the assay is also used to evaluate patients for antibody therapy using Herceptin®. In vivo applications include directed cell therapy and radioimaging of immune system regulation and immune responses. Thus, in certain embodiments, the present disclosure relates to an anti-MSLN antibody and / or pharmaceutical composition of the present disclosure for use as a diagnostic agent. In certain embodiments, the present disclosure relates to an anti-MSLN antibody and / or pharmaceutical composition of the present disclosure for use in a method for predicting, diagnosing and / or monitoring a subject having or suspected of having an immune system dysfunction, and / or for predicting or desired immune system responses, antigen responses or vaccine responses. In another embodiment, the present disclosure relates to the use of an anti-MSLN antibody of the present disclosure for predicting, diagnosing and / or monitoring a subject having or suspected of having an immune system dysfunction, and / or for predicting or desired immune system responses, antigen responses or vaccine responses, by assaying and / or detecting human MSLN protein levels in a biological sample of the subject in vitro.

[0181] In certain embodiments, anti-MSLN antibodies may be used for immunohistochemistry of biopsy samples. In certain embodiments, the method is an in vitro method. In another embodiment, anti-MSLN antibodies may be used to detect levels of MSLN (e.g., human MSLN) or levels of cells containing MSLN (e.g., human MSLN) on their membrane surface, which levels can then be related to specific disease symptoms. The anti-MSLN antibodies described herein may carry a detectable or functional label and / or be conjugated to a radionuclide or detectable label. When using fluorescent labels, specific binding members can be identified and quantified using currently available microscopy and fluorescence activated cell sorting analysis (FACS), or a combination of both procedures known in the art. The anti-MSLN antibodies described herein may carry or be conjugated to a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarins, fluorescein, and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The anti-MSLN antibodies may be radiolabeled or conjugated with radionuclides, e.g., isotopes. 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186Re, etc. When a radioactive label is used, the specific binding of the anti-MSLN antibody to MSLN (e.g., human MSLN) can be identified and quantified using currently available counting procedures known in the art. When the label is an enzyme, detection can be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric methods known in the art. This can be achieved by contacting the sample or control sample under conditions that allow the formation of a complex between the anti-MSLN antibody and MSLN (e.g., human MSLN). Any complex formed between the anti-MSLN antibody and MSLN (e.g., human MSLN) is detected and compared in the sample and the control. In light of the fact that the anti-MSLN antibody described herein specifically binds to MSLN (e.g., human MSLN), the anti-MSLN antibody can be used to specifically detect MSLN (e.g., human MSLN). The anti-MSLN antibodies described herein can also be used to purify MSLN (e.g., human MSLN) via immunoaffinity purification. Also included herein are assay systems that can be prepared in the form of a test kit, kit, or kit-of-parts, for example, to quantitatively analyze the extent to which MSLN (e.g., human MSLN) / MSLN (e.g., human MSLN) ligand complexes are present. The systems, test kits, kits, or kits-of-parts can include a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.

[0182] Polynucleotides, Vectors, and Methods for Producing Antibodies In another aspect, provided herein are polynucleotides comprising nucleotide sequences encoding an antibody or portion thereof or fragment thereof (e.g., VL and / or VH; and light and / or heavy chains) described herein that specifically binds to an MSLN (e.g., human MSLN) antigen, and vectors comprising such polynucleotides for recombinant expression in a host cell (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding the heavy and / or light chains of any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for efficient expression in a host cell, e.g., a mammalian cell.

[0183] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques or chemically synthesized if substantially free of chemical precursors or other chemicals. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In certain embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0184] In certain aspects, provided herein are antibodies that specifically bind to an MSLN (e.g., human MSLN) polypeptide and comprise an amino acid sequence described herein, as well as polynucleotides comprising nucleotide sequences encoding antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to an MSLN (e.g., human MSLN) polypeptide, or antibodies that bind to the same epitope as such antibodies.

[0185] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising the VL FRs and CDRs of an antibody described herein (see, e.g., Table 1), or a nucleotide sequence encoding a heavy chain comprising the VH FRs and CDRs of an antibody described herein (see, e.g., Table 1). In certain embodiments, the polynucleotide encodes a VH, VL, heavy chain, and / or light chain described herein. In another embodiment, the polynucleotide encodes a first VH and a first VL described herein. In another embodiment, the polynucleotide encodes a second VH and a second VL described herein. In another embodiment, the polynucleotide encodes a first heavy chain and a first light chain described herein. In another embodiment, the polynucleotide encodes a second heavy chain and a second light chain described herein. In another embodiment, the polynucleotide encodes a VH and / or VL, or a heavy chain and / or light chain of an antibody described herein.

[0186] Also provided herein are polynucleotides encoding anti-MSLN antibodies that have been optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-MSLN antibodies or fragments thereof (such as light chains, heavy chains, VH domains, VL domains, etc.) for recombinant expression by introducing codon changes and / or removing inhibitory regions in the mRNA can be performed, for example, by applying the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. Such alterations take advantage of the degeneracy of the genetic code, e.g., using alternative codons for the same amino acid. In certain embodiments, conservative mutations, e.g., changing one or more codons to code for similar amino acids having similar chemical structures and properties and / or functions as the original amino acid, may be desirable. Such methods can increase the expression of an anti-MSLN antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the expression of an anti-MSLN antibody encoded by a non-optimized polynucleotide.

[0187] In certain embodiments, an optimized polynucleotide sequence encoding an anti-MSLN antibody or fragment thereof (e.g., VL and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-MSLN antibody or fragment thereof (e.g., VL and / or VH domain) described herein. In certain embodiments, an optimized nucleotide sequence encoding an anti-MSLN antibody or fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-MSLN antibody or fragment thereof described herein. In certain embodiments, an optimized nucleotide sequence encoding an anti-MSLN antibody or fragment thereof described herein hybridizes under high stringency, medium stringency, or low stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-MSLN antibody or fragment thereof described herein. Information regarding hybridization conditions is described in, eg, US Patent Application Publication No. US2005 / 0048549 (eg, paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0188] Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein, such as those described in Table 1, and modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding the antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6, which is incorporated herein by reference in its entirety), which briefly includes the synthesis of overlapping oligonucleotides that include portions of the antibody-encoding sequence, annealing and ligating the oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0189] Alternatively, polynucleotides encoding the antigen-binding regions described herein or antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and further cloning.

[0190] If a clone containing a nucleic acid encoding a particular antigen-binding region or antibody is not available, but the sequence of the antigen-binding region or antibody molecule is known, nucleic acid encoding an immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., nucleic acid isolated from any tissue or cell that expresses an antibody, such as hybridoma cells selected to express an antibody described herein, preferably an antibody cDNA library or a cDNA library generated from polyA+NA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone in a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0191] DNA encoding the anti-MSLN (e.g., human MSLN) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of anti-MSLN (e.g., human MSLN) antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNAs are placed into expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)) that do not otherwise produce immunoglobulin proteins, or myeloma cells, resulting in the synthesis of anti-MSLN antibodies in the recombinant host cells.

[0192] To generate the entire antibody or antigen-binding region, the VH or VL sequence can be amplified in the scFv clone using PCR primers such as the VH or VL nucleotide sequence, restriction sites, and flanking sequences to protect the restriction sites. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., human gamma 1 or human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, e.g., human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing the full-length antibody (e.g., IgG).

[0193] The DNA can also be modified, for example, by substituting human heavy and light chain constant region coding sequences for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0194] Also provided are polynucleotides that hybridize under high stringency, medium stringency, or low stringency hybridization conditions to polynucleotides encoding the antibodies described herein, in certain embodiments, the polynucleotides described herein hybridize under high stringency, medium stringency, or low stringency hybridization conditions to polynucleotides encoding the VH and / or VL domains provided herein.

[0195] Hybridization conditions are described in the art and are known to those of skill in the art. For example, hybridization under stringent conditions can include hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C; hybridization under high stringency conditions can include hybridization to filter-bound nucleic acid in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those of skill in the art and has been described, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, 6.3.1-6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0196] In certain aspects, provided herein are cells (e.g., host cells) and related polynucleotides and expression vectors that express (e.g., recombinant) antibodies described herein that specifically bind to MSLN (e.g., human MSLN). Provided herein are vectors (e.g., expression vectors) that contain a polynucleotide that includes a nucleotide sequence encoding an anti-MSLN antibody or fragment for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell). Also provided herein are host cells that contain such vectors for recombinantly expressing an anti-MSLN antibody (e.g., a human or humanized antibody) described herein. In certain aspects, provided herein are methods for producing the antibodies described herein, the methods comprising expressing the antibody from a host cell.

[0197] Recombinant expression of an antibody described herein (e.g., a full-length antigen-binding region, or an antibody, or a heavy and / or light chain of an antibody described herein) that specifically binds to MSLN (e.g., human MSLN) generally involves constructing an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, an antibody heavy and / or light chain, or a fragment thereof (e.g., a heavy and / or light chain variable region) is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing a protein by expressing a polynucleotide comprising an antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequence. Methods well known to those skilled in the art can be used to construct expression vectors containing an antibody or antibody fragment (e.g., light or heavy chain) coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising nucleotide sequences encoding the antibody molecules described herein, the heavy or light chains of the antibodies, the heavy or light chain variable regions of the antibodies or fragments thereof, or the heavy or light chain CDRs operably linked to a promoter. Such vectors may, for example, comprise nucleotide sequences encoding the constant regions of the antibody molecules (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entireties), and the variable regions of the antibodies may be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0198] In certain embodiments, the vector comprises a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the heavy and light chains of an antibody described herein.

[0199] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce one that contains an antibody or fragment thereof described herein. Thus, provided herein are host cells comprising a polynucleotide that contains and encodes an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell.

[0200] In certain embodiments, the host cell comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the host cell comprises a vector comprising a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the host cell comprises a first polynucleotide encoding the VH of an antibody described herein and a second polynucleotide encoding the VL of an antibody described herein. In another embodiment, the host cell comprises a first vector comprising a first polynucleotide encoding the VH of an antibody described herein and a second vector comprising a second polynucleotide encoding the VL of an antibody described herein.

[0201] In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-MSLN (e.g., human MSLN) antibody described herein. In certain embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0202] In certain embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-MSLN (e.g., human MSLN) antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-MSLN (e.g., human MSLN) antibody described herein.

[0203] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (see, e.g., U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules described herein in situ. These include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems, for example, infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas), for example, infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter). In certain embodiments, the cells for expressing the antibodies described herein are Chinese Hamster Ovary (CHO) cells, e.g., CHO cells from the CHO GS System™ (Lonza).In certain embodiments, the heavy and / or light chains of the antibodies produced by CHO cells may have an N-terminal glutamine or glutamate residue replaced by pyroglutamic acid. In certain embodiments, the cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are used for the expression of recombinant antibody molecules, particularly for the expression of whole recombinant antibody molecules. For example, mammalian cells, such as CHO cells, in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI et al., (1990) Biotechnology 8(7):662-7, each of which is incorporated herein by reference in its entirety). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In certain embodiments, expression of the nucleotide sequence encoding the antibodies described herein that specifically bind to MSLN (e.g., human MSLN) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0204] In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use of the antibody molecule expressed. For example, when producing large quantities of such antibodies, a vector that directs the expression of a high level of fusion protein product that is easily purified to produce a pharmaceutical composition of the antibody molecule may be desirable. Such vectors include, but are not limited to, E. coli expression vector pUR278 (Ruether U&Mueller-Hill B(1983)EMBO J 2:1791-1794), in which a coding sequence can be ligated into the vector in frame with the lacZ coding region to produce a fusion protein such as pIN vector (Inouye S&Inouye M(1985)Nuc Acids Res 13:3101-3109;Van Heeke G&Schuster SM(1989)J Biol Chem 24:5503-5509, all of which are incorporated herein by reference in their entirety). For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0205] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Coding sequences can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0206] In mammalian host cells, several virus-based expression systems can be utilized. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) results in recombinant viruses that are viable and capable of expressing the molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which are incorporated herein by reference in their entireties).

[0207] In addition, a host cell line may be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In certain embodiments, the anti-MSLN (e.g., human MSLN) antibodies described herein are produced in mammalian cells, such as CHO cells.

[0208] In certain embodiments, the antibodies described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that are deficient or lack the ability to fucosylate. In certain examples, a cell line in which both alleles of α1,6-fucosyltransferase are knocked out can be used to produce antibodies with reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0209] For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines can be engineered that stably express the anti-MSLN (e.g., human MSLN) antibodies described herein. In certain embodiments, the cells provided herein stably express the light chain / light chain variable region and the heavy chain / heavy chain variable region that assemble to form the antigen-binding region or antibody described herein.

[0210] In certain embodiments, rather than using expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. Following introduction of the foreign DNA / polynucleotide, the engineered cells can be grown in rich medium for 1-2 days and then switched to a selection medium. The selection marker in the recombinant plasmid confers resistance to the selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. The method can be advantageously used to engineer cell lines that express anti-MSLN (e.g., human MSLN) or fragments thereof as described herein. Such engineered cell lines can be particularly useful for screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0211] Several selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler M et al., (1977) Cell 11(1):223-32), the hypoxanthine guanine phosphoribosyltransferase gene (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and the adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) gene in tk-cells, hgprt-cells, or aprt-cells, respectively, all of which are incorporated herein by reference in their entireties. Also, antimetabolite resistance can be used as a criterion for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56), all of which are incorporated herein by reference in their entireties.Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone, such as those described in, for example, Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entireties.

[0212] The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). If the marker in the vector system is amplifiable, the copy number of the marker gene is increased by increasing the level of inhibitor present in the host cell culture. Since the amplified region is associated with the gene of interest, production of the protein is also increased (Crouse GF et al., (1983) Mol Cell Biol 3:257-66, which is incorporated herein by reference in its entirety)).

[0213] A host cell can be co-transfected with two or more expression vectors described herein, a first vector encoding a heavy chain derived polypeptide, and a second vector encoding a light chain derived polypeptide. The two vectors can contain identical selection markers that allow for equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with a first expression vector and a second expression vector in any one of the following ratios: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0214] Alternatively, one vector can be used that can encode and express both heavy and light chain polypeptides. In such a situation, the light chain must be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot NJ (1986) Nature 322:562-565; and Kohler G (1980) PNAS 77:2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein): In such an expression vector, transcription of both genes can be driven by a promoter, but translation of mRNA from the first gene can be by a cap-dependent scanning mechanism and translation of mRNA from the second gene can be by a cap-independent mechanism, e.g., an IRES.

[0215] The antibody molecules described herein, once produced by recombinant expression, can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen following Protein A, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0216] In certain embodiments, the antibodies described herein are isolated or purified. In certain embodiments, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the antibody (e.g., different post-translationally modified forms of the antibody or other different versions of the antibody (e.g., antibody fragments). When the antibody is recombinantly produced, the antibody is also generally substantially free of culture medium; i.e., culture medium comprises less than about 10% of the volume of the protein preparation. This corresponds to less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1%. When the antibody is produced by chemical synthesis, the antibody is generally substantially free of chemical precursors or other chemicals, i.e., the antibody is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0217] Anti-MSLN (e.g., human MSLN) antibodies or fragments thereof may be produced by any method known in the art for the synthesis of proteins or antibodies, for example, by chemical synthesis or by recombinant expression technology. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature.For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. al.,Current Protocols in Molecular Biology,John Wiley&Sons(1987 and annual updates);Current Protocols in Immunology,John Wiley&Sons(1987 and annual updates)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press;Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entireties.

[0218] In certain embodiments, the antibodies described herein are prepared, expressed, created, or isolated by any means that involves creation, such as synthesis of DNA sequences, genetic engineering, etc. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0219] In one aspect, provided herein is a method of making an anti-MSLN (e.g., human MSLN) antibody, comprising culturing a cell or host cell described herein. In certain embodiments, the method is performed in vitro. In certain aspects, provided herein is a method of making an anti-MSLN (e.g., human MSLN) antibody, comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0220] In certain embodiments, the antibody is produced by expressing a polynucleotide encoding the VH and VL of the antibody described herein in a cell under suitable conditions such that the polynucleotide is expressed and the antibody is produced. In another embodiment, the antibody is produced by expressing a polynucleotide encoding the heavy and light chains of the antibody described herein in a cell under suitable conditions such that the polynucleotide is expressed and the antibody is produced. In certain embodiments, the antibody is produced by expressing a first polynucleotide encoding the VH of the antibody described herein and a second polynucleotide encoding the VL of the antibody described herein in a cell under suitable conditions such that the polynucleotide is expressed and the antibody is produced. In certain embodiments, the antibody is produced by expressing a first polynucleotide encoding the heavy chain of the antibody described herein and a second polynucleotide encoding the light chain of the antibody described herein in a cell under suitable conditions such that the polynucleotide is expressed and the antibody is produced.

[0221] Methods for producing polyclonal antibodies are known in the art (e.g., Short Protocols in Molecular Biology, (2002) 5th Ed., Chapter 11, Ausubel FM et al., eds., John Wiley and Sons, New York, which is incorporated herein by reference in its entirety).

[0222] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, such as using hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, such as those known in the art and taught, for example, in Harlow E&Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells exogenously expressing an antibody or fragment thereof described herein, e.g., the light chain and / or heavy chain of such an antibody.

[0223] In certain embodiments, a "monoclonal antibody" as used herein is an antibody produced by one cell (e.g., a hybridoma or host cell producing recombinant antibody), where the antibody specifically binds to MSLN (e.g., human MSLN) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. In certain embodiments, the monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific antibody or a multispecific antibody (e.g., bispecific antibody). The monoclonal antibodies described herein can be made, for example, by hybridoma methods as described in Kohler G & Milstein C (1975) Nature 256:495, which is incorporated herein by reference in its entirety, or can be isolated, for example, from phage libraries using the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5th Ed., Chapter 11, Ausubel FM et al., supra).

[0224] As used herein, an antibody binds an antigen multivalently (e.g., bivalently) if it contains at least two (e.g., two or more) monovalent binding regions, each of which can bind to an epitope on the antigen. Each monovalent binding region can bind to the same or different epitopes on the antigen.

[0225] The method of producing and screening specific antibodies using hybridoma technology is routine and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization (e.g., MSLN (e.g., human MSLN)). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). Additionally, animals can be immunized using RIMMS (repeated immunization multiple sites) techniques (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0226] In certain embodiments, a mouse (or other animal, e.g., rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., MSLN (e.g., human MSLN)) and once an immune response is detected, e.g., antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well-known techniques with any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of the immunized mouse are harvested and fused with NS0 myeloma cells.

[0227] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances inhibit the growth of HGPRT-deficient cells.

[0228] In certain embodiments, myeloma cells are employed that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, myeloma cell lines are derived from mouse myeloma lines, such as NS0 cell lines available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, or from MOPC-21 and MPC-11 mouse tumors, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0229] The culture medium in which the hybridoma cells grow is assayed for production of monoclonal antibodies directed against MSLN (e.g., human MSLN). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0230] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0231] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0232] The antibodies described herein include, for example, antibody fragments that recognize MSLN (e.g., human MSLN) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds at the hinge region.

[0233] Additionally, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, which is infected with a helper phage. The phages used in these methods are typically filamentous phages, such as fd and M13, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing antigen-binding regions that bind to a particular antigen can be selected or identified using antigen, e.g., labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic Letal., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT Application PCT / GB91 / 001134; International Patent Publications WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 1 1236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; as well as those disclosed in U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein by reference in their entireties.

[0234] As described in the above references, after phage selection, the antibody coding region from the phage can be isolated and used to generate whole antibodies, such as human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, such as mammalian cells, insect cells, plant cells, yeast, and bacteria, such as those described below. For example, techniques for recombinantly producing antibody fragments, such as Fab, Fab', and F(ab')2 fragments, can also be employed using methods known in the art, such as those disclosed in PCT Publication WO92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043, all of which are incorporated herein by reference in their entireties.

[0235] In certain embodiments, to generate whole antibodies, PCR primers such as VH or VL nucleotide sequences, restriction sites, and flanking sequences to protect the restriction sites can be used to amplify VH or VL sequences from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing full-length antibody (e.g., IgG).

[0236] A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229:1202-7; Oi VT & Morrison SL (1986) BioTechniques 4:214-221; Gillies SD et al., (1989) J Immunol Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0237] Humanized antibodies can bind to a given antigen and comprise a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a mouse immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulins, such as IgM, IgG, IgD, IgA, and IgE, and any isotype, such as IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced by a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patents EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), as well as the techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication WO 93 / 17105; Tan P et al., (2002) J Immunol 169:1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3):267-79; Baca M et al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895 904; Couto JR et al. al., (1995) Cancer Res. 55(23 Supp):5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8):1717-22; Sandhu JS (1994) Gene 150(2):409-10; and Pedersen JT et al., (1994) J Mol Biol 235(3):959-73, all of which are incorporated herein by reference in their entireties. See also U.S. Application US2005 / 0042664(A1), filed Feb. 24, 2005, which is incorporated herein by reference in its entirety.

[0238] Methods for making multispecific antibodies (e.g., bispecific antibodies) have been described, see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713, all of which are incorporated herein by reference in their entireties.

[0239] Bispecific, bivalent antibodies and methods for making them are described, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, and U.S. Patent Application Publication Nos. 2003 / 020734 and 2002 / 0155537, each of which is incorporated herein by reference in its entirety. Bispecific, tetravalent antibodies and methods for making them are described, for example, in International Publication Nos. WO 02 / 096948 and WO 00 / 44788, both of which are incorporated herein by reference in their entirety. See generally, International Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of the above is incorporated by reference in its entirety.

[0240] The bispecific antibodies described herein can be produced, for example, according to the DuoBody technology platform (GenmabA / S) described in International Publications WO2011 / 131746, WO2011 / 147986, WO2008 / 119353, and WO2013 / 060867, and Labrijn AF et al., (2013) PNAS110(13):5145-5150. The DuoBody technology can be used to combine a first monospecific antibody or half of a first antigen-binding region, comprising two heavy chains and two light chains, with a second monospecific antibody or half of a second antigen-binding region, comprising two heavy chains and two light chains. The resulting heterodimer comprises one heavy chain and one light chain from the first antibody or first antigen-binding region paired with one heavy chain and one light chain from the second antibody or second antigen-binding region. When both monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0241] The DuoBody technology requires that each of the monospecific antibodies or antigen-binding regions contains a heavy chain constant region with a single point mutation in the CH3 domain. The point mutation allows a stronger interaction between the CH3 domains of the resulting bispecific antibody than between the CH3 domains or antigen-binding regions of either of the monospecific antibodies. The single point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407, or 409 numbered according to the EU numbering system in the CH3 domain of the heavy chain constant region, as described, for example, in International Publication WO2011 / 131746. Furthermore, the single point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to the other monospecific antibody or antigen-binding region. For example, one monospecific antibody or antigen-binding region can contain the mutation F405L (i.e., a phenylalanine to leucine mutation at residue 405) and the other monospecific antibody or antigen-binding region can contain the mutation K409R (i.e., a lysine to arginine mutation at residue 409), numbered according to the EU numbering system. The heavy chain constant regions, or antigen-binding regions, of the monospecific antibodies can be of IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and bispecific antibodies produced by DuoBody technology can retain Fc-mediated effector functions.

[0242] Another method for generating bispecific antibodies is called the "knobs-in-holes" strategy (see, for example, International Publication No. WO 2006 / 028936). Mispairing of Ig heavy chains is reduced in this technique by mutating selected amino acids that form the interface of the CH3 domain in IgG. At positions in the CH3 domain where two heavy chains directly interact, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (knob) is introduced at the position of the interacting residue on the other heavy chain. In some embodiments, the compositions of the present disclosure have immunoglobulin chains whose CH3 domains have been modified by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form bispecific antibodies. Bispecific antibodies can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0243] Bispecific antibodies can optionally comprise IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chain heterodimers. Such heterodimeric heavy chain antibodies can be routinely engineered, for example, by modifying selected amino acids that form the interface of the CH3 domains in human IgG4 and IgG1 or IgG3 to support heterodimeric heavy chain formation.

[0244] In certain embodiments, an antibody described herein that binds to the same epitope of MSLN (e.g., human MSLN) as an anti-MSLN (e.g., human MSLN) antibody described herein is a human antibody. In certain embodiments, an antibody described herein that competitively (e.g., dose-dependently) blocks the binding of any one of the antibodies described herein to MSLN (e.g., human MSLN) is a human antibody. Human antibodies can be produced using any method known in the art. For example, transgenic mice can be used that are incapable of expressing functional endogenous immunoglobulins, but that can express human immunoglobulin genes. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional simultaneously with the introduction of human immunoglobulin loci by homologous recombination, or separately. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, such as all or part of an antigen (e.g., MSLN (e.g., human MSLN)). Monoclonal antibodies directed against this antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, for example, International Publication Nos. WO98 / 24893, WO96 / 34096, and WO96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety.Examples of mice capable of producing human antibodies include the XenoMouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Medarex, Inc. / Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex / Kirin), all of which are incorporated herein by reference in their entireties.

[0245] Human antibodies that specifically bind to MSLN (e.g., human MSLN) can be produced by various methods known in the art, such as the above-mentioned phage display method, using antibody libraries derived from human immunoglobulin sequences.See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741.All of the above are incorporated herein by reference in their entirety.

[0246] In certain embodiments, human antibodies may be produced using mouse-human hybridomas. For example, Epstein-Barr Virus (EBV) transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., MSLN (e.g., human MSLN)). Such methods are known and described in the art. See, e.g., Shinmoto H et al., (2004) Cytotechnology 46:19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31. Each of the above is incorporated herein by reference in its entirety.

[0247] kit Also provided is a kit comprising one or more antibodies, or pharmaceutical compositions, or conjugates thereof, as described herein. In certain embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In certain embodiments, the kit comprises the pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. In certain embodiments, the kit may comprise, for example, a T cell mitogen, such as phytohemagglutinin (PHA) and / or phorbol acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Such container(s) may optionally be accompanied by a notice in a format prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice represents approval by the governmental agency of the manufacture, use, or sale for administration to humans.

[0248] Also provided are kits that can be used in the above methods. In certain embodiments, the kits include the antibodies described herein, preferably purified antibodies, in one or more containers. In certain embodiments, the kits described herein include substantially isolated MSLN (e.g., human MSLN) antigen as a control. In another specific embodiment, the kits described herein further include a control antibody that does not react with MSLN (e.g., human MSLN) antigen. In another specific embodiment, the kits described herein include one or more elements for detecting the binding of the antibody to the MSLN (e.g., human MSLN) antigen (e.g., the antibody can be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In certain embodiments, the kits provided herein can include recombinantly produced or chemically synthesized MSLN (e.g., human MSLN) antigen. The MSLN (e.g., human MSLN) antigen provided in the kit can also be attached to a solid support. In more specific embodiments, the detection means of the above kit comprises a solid support to which MSLN (e.g., human MSLN) antigen is attached. Such kits may also comprise unattached reporter-labeled anti-human or anti-mouse / rat antibodies. In this embodiment, binding of an antibody to MSLN (e.g., human MSLN) antigen may be detected by binding of the reporter-labeled antibody. In certain embodiments, the present disclosure relates to the use of the kit of the present disclosure for assaying and / or detecting MSLN (e.g., human MSLN) antigen in a biological sample in vitro. EXAMPLES

[0249] The following examples are offered by way of illustration and not by way of limitation.

[0250] Example 1: Binding kinetics and optimization of anti-MSLN 1A12 antibody variants Anti-MSLN 1A12 antibody variants (see Table 1 above) were generated to improve properties and optimize the molecule. Variants of the parent 1A12 variable domain were formatted into an aglycosylated (N297A) human kappa IgG1 construct and expressed in 293 Freestyle cells using lentiviral transduction. Culture supernatants were collected and purified using Protein A affinity chromatography and further purified by size exclusion chromatography using an Agilent high pressure liquid chromatography system (HPLC-SEC). Surface plasmon resonance was used to collect kinetic binding data on a Carterra LSA system. Purified 1A12 antibody variants were captured on an LSA biosensor chip (HC30M) using immobilized anti-human IgG, and purified MSLN ectodomain was used as the analyte.

[0251] The "NS" motif of 1A12 CDRL3 (SEQ ID NO:8) was mutated to restore a predicted deamidation site. However, certain mutations had a detrimental effect on antibody off-rate and antibody expression. Mutation of position 92(N) of the light chain (SEQ ID NO:10) to either alanine or serine affected antibody binding, resulting in slower off-rates (Figure 1A). Mutations of N92S and S93A also affected antibody expression (Figure 1B). Mutation of the "DS" motif of heavy chain CDRH2 (SEQ ID NO:4) to restore a predicted isomerization site did not affect antibody binding kinetics. Conservative substitutions at positions 62(N) and 63(N+1) in the heavy chain (SEQ ID NO:X) were well tolerated (Figure 2).

[0252] Pairs of LC / HC variants of the anti-MSLN 1A12 antibody with CDRL3 or CDRH2 mutations to restore predicted deamidation and isomerization sites were evaluated for changes in affinity compared to a control 1A12 antibody. These combinations provided multiple pairwise solutions to improve antibody function and developability (Figure 3). Notably, combinations containing N92A or N92S showed higher affinity (KD ) resulted.

[0253] Example 2: Bispecific antibodies Bispecific IgG-scFv antigen-binding molecules that specifically bind to MSLN and human CD3 were prepared using the Fab domain of the 1A12 anti-MSLN antibody (see Table 1 above) and the anti-CD3 variable domains of Micro194 (VH / VL=SEQ ID NOs: 57 / 58), the humanized version of the OKT3 antibody (VH / VL=SEQ ID NOs: 59 / 60), or the humanized version of the UCHT1 antibody (VH / VL=SEQ ID NOs: 61 / 62) in the structural format shown in FIG. 4.

[0254] Methods for producing these molecules followed standard protein expression protocols described in various references. Briefly, the "Daedalus" human cell line expression platform was used for production and purification of secreted proteins. The expression system utilized suspension-adapted HEK293 Freestyle cells and a highly optimized lentiviral transduction protocol to generate cell lines secreting proteins at high levels. The lentiviral vector contained a cis-linked fluorescent protein reporter driven by an internal ribosome entry site (IRES) that allowed tracking of relative protein expression levels. All mammalian proteins described were purified directly from conditioned media using a HisTrap FF Crude column (GE#17528601) and then on a Superose6 10 / 300 GL SEC column (GE#17517201) using an AKTA Pure 25 instrument.

[0255] The bispecific antigen-binding molecules were purified and characterized using standard techniques. Representative chromatograms showing the purification of IgG-scFv molecules having the structure of Figure 4 are shown in Figures 5A-5B (UCHT1 scFv) and 6A-6B (Micro194 scFv), confirming successful production with minimal aggregates.

[0256] The affinity of the bispecific IgG-scFv molecules for MSLN was assessed using the same method as for the anti-MSLN 1A12 variant antibody described in Example 1.

[0257] As shown in FIG. 7, fusing different CD3-engaging scFv moieties to the light chain of anti-MSLN 1A12 (SEQ ID NO: 10) did not affect affinity for MSLN.

[0258] Example 3: FACS-based cytotoxicity assay to assess T cell-mediated killing of MSLN-expressing cells in the presence of anti-MSLNxanti-CD3 bispecific molecules To observe the specific killing of MSLN-expressing cells, cells with high surface expression MSLN (SKOV3 and OVCAR3 ovarian cancer lines) were used in a T cell killing assay. Briefly, the cytotoxicity assay was performed using an automated platform programmed to simultaneously perform unbiased 96-well assays. In this assay, 100,000 T cells (harvested from healthy donors and qualified by flow cytometry for CD3, CD28, CD4, and CD8 before and after EasySep™ no-touch purification) were mixed with 20,000 Luc-iRFP cancer cells (E:T ratio 5:1) in the presence or absence of 1A12:Micro194 or 1A12:OKT3 bispecific molecules (8 concentrations, ranging from 0 to 1 mg / ml). HPN536 (also called TriTAC, see WO2018 / 209304, the contents of which are incorporated herein by reference), a trispecific antibody that binds MSLN, CD3, and serum albumin, was also assayed for comparison. After contacting the cells with the bispecific molecules, cytotoxicity was determined on day 5 for SKOV3 cells or day 2 for OVCAR3 cells.

[0259] As shown in Figures 8A-B, the 1A12:Micro194 molecule had higher cytotoxicity than the 1A12:OKT3 molecule with humanized OKT3 scFv, especially against SKOV3 cells. The 1A12:Micro194 IgG-scFv molecule was significantly more potent than the HPN536 trispecific antibody against both types of MSLN-expressing cells.

[0260] The ability of IgG-scFv and HPN536 (TriTAC) molecules to induce cytotoxicity in SKOV3 cells was compared in the presence or absence of soluble MSLN (sMSLN). As shown in FIG. 9A, the cytotoxicity of IgG-scFv molecules was significantly reduced in the presence of sMSLN. Without wishing to be bound by theory, the high affinity for both soluble MSLN-associated proteins and cell surface MSLN makes HPN536 molecules more sensitive to the presence of sMSLN, which reduces their cytotoxicity. In contrast, IgG-scFv molecules with the structure shown in FIG. 4 have high affinity for cell surface MSLN and low avidity for soluble MSLN-associated proteins, resulting in a smaller shift in the strength of cytotoxicity (FIG. 9B).

[0261] Example 4: In vivo efficacy NOD / SCID / IL-2Rγc null (NSG) immunodeficient mice were intraperitoneally grafted with luciferase-expressing SKOV3 cells. Mice were treated with 1A12:OKT3 bispecific molecules containing anti-MSLN 1A12 binding domains and humanized anti-CD3 OKT3 binding moieties. Untreated mice and mice treated with activated T cells (ATCs) alone were used as controls. ATCs were CD3+Pan T cells isolated from peripheral blood mononuclear cells (PBMCs) and stimulated for rapid expansion. Figure 10 shows that the 1A12:OKT3 bispecific molecule reduced tumor growth compared to controls over time.

[0262] Example 5: Improved MSLN-CD3 variants Variants and Characteristics Using the Fab domain of the 1A12 anti-MSLN antibody or 1A12 variants described in Example 1 and the Micro194 anti-CD3 scFv, 24 unique variants of bispecific anti-MSLN / anti-CD3 IgG-scFv T cell engager molecules were prepared in the structural format illustrated in Figure 4 (see Table 2). These variants incorporated several different (i) linker lengths between the VH and VL domains, (ii) light chain isomerization repair, (iii) heavy chain deamination repair, and (iv) effector function silencing solutions. The characteristics of the variants and the results of the expression and aggregation assays are shown in Table 3 below.

[0263] The linker region that separates the C-terminus of the light chain from the scFv (LC-linker-scFv) is important for optimizing the potency of bispecific T cell engagers. Another important linker separates the variable domains of the scFv (VH-linker-VL). Optimizing the length and flexibility of these linkers has been found to be important for the function, production, and immunogenicity of bispecific molecules. Novel glycine and serine-containing linkers were designed to maximize the flexibility of both the light chain and the scFv as well as the connection between the variable domains of the scFv. The general structure of this linker is GGGGS-(X)-SGGGG (SEQ ID NO: 85), where X is any combination of common flexible or rigid linker sequences (e.g., glycine-serine, proline-threonine, lysine-serine, human antibody hinge region, etc.). Ultimately, it was determined that the GGGGS-GGGSGGG-SGGGG (SEQ ID NO: 73) linker was the optimal length to prevent the formation of diabodies.

[0264] The expression levels of IgG-scFv molecules were assayed using either an analytical Protein A affinity chromatography titer assay or a variable path length slope spectroscopy SoloVPE A280 measurement, applying the theoretical extinction coefficient of the protein at 1 mg / mL. The equation used to determine the molar extinction coefficient is ε 280= 5500 (number of Trp) + 1490 (number of Tyr) + 125 (number of SS bonds). Size exclusion chromatography (SEC-HPLC) was used to separate IgG-scFv molecules based on differences in hydrodynamic volume. Protein samples were loaded onto a Waters XBridge Protein BEH SEC 200A column (3.5 μm, 7.8x300mm) equilibrated in a running buffer of 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.8. Purity was determined by calculating the percentage of each component separated compared to the total integrated area.

[0265] IgG-scFv variants M-3643, M-3642, M-3648, M-3651, M-3654, and M-3641 (shown in bold in Table 3) had superior expression levels and minimal total aggregates compared to other IgG-scFv molecules tested. Based on this data, these six variants were selected for further analysis. [Table 3]

[0266] heat retention A heat retention assay was performed to evaluate the conformational stability of the IgG-scFv molecules. Samples were placed in a 96-well Biorad PCR plate and heated to various temperatures from 69°C to 74°C for 5 min using a Biorad Thermal Cycler. After heating, protein precipitation was determined by reading the absorbance at 350 nm (A350) using an M5 plate reader. The results showed that M-3654 was more resistant to temperature denaturation (Table 4). [Table 4]

[0267] Chemical unfolding The inflection point of the chemical unfolding curve is believed to be related to the stability of the conformation and the stability during long-term storage, with the inflection point being related to the structurally or conformationally more stable molecule. The chemical unfolding curve of each IgG-scFv molecule was generated by exposing the molecule to increasing concentrations of the denaturing agent guanidine hydrochloride. After 72 hours, the intrinsic fluorescence of the samples was measured using a SUPR-UV plate reader. The collected raw data was then processed, and the chemical unfolding curve and its inflection point were calculated from the processed data corresponding to the denaturing conditions.

[0268] M-3641 (aglycosylated N297A mutant) was found to be less stable than variants incorporating other effector-silencing Fc mutations (Table 5). [Table 5]

[0269] low pH flocculation A low pH aggregation assay can be used to help select candidates suitable for low pH viral inactivation. In this assay, samples containing various IgG-scFv molecules were titrated to pH 3.3 using acetic acid, held for 30 min, and then neutralized to pH 5 with Tris base. Samples were characterized using SEC-HPLC as described above. Samples diluted in PBS with the same amount of acetic acid and Tris base were used as controls. Molecules that showed a significant increase in high molecular weight were deemed unstable during low pH exposure and would require further method development if low pH viral inactivation were to be used.

[0270] Of all the variants tested in this low pH aggregation assay, M-3654 was the most stable at low pH (suggesting that the repair mutations improved the behavior of this molecule) and M-3641 was the least stable (Table 6). [Table 6]

[0271] product quality SEC and non-reduced (NR) and reduced (R) capillary electrophoresis sodium dodecyl sulfate (CE-SDS) showed that the M-3654 IgG-scFv molecule had low levels of high and low molecular weight contaminating species (Table 7). rCE-SDS under denaturing and reducing conditions also showed clear separation of the heavy and light chains of M-3654, indicating that M-3654 behaves as expected as an antibody, even when the isomerization and deamination sites are mutated. [Table 7]

[0272] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

[0273] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety.

[0274] Other embodiments are within the scope of the following claims.

Claims

1. An antibody that specifically binds to human MSLN, wherein the antibody comprises CDRH1, CDRH2, and CDRH3 having the amino acid sequences of SEQ ID NO:3, SEQ ID NO:13, and SEQ ID NO:5, respectively, and CDRL1, CDRL2, and CDRL3 having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:14, respectively, and the antibody does not comprise a CDRH2 having the amino acid sequence of SEQ ID NO:4 or a CDRL3 having the amino acid sequence of SEQ ID NO:

8.

2. (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 30, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 35; (b) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:29, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:35; (c) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:29, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (d) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:29, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:37; (e) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:29, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:38; (f) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 29, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, and CDRL1 comprises SEQ ID NO:

6. CDRL2 comprises the amino acid sequence of SEQ ID NO:7 and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; (g) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:29, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:40; (h) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:30, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 30, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 37; (j) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:30, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:38; (k) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:30, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; (l) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:30, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:40; (m) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:31, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:35; (n) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:31, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (o) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 37; (p) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 31, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 38; (q) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:31, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; (r) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:31, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:40; (s) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:32, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:35; (t) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:32, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (u) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 32, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 37; (v) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:32, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:38; (w) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:32, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; (x) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 32, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 40; (y) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:33, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:35; (z) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:33, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (aa) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 33, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 37; (bb) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:33, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:38; (cc) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:33, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; (dd) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 33, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 40; (ee) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 34, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, and CDRL1 comprises the amino acid sequence of SEQ ID NO: No. 6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7 and CDRL3 comprises the amino acid sequence of SEQ ID NO:35; (ff) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:34, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:36; (gg) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 34, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 37; (hh) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:34, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:38; (ii) CDRH1 comprises the amino acid sequence of SEQ ID NO:3, CDRH2 comprises the amino acid sequence of SEQ ID NO:34, CDRH3 comprises the amino acid sequence of SEQ ID NO:5, CDRL1 comprises the amino acid sequence of SEQ ID NO:6, CDRL2 comprises SEQ ID NO:7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:39; or (jj) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 34, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 40; The antibody described in claim 1.

3. The antibody of claim 1, wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 3, CDRH2 comprises the amino acid sequence of SEQ ID NO: 30, CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, CDRL1 comprises the amino acid sequence of SEQ ID NO: 6, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of SEQ ID NO:

35.

4. the antibody comprises a variable heavy chain region (VH) and a variable light chain region (VL); (a) the VH comprises the amino acid sequence of SEQ ID NO: 18 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (b) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (c) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 24; (d) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 25; (e) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 26; (f) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 27; (g) the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 28; (h) VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 24; (i) VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 25; (j) VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 26; (k) VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 27; (l) VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 28; (m) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (n) VH comprises the amino acid sequence of SEQ ID NO: 19 and VL comprises the amino acid sequence of SEQ ID NO: 24; (o) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 25; (p) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 26; (q) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 27; (r) the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 28; (s) the VH comprises the amino acid sequence of SEQ ID NO: 20 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (t) VH comprises the amino acid sequence of SEQ ID NO: 20 and VL comprises the amino acid sequence of SEQ ID NO: 24; (u) the VH comprises the amino acid sequence of SEQ ID NO: 20 and the VL comprises the amino acid sequence of SEQ ID NO: 25; (v) VH comprises the amino acid sequence of SEQ ID NO: 20 and VL comprises the amino acid sequence of SEQ ID NO: 26; (w) VH comprises the amino acid sequence of SEQ ID NO: 20 and VL comprises the amino acid sequence of SEQ ID NO: 27; (x) the VH comprises the amino acid sequence of SEQ ID NO: 20 and the VL comprises the amino acid sequence of SEQ ID NO: 28; (y) the VH comprises the amino acid sequence of SEQ ID NO: 21 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (z) VH comprises the amino acid sequence of SEQ ID NO: 21 and VL comprises the amino acid sequence of SEQ ID NO: 24; (aa) VH comprises the amino acid sequence of SEQ ID NO: 21 and VL comprises the amino acid sequence of SEQ ID NO: 25; (bb) the VH comprises the amino acid sequence of SEQ ID NO: 21 and the VL comprises the amino acid sequence of SEQ ID NO: 26; (cc) the VH comprises the amino acid sequence of SEQ ID NO: 21 and the VL comprises the amino acid sequence of SEQ ID NO: 27; (dd) the VH comprises the amino acid sequence of SEQ ID NO: 21 and the VL comprises the amino acid sequence of SEQ ID NO: 28; (ee) the VH comprises the amino acid sequence of SEQ ID NO: 22 and the VL comprises the amino acid sequence of SEQ ID NO: 23; (ff) the VH comprises the amino acid sequence of SEQ ID NO: 22 and the VL comprises the amino acid sequence of SEQ ID NO: 24; (gg) VH comprises the amino acid sequence of SEQ ID NO: 22 and VL comprises the amino acid sequence of SEQ ID NO: 25; (hh) VH comprises the amino acid sequence of SEQ ID NO: 22 and VL comprises the amino acid sequence of SEQ ID NO: 26; (ii) the VH comprises the amino acid sequence of SEQ ID NO: 22 and the VL comprises the amino acid sequence of SEQ ID NO: 27; or (jj) VH comprises the amino acid sequence of SEQ ID NO: 22 and VL comprises the amino acid sequence of SEQ ID NO: 28; The antibody described in claim 1.

5. The antibody of claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO:

23.

6. The antibody is optionally a human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 a heavy chain constant region selected from the group consisting of: Optionally, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region binds to the FcγR; 2. The antibody of claim 1, wherein the heavy chain constant region optionally comprises the amino acid sequence of SEQ ID NO: 55, 53, 54, 56, or 72.

7. the antibody comprises a heavy chain (HC) and a light chain (LC); (a) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (b) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (c) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (d) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (e) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (f) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (g) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (h) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (i) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (j) the HC comprises the amino acid sequence of SEQ ID NO: 83 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (k) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (l) the HC comprises the amino acid sequence of SEQ ID NO: 84 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (m) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (n) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (o) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (p) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (q) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (r) the HC comprises the amino acid sequence of SEQ ID NO: 41 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (s) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (t) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (u) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (v) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (w) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (x) the HC comprises the amino acid sequence of SEQ ID NO: 42 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (y) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (z) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (aa) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (bb) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (cc) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (dd) the HC comprises the amino acid sequence of SEQ ID NO: 43 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (ee) the HC comprises the amino acid sequence of SEQ ID NO: 44 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (ff) the HC comprises the amino acid sequence of SEQ ID NO: 44 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (gg) HC comprises the amino acid sequence of SEQ ID NO: 44 and LC comprises the amino acid sequence of SEQ ID NO: 49; (hh) the HC comprises the amino acid sequence of SEQ ID NO: 44 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (ii) the HC comprises the amino acid sequence of SEQ ID NO:44 and the LC comprises the amino acid sequence of SEQ ID NO:51; (jj) the HC comprises the amino acid sequence of SEQ ID NO: 44 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (kk) HC comprises the amino acid sequence of SEQ ID NO: 45 and LC comprises the amino acid sequence of SEQ ID NO: 47; (ll) the HC comprises the amino acid sequence of SEQ ID NO: 45 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (mm) HC comprises the amino acid sequence of SEQ ID NO:45 and LC comprises the amino acid sequence of SEQ ID NO:49; (nn) the HC comprises the amino acid sequence of SEQ ID NO: 45 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (oo) the HC comprises the amino acid sequence of SEQ ID NO: 45 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (pp) HC comprises the amino acid sequence of SEQ ID NO:45 and LC comprises the amino acid sequence of SEQ ID NO:52; (qq) HC comprises the amino acid sequence of SEQ ID NO: 46 and LC comprises the amino acid sequence of SEQ ID NO: 47; (rr) HC comprises the amino acid sequence of SEQ ID NO: 46 and LC comprises the amino acid sequence of SEQ ID NO: 48; (ss) HC comprises the amino acid sequence of SEQ ID NO: 46 and LC comprises the amino acid sequence of SEQ ID NO: 49; (tt) HC comprises the amino acid sequence of SEQ ID NO:46 and LC comprises the amino acid sequence of SEQ ID NO:50; (uu) the HC comprises the amino acid sequence of SEQ ID NO: 46 and the LC comprises the amino acid sequence of SEQ ID NO: 51; (vv) the HC comprises the amino acid sequence of SEQ ID NO: 46 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (ww) HC comprises the amino acid sequence of SEQ ID NO: 66 and LC comprises the amino acid sequence of SEQ ID NO: 47; (xx) the HC comprises the amino acid sequence of SEQ ID NO: 66 and the LC comprises the amino acid sequence of SEQ ID NO: 48; (yy) HC comprises the amino acid sequence of SEQ ID NO: 66 and LC comprises the amino acid sequence of SEQ ID NO: 49; (zz) HC comprises the amino acid sequence of SEQ ID NO: 66 and LC comprises the amino acid sequence of SEQ ID NO: 50; (aaa) HC comprises the amino acid sequence of SEQ ID NO: 66 and LC comprises the amino acid sequence of SEQ ID NO: 51; (bbb) the HC comprises the amino acid sequence of SEQ ID NO: 66 and the LC comprises the amino acid sequence of SEQ ID NO: 52; (ccc) the HC comprises the amino acid sequence of SEQ ID NO: 67 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (ddd) HC comprises the amino acid sequence of SEQ ID NO:67 and LC comprises the amino acid sequence of SEQ ID NO:48; (eee) the HC comprises the amino acid sequence of SEQ ID NO: 67 and the LC comprises the amino acid sequence of SEQ ID NO: 49; (fff) HC comprises the amino acid sequence of SEQ ID NO: 67 and LC comprises the amino acid sequence of SEQ ID NO: 50; (ggg) HC comprises the amino acid sequence of SEQ ID NO: 67 and LC comprises the amino acid sequence of SEQ ID NO: 51; (hhh) HC comprises the amino acid sequence of SEQ ID NO: 67 and LC comprises the amino acid sequence of SEQ ID NO: 52; (iii) the HC comprises the amino acid sequence of SEQ ID NO: 68 and the LC comprises the amino acid sequence of SEQ ID NO: 47; (jjj) HC comprises the amino acid sequence of SEQ ID NO: 68 and LC comprises the amino acid sequence of SEQ ID NO: 48; (kkk) HC comprises the amino acid sequence of SEQ ID NO: 68 and LC comprises the amino acid sequence of SEQ ID NO: 49; (lll) the HC comprises the amino acid sequence of SEQ ID NO: 68 and the LC comprises the amino acid sequence of SEQ ID NO: 50; (mmm) HC comprises the amino acid sequence of SEQ ID NO: 68 and LC comprises the amino acid sequence of SEQ ID NO: 51; (nnn) HC comprises the amino acid sequence of SEQ ID NO: 68 and LC comprises the amino acid sequence of SEQ ID NO: 52; (ooo) HC comprises the amino acid sequence of SEQ ID NO: 69 and LC comprises the amino acid sequence of SEQ ID NO: 47; (ppp) HC comprises the amino acid sequence of SEQ ID NO: 69 and LC comprises the amino acid sequence of SEQ ID NO: 48; (qqq) HC comprises the amino acid sequence of SEQ ID NO:69 and LC comprises the amino acid sequence of SEQ ID NO:49; (rrr) HC comprises the amino acid sequence of SEQ ID NO:69 and LC comprises the amino acid sequence of SEQ ID NO:50; (sss) HC comprises the amino acid sequence of SEQ ID NO: 69 and LC comprises the amino acid sequence of SEQ ID NO: 51; (ttt) HC comprises the amino acid sequence of SEQ ID NO:69 and LC comprises the amino acid sequence of SEQ ID NO:52; (uuu) HC comprises the amino acid sequence of SEQ ID NO: 70 and LC comprises the amino acid sequence of SEQ ID NO: 47; (vvv) HC comprises the amino acid sequence of SEQ ID NO: 70 and LC comprises the amino acid sequence of SEQ ID NO: 48; (www) HC comprises the amino acid sequence of SEQ ID NO: 70 and LC comprises the amino acid sequence of SEQ ID NO: 49; (xxx) HC comprises the amino acid sequence of SEQ ID NO: 70 and LC comprises the amino acid sequence of SEQ ID NO: 50; (yyy) HC comprises the amino acid sequence of SEQ ID NO: 70 and LC comprises the amino acid sequence of SEQ ID NO: 51; (zzz) HC comprises the amino acid sequence of SEQ ID NO:70 and LC comprises the amino acid sequence of SEQ ID NO:52; (aaaa) HC comprises the amino acid sequence of SEQ ID NO:71 and LC comprises the amino acid sequence of SEQ ID NO:47; (bbbb) HC comprises the amino acid sequence of SEQ ID NO:71 and LC comprises the amino acid sequence of SEQ ID NO:48; (cccc) the HC comprises the amino acid sequence of SEQ ID NO:71 and the LC comprises the amino acid sequence of SEQ ID NO:49; (dddd) HC comprises the amino acid sequence of SEQ ID NO:71 and LC comprises the amino acid sequence of SEQ ID NO:50; (eeee) the HC comprises the amino acid sequence of SEQ ID NO: 71 and the LC comprises the amino acid sequence of SEQ ID NO: 51, or (ffff) HC comprises the amino acid sequence of SEQ ID NO: 71 and LC comprises the amino acid sequence of SEQ ID NO: 52; The antibody described in claim 1.

8. The antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the light chain comprises the amino acid sequence of SEQ ID NO:

47.

9. The antibody of claim 1, further comprising a CD3 binding portion.

10. The antibody of claim 9, wherein the CD3 binding portion is a single chain fragment variable (scFv).

11. The antibody of claim 10, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

76.

12. the antibody comprises a polypeptide comprising a CD3-binding portion covalently linked to a light chain of an antibody that binds to human MSLN; optionally, the CD3 binding moiety is covalently linked to the C-terminus of the light chain of the antibody that binds to human MSLN; Optionally, the CD3 binding moiety is a peptide at the C-terminus of the light chain of the antibody that binds to human MSLN. covalently linked via a peptide linker, optionally wherein the peptide linker comprises an amino acid sequence represented by SEQ ID NO: 73, 63, 64, 74, or 75; The antibody described in claim 9.

13. The antibody of claim 12, wherein the polypeptide comprising the CD3 binding portion covalently linked to the light chain of the antibody that binds to human MSLN comprises the amino acid sequence represented by SEQ ID NO: 77, 78, 79, 80, 81, or 82.

14. An antibody that binds to human MSLN and CD3, the antibody comprising a polypeptide comprising a heavy chain and a polypeptide comprising a light chain; (a) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (b) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (c) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (d) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (e) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (f) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (g) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (h) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:77; (i) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (j) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (k) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (l) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 69 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (m) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (n) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 77; (o) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (p) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (q) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (r) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (s) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (t) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (u) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (v) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:78; (w) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (x) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (y) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (z) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 69 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (aa) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (bb) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 78; (cc) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (dd) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (ee) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (ff) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (gg) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (hh) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (ii) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:79; (jj) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (kk) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (ll) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (mm) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (nn) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 69, and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (oo) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (pp) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 79; (qq) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (rr) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (ss) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (tt) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (uu) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (vv) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (ww) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (xx) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (yy) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (zz) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (aaa) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (bbb) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 69 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (ccc) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 80; (ddd) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:80; (eee) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (fff) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (ggg) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:81; (hhh) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (iii) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (jjj) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:81; (kkk) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:81; (lll) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (mmm) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (nnn) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (ooo) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (ppp) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 69 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (qqq) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70, and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 81; (rrr) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:81; (sss) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (ttt) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 84 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (uuu) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 41 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (vvv) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (www) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 43 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (xxx) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (yyy) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 45 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (zzz) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 46 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (aaaa) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 66 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (bbbb) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 67 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (cccc) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 68 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; (dddd) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO:69 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:82; (eeee) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 70 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; or (ffff) the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 71 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO: 82; The antibody.

15. The antibody of claim 14, wherein the polypeptide comprising the heavy chain comprises the amino acid sequence of SEQ ID NO: 83 and the polypeptide comprising the light chain comprises the amino acid sequence of SEQ ID NO:

77.

16. A pharmaceutical composition comprising the antibody of any one of claims 1 to 15 and a pharmaceutically acceptable carrier or excipient.

17. 16. Use of an antibody according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of cancer in a subject in need thereof, comprising: Optionally, the cancer is lung cancer, melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, ovarian cancer, colon cancer, pancreatic cancer, head and neck cancer, anal cancer, gastroesophageal cancer, mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer, or lymphoproliferative disease.

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