Antimesothelin antibodies

Abstract

Antibodies and antigen-binding fragments thereof that bind to mesothelin, and uses thereof, nucleic acids encoding the antibodies and antigen-binding fragments, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors are provided. Pharmaceutical compositions and conjugates comprising the antibodies, and methods of treatment by administering the antibodies are also provided.

Description

[Technical Field]

[0001] The present invention relates to antibodies and antigen-binding fragments thereof that bind to mesothelin (MSLN). [Background technology]

[0002] Cancers such as mesothelioma, pancreatic adenocarcinoma, ovarian cancer, and lung adenocarcinoma are highly destructive and extremely difficult to treat. For example, pancreatic ductal adenocarcinoma accounts for 90% of all pancreatic tumors, and its incidence is on the rise, while its prognosis is very poor. The lack of available specific diagnostic tests and very limited treatment opportunities pose serious health challenges.

[0003] Mesothelin (MSLN) is a cell surface molecule expressed as a 71-kD precursor protein, which is further processed into a 40-kD glycoprotein and present on the cell surface as a glycosylphosphatidylinositol (GPI)-anchored form. MSLN shows very limited expression in normal tissues. It is expressed in mesothelial cells lining the pleura, pericardium, and peritoneum, where it appears to be involved in cell adhesion (Chang et al. (1996) PNAS 93:136-140). A soluble truncated form of MSLN has also been proposed to be involved in megakaryocyte stimulation, but its biological role remains unclear because knockout mice show no abnormalities in development (Yamaguchi et al. (1994) 269(2):805-8; Bera et al. (2000) 20(8):2902-6).

[0004] In contrast, MSLN is highly expressed in several human cancers, including virtually all mesotheliomas and pancreatic adenocarcinomas, approximately 70% of ovarian cancers, and 50% of lung adenocarcinomas (Hassan and Ho (2008) Eur. J. Cancer 44:46-53; Miettinen and Sarlomo-Rikala (2003) Am. J. Surg. Pathol. 27:150-8; Ordonez (2003) Am. J. Surg. Pathol. 27:1418-1428; Ho et al. (2007) Clin. Cancer Res. 13:1571-5). The high expression level of MSLN makes it an attractive candidate for targeted therapy due to its important role in promoting tumor growth and invasion (Servais et al. (2012) Clin. Cancer Res. 18(9):2478-2489).

[0005] MSLN interacts with MUC16 to mediate cell adhesion, playing an important role in peritoneal implantation of ovarian cancer cells and increasing the motility and invasion of pancreatic cancer cells (Rump et al. (2004) J Biol Chem. 279(10):9190-8; Gubbels et al. (2006) Mol Cancer 5(1):50; Coehlo et al. Expert Rev Anticancer Ther. 18(2):177-186; Chen et al. (2013) Sci Rep. 3:1870). Pancreatic tumors, in particular, are often advanced before patients experience symptoms, resulting in a short median survival time (often less than one year). This poor prognosis is due to the tumors often being unable to be surgically removed (completely) and having already metastasized (often undetected). Chemotherapy has also not significantly improved survival or curative outcomes. Despite several attempts, no successful immunotherapy has yet been demonstrated. Even the best-known monoclonal antibody against mesothelin (amatuximab), which binds to the soluble form of mesothelin in MSLN, has not been particularly successful (Baldo and Cecco (2017) Onco. Targets Ther. 10:5337-5353; Nicolaides et al. (2018) Cancer Biology & Therapy 19(7):622-630). Unfortunately, although mesothelin can be used as a biomarker for disease, tumor shedding of mesothelin poses numerous challenges (Hassan et al. (2006) Clin. Cancer Res. 12:447-453). This shedding poses problems for tumor imaging and potential radiotherapy. When MSLN is excreted into the bloodstream, imaging with radiolabeled antibodies exhibits high background levels, and radiolabeled antibodies are toxic due to binding to systemically excreted mesothelin. This necessitates the administration of high doses of anti-mesothelin antibodies, which can be toxic. Summary of the Invention

[0006] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to mesothelin, uses thereof, nucleic acids encoding the antibodies and antigen-binding fragments, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. Pharmaceutical compositions and conjugates comprising the antibodies, and methods of treatment by administering the antibodies, are also disclosed.

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 26, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) VH comprises HCDR1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 69.

[0008] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the integrated Kabat and Chothia numbering system.

[0009] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (2) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 18, respectively; (3) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 17, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (4) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (5) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 20, respectively; (6) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 21, respectively; (7) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 22, respectively; (8) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 23, respectively; (9) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (10) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (11) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (12) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (13) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (14) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (15) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (16) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (17) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (18) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; or (19) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively.

[0010] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (10) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0011] In some embodiments, the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to mesothelin, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), (1) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (2) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0012] In some preferred embodiments, the antibody or antigen-binding fragment thereof specifically binds to membrane-bound mesothelin. In some preferred embodiments, the antibody or antigen-binding fragment thereof binds to membrane-bound mesothelin with higher affinity than to soluble mesothelin. In some preferred embodiments, the antibody or antigen-binding fragment thereof binds to membrane-bound mesothelin with an affinity that is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold greater than its binding affinity to soluble mesothelin. In some embodiments, the antibody or antigen-binding fragment thereof does not substantially bind to soluble MSLN. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to soluble MSLN.

[0013] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In preferred embodiments, the antibody is a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0014] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv, and dAb. In some embodiments, the antibody is a monoclonal antibody, a bispecific or a multispecific antibody.

[0015] In some embodiments, the antibody is monovalent, bivalent, or multivalent. In some embodiments, the antibody or antigen-binding fragment is conjugated to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0016] In a second aspect, the present invention provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof of the first aspect of the invention and a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

[0017] In a third aspect of the invention, the invention provides a nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the first aspect of the invention, or the bispecific antibody of the second aspect of the invention.

[0018] In a fourth aspect, the present invention provides a vector comprising a nucleic acid of the third aspect of the invention. In a fifth aspect, the present invention provides a host cell comprising a nucleic acid of the third aspect of the invention or a vector of the fourth aspect of the invention.

[0019] In a sixth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof of the first aspect of the invention, or the bispecific antibody of the second aspect of the invention.

[0020] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a bispecific antibody according to the second aspect of the invention, or a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a host cell according to the fifth aspect of the invention, or an antibody-drug conjugate according to the sixth aspect of the invention, and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0021] In some embodiments, the composition further comprises a second therapeutic agent selected from the group consisting of an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0022] In an eighth aspect, the present invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition of the present invention.

[0023] In some particular embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0024] In some embodiments, the method further comprises administering to the subject a second therapeutic agent. Preferably, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0025] In a ninth aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition of the invention in the manufacture of a medicament for treating cancer in a subject.

[0026] In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0027] In some embodiments, the agent comprises a second therapeutic agent, optionally wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0028] In some embodiments, the agent is administered in combination with a second therapeutic agent, optionally wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0029] In a tenth aspect, the present invention provides an antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition of the present invention for use in a method for treating cancer in a subject.

[0030] In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0031] In some embodiments, the subject is further administered a second therapeutic agent, optionally selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0032] In an eleventh aspect, the present invention provides a method for producing a composition comprising: (a) obtaining a biological sample from a subject; (b) contacting the sample with an antibody or antigen-binding fragment thereof of the invention; (c) detecting binding of the antibody to the sample; wherein increased binding of the antibody or antigen-binding fragment thereof to the sample compared to binding of the antibody or antigen-binding fragment thereof to a control sample identifies the subject as having the mesothelin-positive cancer.

[0033] In a twelfth aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) administering to a subject an antibody of the present invention or an antigen-binding fragment thereof conjugated to a detectable marker; (b) detecting the presence of the marker;

[0013] The present invention provides a method for imaging mesothelin-positive cancer in a subject, comprising:

[0034] In some preferred embodiments, (a) the detectable marker is 111 In, preferably wherein detection of the marker is single photon emission computed tomography, or (b) Detectable markers are 89 Zr, and preferably the detection of the marker is by positron emission tomography. [Brief explanation of the drawings]

[0035] [Figure 1] This is a scheme for generating anti-mesothelin antibodies. Harbor mouse H2L2 transgenic mice were immunized with repeated injections of recombinant mesothelin protein, followed by hybridoma generation and clone screening to identify MSLN-specific tetramer (H2L2) antibodies. [Figure 2] Screening strategy and process workflow for single B cell cloning screening. [Figure 3] Binding of antibodies PR300159 and PR300186 to membrane-bound human MSLN on the surface of CHOK1 human MSLN cells. [Figure 4] Binding of antibodies PR300159 and PR300186 to membrane-bound cynomolgus MSLN on the surface of CHOK1 cynomolgus MSLN cells. [Figure 5] Binding of antibodies PR300159 and PR300186 to membrane-bound human MSLN endogenously expressed on the COV644 cell line. [Figure 6] Binding of PR300186 and PR300186 PTM-removed antibody to human MSLN protein. [Figure 7] Binding of PR300186 and PR300186 PTM-removed antibody to cynomolgus monkey MSLN protein. [Figure 8] Binding of PR300159 and PR300159 PTM-depleted antibody to COV644 cells. [Figure 9] Binding of PR300186 and PR300186 PTM-depleted antibody to COV644 cells. [Figure 10] Internalization of PR300159 and PR300159 PTM-depleted antibody on COV644 cells. [Figure 11] Internalization of PR300186 and PR300186 PTM-deleted antibodies on COV644 cells. [Figure 12] Binding of antibody PR300159 to COV644 cells in the presence or absence of soluble MSLN (sMSLN): (A) without sMSLN, (B) with 90 nM sMSLN. [Figure 13] Binding of antibody PR300186 to COV644 cells in the presence or absence of soluble MSLN (sMSLN): (A) without sMSLN, (B) with 90 nM sMSLN. [Figure 14] Binding of antibody PR300159-8 to COV644 cells in the presence or absence of soluble MSLN (sMSLN). [Figure 15] Binding of antibody PR300186-10 to COV644 cells in the presence or absence of soluble MSLN (sMSLN). DETAILED DESCRIPTION OF THE INVENTION

[0036] array The amino acid sequences of the light chain, heavy chain, light chain variable region (VL), and heavy chain variable region (VH), as well as the CDRs and FWRs of the light chain and heavy chain are shown in Tables 1 to 4 below.

[0037] [Table 1-1]

[0038] [Table 1-2]

[0039] [Table 1-3]

[0040] [Table 1-4]

[0041] [Table 1-5]

[0042] [Table 1-6]

[0043] [Table 1-7]

[0044] [Table 1-8]

[0045] [Table 1-9]

[0046]

Table 2-1

[0047]

Table 2-2

[0048]

Table 2-3

[0049]

Table 2-4

[0050]

Table 3-1

[0051]

Table 3-2

[0052]

Table 3-3

[0053]

Table 4-1

[0054]

Table 4-2

[0055]

Table 4-3

[0056] Detailed Description of the Invention The foregoing features and advantages of the present invention, as well as further features and advantages of the present invention, will be more clearly understood hereinafter as a result of the following detailed description of the embodiments taken in conjunction with the drawings.

[0057] The embodiments described herein with reference to the drawings are illustrative and exemplary and are used to generally understand the present invention. The embodiments should not be construed as limiting the scope of the present invention. Identical or similar elements, and elements having identical or similar functions, are designated by like reference numerals throughout the description.

[0058] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which is apparent to one of ordinary skill in the art. For example, Leuenberger, HGW, Nagel, B. and Klbl, H. eds., “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley InterScience, New York (1987); Roitt et al., “Immunology (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., “Immunobiology” (6th Ed.), Garland Science Publishing / Churchill Livingstone,New Reference is made to standard handbooks such as York (2005), as well as the general background art cited above.

[0059] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the reference to "an antibody" includes a plurality of antibodies.

[0060] Unless otherwise indicated or defined, the term "comprise" and variations thereof, such as "comprises" and "comprising," should be understood to mean the inclusion of the stated element or step or group of elements or steps, but not the exclusion of other elements or steps or groups of elements or steps. The term "comprising" encompasses the meanings of "comprise" and "consist of," e.g., a composition "comprising" X may consist solely of X, or it may include additional elements such as X+Y.

[0061] The term "about" in reference to a numerical value x is arbitrary and means, for example, x+10% or x±5%. As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific antigen. Antibodies often comprise variable and constant regions in each of their heavy and light chains. The variable regions of the antibody's heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or elements, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as CIq, the first component of the classical pathway of complement activation. Most antibodies contain a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antigen-binding portion.

[0062] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of four "framework" regions interrupted by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs for specific binding to an antigen epitope. The CDRs contain the amino acid residues of an antibody primarily responsible for antigen binding. Both VL and VH domains are composed of framework (FR) and CDR regions, from amino- to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, 2, and 3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively, and CDR1, 2, and 3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0063] The assignment of amino acids to each VL and VH domain follows the conventional CDR definitions. Classic definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)); the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); Chothia-Kabat CDR combinations (also called Chothia and Kabat combined CDRs), in which each CDR is a combination of a Chothia CDR and a Kabat CDR; the AbM definition used in Oxford Molecular's antibody modeling software; and the contact definition of Martin et al. (worldwidewebbioinfo.org.uk / abs). Kabat provided a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or different light chains are assigned the same number.

[0064] Although the present disclosure includes CDRs defined according to any of these numbering systems, preferred embodiments include the combined Chothia and Kabat defined CDRs.

[0065] [Table 5]

[0066] In Table 5, Laa-Lbb may refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain; Haa-Hbb may refer to the amino acid sequence from position aa (according to the Chothia numbering system) to position bb (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from positions 24 to 34 (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain; H26-H32 may refer to the amino acid sequence from positions 26 to 32 (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain.

[0067] The term "antibody" as used herein should be understood in the broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen-binding regions. Antibodies may contain further modifications such as unnatural amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of the antibody, and immunoglobulin molecules containing other modifications in the antigen recognition site, so long as the antibody exhibits the desired biological activity.

[0068] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., MSLN). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0069] Examples of antigen-binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed. 1993)); (iv) an Fd fragment consisting of the VH and CH1 domains; (v) an Fd' fragment comprising the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (vii) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (viii) isolated complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing one variable domain and two constant domains. Furthermore, although the two domains of an Fv fragment, the VL and VH, are encoded by separate genes, they can be linked using recombinant techniques by a synthetic linker that allows the VL and VH domains to pair into a single protein chain, forming a monovalent molecule known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Additionally, the term also includes "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which form an antigen-binding region together with complementary light chain polypeptides, and modified versions of any of the foregoing fragments that retain antigen-binding activity.

[0070] These antigen-binding fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. As used herein, the terms "binding" or "specifically bind" refer to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium constant (KD) of the dissociation of an antigen with an antibody, is a measure of the binding strength between an antigenic determinant (epitope) and an antigen-binding site on an antibody: the lower the KD value, the stronger the binding strength between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can be expressed as an affinity constant (KA), which is 1 / KD.

[0071] Avidity is a measure of the strength of binding between an antibody and the relevant antigen. Avidity is related to both the affinity between the antigenic determinant (epitope) and the antigen-binding site on the antibody, and the number of suitable binding sites present on the antibody. Typically, antibodies have a binding affinity of 10 -5 ~10 -12 M or less, preferably 10 -7 ~10 -12 M or less, preferably 10 -8 ~10 -12 with a dissociation constant (KD) of M and / or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , at least 10 12 M -1 It binds with a binding affinity of 10 -4 Any K greater than M DThe values are generally considered to be indicative of non-specific binding. In particular, the binding of an antibody to an antigen or antigenic determinant can be determined by any suitable method known per se in the art, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), biolayer interferometry (BLI) assays, and sandwich competition assays, and their different modifications known per se in the art.

[0072] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding (also called conformational epitopes) are typically lost upon treatment with denaturing solvents. An epitope typically contains at least three, more usually at least five or 8-10, amino acids in a unique spatial or conformational configuration. An epitope defines the minimal binding site of an antibody and thus serves as a specific target for the antibody or its antigen-binding fragment.

[0073] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have the same residues at the same positions in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity between an amino acid sequence and SEQ ID NO: Y, meaning that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in SEQ ID NO: Y. Generally, computer programs are used for such calculations. Examples of programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and Gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0074] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art can also take into account so-called "conservative" amino acid substitutions, which are generally described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, and have little or no effect on the function, activity or other biological properties of a polypeptide.Such conservative amino acid substitutions are well known in the art, for example, from WO 04 / 037999, GB-A-2357768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0075] Such conservative substitutions are preferably those in which one amino acid residue within the following groups (a) to (e) is substituted with another amino acid residue within the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, Gln; (c) polar, positively charged residues: His, Arg, Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val, Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0076] Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0077] Any amino acid substitutions applied to the polypeptides described herein may be based on the following: the frequency analysis of amino acid mutations between homologous proteins of different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978; the structure-forming ability analysis, developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol., 47:45-149, 1978; the hydrophobicity pattern analysis of proteins, developed by Eisenberg et al., Proc. Nat. Acad Sci. USA 81:140-144, 1984; Kyte & Doolittle, J Mol. Biol. 157:105-132, 1981; and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986; all of which are incorporated herein by reference in their entireties.

[0078] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population; that is, each antibody comprising the population is identical except for minor variations that may occur naturally. Monoclonal antibodies are highly specific, being directed against a single antigen. The use of the term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring production of the antibody by any particular method.

[0079] The term "bispecific antibody" in the context of the present invention is understood as an antibody having two different antigen-binding regions defined by different antibody sequences, which bind to different targets, but can also be understood to include binding to different epitopes on one target.

[0080] As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and therefore can be used to target cancer cells. As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0081] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or that such carrier or adjuvant is approved or licensed for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0082] As used herein, the terms "therapy," "treatment," and the like refer to administering a drug or performing a procedure with the intent to achieve an effect. The effect may be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or its symptoms are partially or completely cured. As used herein, "therapy" includes the treatment of a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the development of a disease or disease symptoms in a subject who is predisposed to the disease but has not yet been diagnosed with the disease (e.g., including diseases associated with or caused by a primary disease); (b) inhibiting the disease, i.e., arresting the progression of the disease; and (c) palliating the disease, i.e., causing regression of the disease. Treatment may refer to any indicator of success in treating, ameliorating, or preventing cancer, including objective or subjective parameters such as palliation; remission; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of degeneration or decline; or reducing decline in the final stages of degeneration. The treatment or amelioration of symptoms may be based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treatment" includes administering an antibody or composition or conjugate disclosed herein to prevent, delay, alleviate, or arrest or inhibit the onset of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0083] As used herein, the term "effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease. The term "subject," as used herein, refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, farm animals, farm animals, laboratory animals, zoo animals, sport animals, or pet animals (dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.).

[0084] The terms "cyno," "cynomolgus," and "Cynomolgus macaques" are used interchangeably herein to refer to cynomolgus MSLN. This term includes variants, isoforms, and species homologs of MSLN naturally expressed by cynomolgus cells or expressed on cells transfected with a gene or cDNA encoding cynomolgus MSLN naturally expressed in cynomolgus cells.

[0085] Anti-MSLN antibody The present invention provides anti-mesothelin antibodies. In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 26, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) VH comprises HCDRs 1 to 3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1 to 3 of VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) The VH comprises HCDRs 1 to 3 of the VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDRs 1 to 3 of the VL having the amino acid sequence set forth in SEQ ID NO: 69, and specifically binds to mesothelin.

[0086] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined according to the integrated Kabat and Chothia numbering system.

[0087] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (2) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 18, respectively; (3) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 17, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (4) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (5) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 20, respectively; (6) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 21, respectively; (7) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 22, respectively; (8) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 23, respectively; (9) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (10) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (11) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (12) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (13) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (14) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (15) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (16) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (17) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (18) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; or (19) VH comprises HCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDR1 to 3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively.

[0088] In some embodiments, the CDRs are determined by the Kabat Chothia integrated system. In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (10) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0089] In some embodiments, the VL comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 27-33 and 67-69 formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that antibodies comprising the VL comprising the functional variant retain the ability to bind to MSLN. In some embodiments, the VH comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 24-26 and 63-66 formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that antibodies comprising the VH comprising the functional variant retain the ability to bind to MSLN.

[0090] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide. For example, a functional variant of any one of SEQ ID NOs: 27 to 33 and 67 to 69 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 27 to 33 and 67 to 69, respectively. For example, a functional variant of any one of SEQ ID NOs: 24 to 26 and 63 to 66 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 24 to 26 and 63 to 66.

[0091] In some embodiments, a functional variant of any one of SEQ ID NOs: 27-33 and 67-69 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 27-33 and 67-69, formed by insertion, deletion, and / or substitution of one or more amino acids in any one of SEQ ID NOs: 27-33 and 67-69. In some embodiments, a functional variant of any one of SEQ ID NOs: 24-26, and 63-66 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity thereto, formed by insertion, deletion, and / or substitution of one or more amino acids in any one of SEQ ID NOs: 24-26, and 63-66.

[0092] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the parent amino acid sequence, more preferably 35% or less, even more preferably 1 to 33%, even more preferably 5 to 30%, even more preferably 10 to 25%, and even more preferably 15 to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0093] In some embodiments, insertions, deletions and / or substitutions may be made in framework (FR) regions, such as, for example, FR1, FR2, FR3, and / or FR4. In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid residue within the following groups (a) to (e) is replaced with another amino acid residue within the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, Gln; (c) polar, positively charged residues: His, Arg, Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val, Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0094] Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0095] In a preferred embodiment, the VL comprises the amino acid sequence set forth in SEQ ID NO:27, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:28, and the VH comprises the amino acid sequence set forth in SEQ ID NO:25; or the VL comprises the amino acid sequence set forth in SEQ ID NO:27, and the VH comprises the amino acid sequence set forth in SEQ ID NO:26; or the VL comprises the amino acid sequence set forth in SEQ ID NO:29, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:30, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:31, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:32, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:33, and the VH comprises the amino acid sequence set forth in SEQ ID NO:24; or the VL comprises the amino acid sequence set forth in SEQ ID NO:29, and the VH comprises the amino acid sequence set forth in SEQ ID NO:25. or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 63; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 64; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 68 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 68 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 63; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 67 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 66; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 64; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 66; or the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 63;or the VL comprises the amino acid sequence set forth in SEQ ID NO: 69 and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65;

[0096] Based on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulin molecules are classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, which may be further classified into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chain of an antibody may be classified as a lambda (λ) chain or a kappa (κ) chain based on the amino acid sequence of the light chain. The antibodies disclosed herein may be of any of the above classes or subtypes.

[0097] In some embodiments, the antibody may be of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody may be of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgG1 antibody.

[0098] The antibodies disclosed herein may be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment may be any fragment of an antibody that retains the ability to specifically bind to MSLN. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity-determining regions (CDRs); nanobodies; linear antibodies comprising a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified versions of any of the foregoing fragments that retain antigen-binding activity.

[0099] In some embodiments, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In preferred embodiments, the antigen-binding fragment is a Fab or scFv.

[0100] In some embodiments, the invention provides an antibody, or antigen-binding fragment thereof, that specifically binds to mesothelin, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), (1) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (2) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0101] In some embodiments, the light chain comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 37-43 and 74-76 formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that an antibody comprising a light chain comprising the functional variant retains the ability to bind to MSLN. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 34-36 and 70-73 formed by the insertion, deletion, and / or substitution of one or more amino acids therein, provided that an antibody comprising a heavy chain comprising the functional variant retains the ability to bind to MSLN.

[0102] For example, a functional variant of any one of SEQ ID NOs: 37 to 43 and 74 to 76 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 37 to 43 and 74 to 76, respectively. For example, a functional variant of any one of SEQ ID NOs: 34 to 36 and 70 to 73 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 34 to 36 and 70 to 73, respectively.

[0103] In some embodiments, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the parent amino acid sequence, more preferably 35% or less, even more preferably 1 to 33%, even more preferably 5 to 30%, even more preferably 10 to 25%, and even more preferably 15 to 20%. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In a preferred embodiment, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0104] In some embodiments, insertions, deletions and / or substitutions may be made in framework (FR) regions, such as, for example, FR1, FR2, FR3 and / or FR4; and / or constant regions, such as, for example, CL, CH1, CH2 and / or CH3.

[0105] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above. In a preferred embodiment, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 38 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 35; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 36; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 40 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 41 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 42 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 43 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 34; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 39 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 35. or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 71; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 75 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 75 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 74 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 73; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 71; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 73; or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 70;or the light chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 72;

[0106] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region is of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0107] In one embodiment, the Fc region is effector function-deficient. For example, the Fc region may be of the IgG1 isotype, or a non-IgG1 type such as IgG2, IgG3, or IgG4 that has been mutated to reduce or eliminate its ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001). In some embodiments, the antibody Fc region comprises a wild-type IgG1 Fc with the following mutations: L234A, L235A, and G237A.

[0108] In some embodiments, the antibody is mutated at one or more post-translational modification sites, hi one embodiment, the Fc region comprises a mutation that removes acceptor sites for Asn-linked glycosylation or is engineered to eliminate an effector function of the antibody.

[0109] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. Apart from conserved PTM sites in antibodies, such as the conserved N-glycosylation site on the CH2 domain of an IgG1 antibody, other PTM sites occurring in the antigen-binding site (i.e., CDR region) of an antibody may reduce antigen-binding activity and chemical stability. For example, deamidation or isomerization may lead to instability or heterogeneity of the molecule. To reduce sequence burden, PTM motifs can be removed by mutation. VH or VL sequences were scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). Next, "hotspot" residues (e.g., D or G in a DG motif) were mutated to their germline counterparts or other residues with similar biophysical properties.

[0110] bispecific antibody In a second aspect, the application provides a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen may be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0111] Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that have the potential to stimulate distinct tumor-specific immune responses. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at specific stages of differentiation, or those that are expressed over time, such as embryonic or fetal antigens. Other cancer antigens are encoded by mutated cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutated p53), and fusion proteins generated by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies thereto are known and / or commercially available, and can be produced by those skilled in the art.

[0112] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, CD200R1, MUC-1, mutant p53, mutant ras, ROR1, VEGFR2, and combinations thereof.

[0113] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen can be selected from the group consisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof.

[0114] In some embodiments, the second antigen is an immune checkpoint molecule, hi some embodiments, the immune checkpoint molecule may be selected from the group consisting of PD-1, PD-L1, CTLA-4, etc.

[0115] In some embodiments, a bispecific antibody comprises a single polypeptide chain comprising a first antigen-binding region and a second antigen-binding region, and optionally an Fc region. The Fc region may be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0116] In one embodiment, the Fc region is effector-function-deficient. For example, the Fc region may be of the IgG1 isotype, or a non-IgG1 type such as IgG2, IgG3, or IgG4 that has been mutated to reduce or eliminate its ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).

[0117] In one embodiment, the Fc region comprises a mutation that removes an acceptor site for Asn-linked glycosylation or is engineered to alter glycosylation characteristics. For example, in an IgG1 Fc region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence with an N297Q mutation. For example, in an IgG1 Fc region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence with an N297Q mutation.

[0118] In further embodiments, the Fc region has been glycoengineered to reduce fucose and thus enhance ADCC, e.g., by adding compounds to the culture medium during antibody production, as described in U.S. Patent No. 2009317869, or by using FUT8 knockout cells, as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87:614. Alternatively, ADCC may be optimized using the methods described in Umana et al. (1999) Nature Biotech 17:176. In further embodiments, the Fc region has been engineered to enhance complement activation, as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0119] nucleic acid In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0120] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides that make up a nucleic acid can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0121] For example, the invention provides a nucleic acid molecule encoding any one of the heavy chain variable region sequences disclosed herein. The invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the heavy chain variable region sequences disclosed herein.

[0122] For example, the invention provides nucleic acid molecules encoding any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the heavy chain variable region sequences disclosed herein.

[0123] For example, the invention provides nucleic acid molecules encoding: (i) any one of the heavy chain variable region sequences disclosed herein, and (ii) any one of the light chain variable region sequences disclosed herein. The invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding: (i) any one of the heavy chain variable region sequences disclosed herein, and (ii) any one of the light chain variable region sequences disclosed herein.

[0124] For example, the invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising the CDR sequences of any one of the heavy chain variable region sequences disclosed herein. In some embodiments, the invention provides nucleic acid molecules encoding a heavy chain variable region sequence comprising any one of the three groups of CDR sequences disclosed herein.

[0125] The present invention also provides nucleic acid molecules encoding heavy chain variable region sequences comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequences of any of the heavy chain variable region sequences disclosed herein.

[0126] In some embodiments, the present invention provides nucleic acid molecules encoding heavy chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical, respectively, to the CDR1, CDR2, and CDR3 of any one of the three groups of CDR sequences disclosed herein.

[0127] For example, the invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising the CDR sequences of any one of the light chain variable region sequences disclosed herein. In some embodiments, the invention provides nucleic acid molecules encoding a light chain variable region sequence comprising any one of the three groups of CDR sequences disclosed herein.

[0128] The present invention also provides nucleic acid molecules encoding light chain variable region sequences comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequences of any of the light chain variable region sequences disclosed herein.

[0129] In some embodiments, the present invention provides nucleic acid molecules encoding light chain variable region sequences comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical, respectively, to the CDR1, CDR2, and CDR3 of any one of the three groups of CDR sequences disclosed herein.

[0130] For example, the present invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence comprising the CDR sequences of any one of the heavy chain variable region sequences disclosed herein, and (ii) a light chain variable region sequence comprising the CDR sequences of any one of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein, and (ii) a light chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein. The present invention also provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequences of any of the heavy chain variable region sequences disclosed herein, and (i) a light chain variable region sequence comprising CDR sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR sequences of any of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical, respectively, to the CDR1, CDR2, and CDR3 of any one of the three groups of CDR sequences disclosed herein; and (i) a light chain variable region sequence comprising CDR1, CDR2, and CDR3 sequences that are at least 90%, at least 95%, at least 98%, or at least 99% identical, respectively, to the CDR1, CDR2, and CDR3 of any one of the three groups of CDR sequences disclosed herein.

[0131] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the invention provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the invention provides a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0132] Thus, deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, is used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0133] In some embodiments, deoxyribonucleic acid (DNA) may be introduced into cells of the human body in vivo. In some embodiments, deoxyribonucleic acid (DNA) of the invention is contained in a vector or delivery agent. In some embodiments, deoxyribonucleic acid (DNA) of the invention is integrated into the genome of a cell.

[0134] Thus, ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, may be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0135] In some embodiments, the ribonucleic acid (RNA) may be introduced into cells of the human body in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0136] In certain embodiments, the ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, is mRNA. In some embodiments, the mRNA of the present invention is contained in a vector or delivery system (e.g., lipidosome). In some embodiments, the mRNA may be introduced into cells of the human body in vivo via a vector or delivery system (e.g., lipidosome), resulting in expression of the MSLN antibody of the present invention in vivo.

[0137] The mRNA of the present invention may be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

[0138] vector In a fourth aspect, the present invention further provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding an anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0139] In some embodiments, the vector is a recombinant expression vector capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-MSLN antibody. For example, the invention provides recombinant expression vectors comprising any of the above-described nucleic acid molecules.

[0140] Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neolargeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCVPIG (PuroIRESGFP emptyplasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCVIRESLuciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherryFP, pRetroXGFPT2ACre, pRXTN, pLncEXP, and pLXIN-Luc.

[0141] The recombinant expression vector may be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion, or for expression, or both, such as plasmids and viruses. For example, vectors may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0142] Recombinant expression vectors may be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs may be prepared to contain an autonomously replicating system that functions in prokaryotic or eukaryotic host cells. Autonomous replicating systems may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0143] Thus, the vectors may be used to treat diseases. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer. The vectors of the present invention may be introduced into cells. In some embodiments, the vectors of the present invention may be introduced into cells in vitro or ex vivo. Optionally, the cells transfected with the vector may subsequently be administered to the body of a subject. In some embodiments, the vectors of the present invention may be introduced into cells in vivo.

[0144] For example, the vector may be an adenovirus vector comprising a nucleotide sequence encoding the anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or the bispecific antibody or antigen-binding fragment thereof disclosed herein. The vector may be administered to a subject and then enter the subject's cells in vivo, whereby the nucleotide sequence encoding the anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, or the bispecific antibody or antigen-binding fragment thereof disclosed herein, is integrated into the genome of the cells, and the cells subsequently express the anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, thereby treating the disease disclosed herein.

[0145] host cell In a fifth aspect, the present invention further provides a host cell comprising a nucleic acid as disclosed herein or a vector as disclosed herein.

[0146] Any cell may be used as a host cell for the nucleic acids or vectors of the present disclosure, hi some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotes include, but are not limited to, eubacteria such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans; and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas aeruginosa (P. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHOK1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0147] The host cells of the present invention are prepared by introducing the vectors or nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present invention may be administered to a subject, whereby the host cells express the anti-MSLN antibodies or antigen-binding fragments thereof disclosed herein in vivo, thereby treating the diseases disclosed herein.

[0148] The present invention further provides host cells into which any of the above vectors have been introduced. The present invention further provides methods for producing the antibodies and antibody fragments of the invention by culturing host cells under conditions that allow for the production of antibodies or antibody fragments, and recovering the produced antibodies and antibody fragments.

[0149] antibody-drug conjugates In a sixth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof of the first aspect of the invention, or the bispecific antibody of the second aspect of the invention.

[0150] In the context of this disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody bound to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC." Other antibody conjugates include, for example, multispecific (e.g., bispecific or trispecific) antibodies.

[0151] In some embodiments, the effector molecule may be a detectable label or an immunotoxin. Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxins (such as PE, PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic substances that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds, usually resulting in death through liver toxicity. However, PE and DT can be modified into a form for use as an immunotoxin by removing the toxin's original targeting component (such as domain 1a of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. The terms "conjugated" or "linked" may refer to the assembly of two polypeptides into a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to an effector molecule. In another embodiment, the antibody linked to the effector molecule is further linked to a lipid or other molecule, protein, or peptide to extend its half-life in the body. Linkage can be achieved by either chemical or recombinant means. In one embodiment, the linkage is chemical, where a reaction between the antibody moiety and the effector molecule forms a covalent bond between the two molecules, forming a single molecule. Optionally, a peptide linker (a short peptide sequence) can be included between the antibody and the effector molecule.

[0152] The present invention provides immunoconjugates comprising the monoclonal antibodies or antigen-binding fragments disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or a variant thereof. In other embodiments, the effector molecule is a detectable label, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope.

[0153] The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, a molecule in which a therapeutic agent is covalently linked to an antibody. A therapeutic agent is an agent with a specific biological activity directed against a specific target molecule or a cell bearing the target molecule. Those skilled in the art will recognize that therapeutic agents can be vinblastine; daunomycin, etc.; cytotoxins, such as natural or modified Pseudomonas exotoxin or diphtheria toxin; encapsulating media (such as liposomes) containing pharmacological compositions; 125 I, 32 P, 14 C. 3 H, and 35 It will be understood that the present invention may include a variety of agents, such as radiopharmaceuticals, such as S; and other labels, targeting moieties, and ligands.

[0154] The selection of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, a therapeutic agent may be a cytotoxin used to cause the death of a particular target cell (such as a tumor cell). Conversely, if it is desired to elicit a non-lethal biological response, the therapeutic agent may be attached to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0155] Using the therapeutic agents and antibodies described herein, one of skill in the art can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids that vary in sequence but encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies, as well as conjugates and fusion proteins thereof.

[0156] Effector molecules can be linked to the antibody of interest using several means known to those of skill in the art. Both covalent and non-covalent means may be used. The procedure for attaching an effector molecule to an antibody varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which react with appropriate functional groups on the antibody to allow attachment of the effector molecule. Alternatively, the antibody can be derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any of several known linker molecules. The linker can be any molecule used to attach an antibody to an effector molecule. The linker can form covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule are polypeptides, the linkers may be attached to the constituent amino acids through their side groups (such as a disulfide bond to cysteine) or may be linked to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0157] In some situations, it is desirable to release the effector molecule from the antibody once the immunoconjugate has reached the target site, and therefore, in these situations, the immunoconjugate comprises a cleavable linkage in the vicinity of the target site.

[0158] Cleavage of the linker to release the effector molecule from the antibody may be facilitated by enzymatic activity or conditions to which the immunoconjugate is subjected within the target cell or near the target site. In view of the numerous reported methods for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art would be able to determine an appropriate method for conjugating a given agent to an antibody or other polypeptide.

[0159] The antibodies disclosed herein may be derivatized or conjugated to another molecule (such as another peptide or protein). Generally, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, the antibody may be functionally linked (by chemical bonding, genetic fusion, non-covalent bonding, etc.) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate binding of the antibody or antibody portion to another molecule (such as a streptavidin core region or a polyhistidine tag).

[0160] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or different types, so as to create bispecific antibodies). Suitable crosslinkers include heterobifunctional crosslinkers (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), which have two distinct reactive groups separated by a suitable spacer, or homobifunctional crosslinkers (such as disuccinimidyl suberate). Such linkers are commercially available.

[0161] The antibody may be conjugated to a detectable marker, such as a detectable marker that can be detected by, for example, ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. Antibodies or antigen-binding fragments can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to generate a discernible reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine generates a visually detectable colored reaction product. Antibodies or antigen-binding fragments can also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0162] The antibody may be fused with a self-labeling protein tag (e.g., HaloTag). For example, the protein tag can be cloned at the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) and is designed to be covalently bound to a synthetic ligand. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0163] The antibody may be labeled with a magnetic agent such as gadolinium. The antibody may also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles, such as superparamagnetic iron oxide, are also useful as labels. Antibodies may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, etc.). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0164] Antibodies can also be labeled with radioactively labeled amino acids. Radioactive labels can be used for both diagnostic and therapeutic purposes. For example, radioactive labels can be used to detect the expression of target antigens by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0165] Antibodies can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl groups, ethyl groups, or carbohydrate groups. These groups may be useful to improve the biological properties of the antibody, such as increasing serum half-life or enhancing tissue binding.

[0166] Toxins can be used in combination with the monoclonal antibodies described herein to generate immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and its subunits, and botulinum toxins A-F. These toxins are readily available commercially (e.g., from Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include mutant forms of the toxins described herein (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length native (naturally occurring) PE or modified PE. Such modifications include, but are not limited to, removal of domain Ia, deletion of various amino acids in domains Ib, II, and III, substitution of single amino acids, addition of one or more sequences to the carboxyl terminus, etc. (See, e.g., Siegall et al. Biol. Chem. 264:14256-14261, 1989).

[0167] PE for use in the monoclonal antibodies described herein includes native sequences, cytotoxic fragments of native sequences, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include fragments that exhibit cytotoxicity with or without subsequent proteolytic or other processing within target cells. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see, e.g., U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; WO 99 / 51643; and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0168] Also contemplated herein are protease-resistant and reduced immunogenicity PE variants, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, which are incorporated herein by reference).

[0169] In some instances, the PE is a mutant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; WO 2009 / 032954). In other instances, the PE is a mutant designated PE-LR / 6X (WO 2011 / 032022). In other instances, the PE mutant is a PE with reduced immunogenicity. In yet other instances, the PE is a mutant designated PE-LR / 8M (WO 2011 / 032022).

[0170] Modification of PE may occur in any of the variants described above, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). Modified PEs include any substitution, such as the substitution of one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or the deletion of one or more T cell epitopes and / or B cell epitopes (see, e.g., U.S. Patent Application Publication No. 2015 / 0099707). Contemplated forms of PE also include deimmunized forms, such as PE lacking domain II (e.g., PE24). Deimmunized forms of PE are described, for example, in WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0171] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing tumor or viral antigens on their surface. Thus, the antibodies of the present disclosure can be conjugated directly or via a linker to drugs that are delivered directly to cells expressing the cell surface antigen. This can be done for therapeutic, diagnostic, or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or their derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single- or double-stranded DNA, and triplex-forming oligonucleotides.

[0172] Alternatively, the molecule linked to the antibody can be an encapsulation system such as a nanoparticle, liposome, or micelle containing a therapeutic composition such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety, preferably shielded from direct exposure to the circulatory system. Methods for preparing antibody-bound liposomes are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0173] The antibodies described herein may be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (such as horseradish peroxidase and alkaline phosphatase commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, or latex) beads.

[0174] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radioactive labels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0175] Pharmaceutical Composition In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a bispecific antibody according to the second aspect of the invention, or a nucleic acid according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a host cell according to the fifth aspect of the invention, or an ADC according to the sixth aspect of the invention, and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0176] The present invention provides pharmaceutical compositions comprising an antibody of the present invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer.

[0177] The antibodies or agents of the present invention (also referred to herein as "active compounds"), and their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference textbook in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0178] The pharmaceutical compositions of the present invention are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetic acid, citric acid, or phosphate, and an agent for adjusting osmolality such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0179] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Carriers suitable for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0180] Sterile injectable solution can be prepared by mixing the required amount of active compound in a suitable solvent containing one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.Generally, dispersion is prepared by mixing the active compound in a sterile vehicle containing a basic dispersion medium and other necessary ingredients as listed above.For the preparation of sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0181] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated into an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is orally applied, swished, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0182] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0183] Systemic administration can also be carried out by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as is generally known in the art.

[0184] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared using carriers that protect the compound from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes bearing monoclonal antibodies against viral antigens to target infected cells) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0185] For ease of administration and uniformity of dosage, it is particularly advantageous to prepare oral or parenteral compositions in dosage unit form.Dosage unit form, as used herein, refers to a physically discrete unit suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect together with the required pharmaceutical carrier.The specifications of the dosage unit form of the present invention are determined and directly depend on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations of the technology of preparing such active compound for individual treatment.

[0186] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The present invention provides therapeutic compositions comprising the anti-MSLN antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions of the present invention are administered with suitable carriers, excipients, and other agents that are incorporated into the drug to improve transport, delivery, tolerance, etc. Many suitable formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary known to all pharmaceutical chemists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., Lipofectin™), DNA complexes, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0187] Production Method Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to produce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0188] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. Generally, peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. Hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0189] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[0190] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, 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 immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be measured, for example, by Scatchard analysis (Munson and Pollard, Anal. Biochem., 107:220 (1980)). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0191] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Culture media suitable for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0192] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0193] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or for the variable domains of one antigen-binding site of an antibody of the invention, to generate a chimeric bivalent antibody.

[0194] A fully human antibody is an antibody molecule in which the entire sequences of both the light and heavy chains, including the CDRs, arise from human genes. Such antibodies are referred to herein as "humanized antibodies," "human antibodies," or "fully human antibodies." Human monoclonal antibodies can be prepared using trioma technology; human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72); and EBV hybridoma technology for producing human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be used and may be produced using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0195] Furthermore, humanized antibodies can be produced in genetically modified plants as an inexpensive alternative to existing mammalian systems. For example, transgenic plants can be tobacco plants, i.e., Nicotiania benthamiana and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable plant transformation can be achieved using Agrobacterium tumefaciens or particle bombardment. For example, nucleic acid expression vectors containing at least the heavy and light chain sequences can be expressed in bacterial cultures, i.e., A. tumefaciens strain BLA4404, via transformation. Infiltration of plants can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and mortar and centrifuging. Antibody isolation and purification can be easily performed by a number of methods known to those skilled in the art. Other methods for producing antibodies in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5; and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Accordingly, the present invention further provides any cell or plant comprising a vector encoding an antibody of the present invention or a vector that produces an antibody of the present invention.

[0196] Additionally, the (human) antibody of interest may be produced in a fungus, for example, Myceliophthora thermophila (e.g., Myceliophthora thermophila strain C1; Visser et al. (2011) Industrial Biotechnology 7(3):214-223). Other examples include Aspergillus species (e.g., A. oryzae (Huynh et al. (2020) Fungal Biology and Biotechnology 7:7), A. niger (Ward et al. (2004) Environ. Microbiol. 70:2567-76), or A. awamori (Joosten et al. (2003) Microb. Cell Fact 2:1)), and Trichoderma species (e.g., T. reesei (Nyyssonen et al. (1993) Biotechnology 11:591-595)). In other cases, the fungus may be a yeast such as Saccharomyces cerevisiae, Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis, or Ogataea minuta (Joosten et al. (2003); Suzuki et al. (2017) J Biosci Bioeng. 124:156-63).

[0197] Moreover, human antibodies can also be produced using additional technologies, such as phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that observed for human antibodies in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in WO 2006 / 008548, WO 2007 / 096779, WO 2010 / 109165, WO 2010 / 070263, WO 2014 / 141189, and WO 2014 / 141192.

[0198] One method for producing a desired antibody, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0199] A further refinement of this procedure, a method for identifying clinically relevant epitopes on immunogens and a correlated method for selecting antibodies that immunospecifically bind to the relevant epitopes with high affinity, is disclosed in WO 99 / 53049.

[0200] The antibodies can be expressed by vectors containing DNA segments encoding the single chain antibodies described above. These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins such as those described in PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid-binding moiety (e.g., protamine); plasmids; phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[0201] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include smallpox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci. USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)), and adeno-associated virus vectors (Kaplitt, M. Get al., Nat. Genet. 8:148 (1994)).

[0202] Poxvirus vectors deliver genes into the cytoplasm. Avipoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus, adeno-associated virus, and herpes simplex virus (HSV) vectors are preferred for delivering nucleic acids to neural cells. Adenovirus vectors deliver expression for a shorter period (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn results in a shorter period than HSV vectors. The specific vector selected depends on the target cell and the condition being treated. Introduction can be achieved by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene transfer include naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0203] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to guide vectors (e.g., adenovirus, HSV) to the desired location. Furthermore, particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as the SynchroMed infusion system. A bulk flow-based method called convection has also proven effective in delivering large molecules to a wide area of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be utilized include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known administration routes.

[0204] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, such as to detect the presence of MSLN in a sample, or to bind to MSLN and disrupt the interaction between MSLN and MUC16.

[0205] In a preferred embodiment, the antibodies of the invention are full-length antibodies that comprise an Fc region similar to a wild-type Fc region that binds to an Fc receptor. treatment The antibodies provided herein can be administered to slow or inhibit the progression of mesothelin-positive cancers or to inhibit the metastasis of mesothelin-positive cancers. In these applications, a therapeutically effective amount of the composition is administered to a subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to inhibit a sign or symptom of cancer. Suitable subjects include patients diagnosed with a cancer that expresses mesothelin, such as mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), or ovarian cancer.

[0206] Provided herein are methods of treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody described herein. Also provided herein are methods of inhibiting metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody described herein. In some embodiments, the mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (e.g., triple-negative breast cancer), or ovarian cancer.

[0207] Administration of the antibodies disclosed herein may also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical removal of tumors). Any suitable anti-cancer agent may be administered in combination with the antibodies disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents such as antimitotic agents, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g., anti-androgens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibody therapies that specifically target cancer cells.

[0208] Another common treatment for some types of cancer is surgical removal of, for example, metastatic tumors. Another example of a treatment is radiation therapy, which is the administration of radioactive material or energy (such as external beam radiation therapy) to the tumor site to eradicate or shrink the tumor before surgical removal.

[0209] Diagnosis and detection methods Provided herein are methods for detecting mesothelin protein in vitro or in vivo. In some cases, mesothelin expression is detected in a biological sample. The sample can be any sample, including, but not limited to, blood samples, tissue from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples also include bodily fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0210] Provided herein is a method for determining whether a subject has a mesothelin-positive cancer by contacting a sample from the subject with a mesothelin-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. If binding of the antibody to the sample is increased compared to binding of the antibody to a control sample, the subject is identified as having a mesothelin-positive cancer.

[0211] In another embodiment, a method is provided for confirming the diagnosis of mesothelin-positive cancer in a subject by contacting a sample from the subject diagnosed with mesothelin-positive cancer with a mesothelin-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. If binding of the antibody to the sample is increased compared to binding of the antibody to a control sample, the diagnosis of mesothelin-positive cancer in the subject is confirmed.

[0212] In some examples of the disclosed methods, the monoclonal antibody is directly labeled. In other examples, the method further includes contacting the sample with a second antibody that specifically binds to the monoclonal antibody and detecting binding of the second antibody. Increased binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects mesothelin-positive cancer in the subject or confirms a diagnosis of mesothelin-positive cancer in the subject.

[0213] In some cases, the cancer is mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), or ovarian cancer. In some instances, the control sample is a sample from a subject without cancer. In particular instances, the sample is a blood or tissue sample.

[0214] In some embodiments of the diagnostic and detection methods, the anti-MSLN antibody is directly labeled with a detectable label. In other embodiments, the anti-MSLN antibody (first antibody) is unlabeled, and a secondary antibody or other molecule capable of binding to the primary antibody is labeled. As is well known to those skilled in the art, the secondary antibody is selected to be capable of specifically binding to a particular species and class of the primary antibody. For example, if the primary antibody is human IgG, the secondary antibody may be an anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, Protein A and Protein G, both of which are commercially available.

[0215] Suitable labels for antibodies or secondary antibodies include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting example of a luminescent material is luminol; a non-limiting example of a magnetic agent is gadolinium; and a non-limiting example of a radioactive label are 125 I, 131 I, 35 S, or 3 H is one example.

[0216] In an alternative embodiment, mesothelin in a biological sample can be assayed by a competitive immunoassay utilizing a mesothelin protein standard labeled with a detectable substance and an unlabeled anti-MSLN antibody. In this assay, the biological sample, labeled MSLN protein standard, and anti-MSLN antibody are combined, and the amount of labeled MSLN protein standard bound to the unlabeled antibody is measured. The amount of MSLN in the biological sample is inversely proportional to the amount of labeled MSLN protein standard bound to the anti-MSLN antibody.

[0217] The immunoassays and methods disclosed herein can be used for several purposes. In one embodiment, an anti-MSLN antibody can be used to detect MSLN production in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of MSLN in a biological sample, such as a tissue sample, blood sample, or serum sample. In some examples, the MSLN is cell surface MSLN. In other examples, the MSLN protein is soluble (e.g., in a cell culture supernatant or in a body fluid sample, such as a blood or serum sample).

[0218] In one embodiment, a kit is provided for detecting MSLN in a biological sample, such as a blood sample or a tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. Kits for detecting polypeptides typically comprise a monoclonal anti-MSLN antibody, such as any of the monoclonal antibodies disclosed herein. In a further embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic label).

[0219] In one embodiment, the kit includes instructional materials disclosing methods for using the anti-MSLN antibody. The instructional materials may be in written, electronic format (e.g., a computer diskette or compact disc), or visual format (e.g., a video file). The kit may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally include means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit may further include buffers and other reagents routinely used in carrying out the particular method. Such kits and suitable contents are well known to those of skill in the art.

[0220] In one embodiment, the diagnostic kit comprises an immunoassay. While the details of the immunoassay may vary depending on the particular format employed, methods for detecting MSLN in a biological sample generally involve contacting the biological sample with an anti-MSLN antibody. The antibody specifically binds under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0221] The antibodies disclosed herein can also be utilized in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. The antibodies can also be used for fluorescence-activated cell sorting (FACS). FACS employs more sophisticated levels of detection, such as multiple color channels, low-angle and obtuse-angle light scatter detection channels, and impedance channels, to separate or sort cells (see U.S. Pat. No. 5,061,620). Any of the monoclonal antibodies that bind to mesothelin disclosed herein can be used in these assays. Thus, the antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation. [Example]

[0222] Example 1. Animal immunization scheme To obtain MSLN-specific antibodies, we immunized Harbor H2L2 transgenic mice (https: / / harbourantibodies.com / ) using different approaches. These immunizations produced numerous antibodies that bound to MSLN extracellular CD proteins and MSLN-expressing cells, including COV644 cells (an ovarian epithelial mucinous carcinoma cell line expressing MSLN).

[0223] Specifically, recombinant human MSLN ECDHis-tag protein (Acro Biosystem, catalog number MSN-H5223) or recombinant cynomolgus macaque MSLN ECDHis-tag protein (having the sequence shown below, SEQ ID NO: 77) was used as an immunogen to immunize Harbor H2L2 transgenic mice.

[0224] [ka]

[0225] Figure 1 shows the scheme for generating anti-mesothelin antibodies by antigen immunization and screening of hybridoma clones. An example of an immunization scheme is listed in Table 6 below. Briefly, each mouse received 50 μg of immunogen as the first boost, followed by 25 μg of immunogen for subsequent boosts, subcutaneously or intraperitoneally, along with an adjuvant (Sigma, S6322). Immunizations were administered every other week for a total of six times. The final immunization was administered intraperitoneally with the immunogen diluted in PBS. Serum titers against recombinant human MSLN ECD His-tagged protein (Acro Biosystem, catalog number MSN-H5223) were tested using ELISA and FACS.

[0226] [Table 6]

[0227] Example 2. Screening for MSLN-specific antibodies 2.1 Hybridoma generation and screening for MSLN-specific antibodies Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, lymphocytes are fused with an immortalized cell line to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a rodent myeloma cell. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. Antibody supernatants produced by hybridoma cells can be screened for binding specificity to the target MSLN by in vitro assays such as enzyme-linked immunosorbent assay (ELISA). After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).

[0228] 2.2 Single B cell screening for MSLN-specific antibodies For single B cell screening, the Beacon® Optofluidic System was used. The system uses photoelectric positioning (OEP™) technology to move individual cells, allowing for simultaneous biological function testing, experimental analysis, positive clone selection, and other manipulations under cell culture conditions. The Beacon platform can perform these tasks on thousands of cells in a massively parallel and automated manner.

[0229] In this example, we used a plasma cell discovery workflow. In each experiment, up to 14,000 plasma cells were screened for the secretion of MSLN-specific antibodies. Plasma cells secreting antigen-specific antibodies were then transferred to a 96-well plate and subjected to single B cell sequencing to identify the heavy and light chains of the antibodies produced by the single B cells (monoclonal). Figure 2 illustrates the screening strategy and process.

[0230] In the examples, antibody heavy and light chain sequences were obtained from single plasma cells using single B cell sequencing. The general procedure includes extraction and purification of total RNA from single plasma cell lysates, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of DNA sequences encoding the antibody heavy and light chains, cloning and transfection, and Sanger sequencing. The obtained sequences were subjected to uniqueness analysis and cluster analysis, and DNA sequences encoding antibody heavy and light chain pairs were synthesized.

[0231] Example 3. Antibody production and purification Recombinant plasmids encoding target antibodies were transiently transfected into HEK293-6E or 293-F cells using PEI (Polyscience, 24885). After transfection, cells were cultured at 37°C, 5% CO2, and 120 rpm with shaking. Cell culture supernatants containing target antibodies were harvested by centrifugation and filtration 6–7 days after transfection. Monoclonal antibodies were purified using Protein A magnetic beads (AmMag Protein A Magnetic Beads, Genscript, L00695).

[0232] The purity of the antibody was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC, Welch Xtimate SEC-300 column, 1x PBS pH 7.4 as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10x8, 4-12%, 12-well, Genscript, M00653). The recombinant antibody was successfully expressed and purified for further characterization.

[0233] Two antibodies were obtained: PR300159 and PR300186. The amino acid sequences of antibodies PR300159 and PR300186 are listed in Tables 1 to 4 above. Meanwhile, the anti-MSLN antibody amatuximab was also generated according to the procedure described above using sequence information from IMGT (http: / / www.imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=9343). This antibody was used as a control in subsequent studies and was assigned the code PR000685.

[0234] Example 4. Binding activity of antibodies to MSLN-expressing cells The binding of recombinant anti-MSLN antibodies to human or cynomolgus monkey MSLN-expressing cells was examined by flow cytometry. In this example, the MSLN-expressing cell lines were the CHOK1 cell line transfected to express human MSLN (CHOK1-huMSLN, supplier: Kyinno, catalog number: KC-1152) or cynomolgus monkey MSLN (CHOK1-cynoMSLN, supplier: Kyinno, catalog number: KC-1174) on their surface, and the COV644 cell line (ECACC, catalog number: 07071908).

[0235] Briefly, anti-MSLN antibodies were serially diluted in staining buffer (PBS containing 2% FBS) from 1 to 2 × 10 5 50 μL of diluted antibody solution was added to 50 μL of cell suspension containing 100 cells and incubated at 4°C for 1 hour. The cells were washed twice with staining buffer (PBS containing 2% FBS), and 100 μL of a 1:1000 diluted florescent-labeled anti-human IgG antibody (AlexaFluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation at 4°C for 1 hour, the cells were washed twice with staining buffer and subjected to flow cytometry. PR000685 (amatuximab) and an unrelated IgG isotype control (Crownbio) were used as positive and negative controls, respectively.

[0236] The results are shown in Figures 3 to 5 below. The results show that both PR300159 and PR300186 exhibited strong binding activity to both human and cynomolgus monkey MSLN-expressing cells, with EC50 values comparable to those of PR000685 (amatuximab). These results indicate that the anti-MSLN antibodies PR300159 and PR300186 can bind with high affinity to human and cynomolgus monkey MSLN on the cell membrane.

[0237] Example 5. Antibody Engineering The VH and VL sequences of the anti-MSLN antibodies PR300159 and PR300186 were further optimized by a PTM removal procedure.

[0238] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. Apart from conserved PTM sites in antibodies, such as the conserved N-glycosylation site on the CH2 domain of an IgG1 antibody, other PTM sites in the antigen-binding site (i.e., CDR region) of an antibody may reduce antigen-binding activity and chemical stability. For example, deamidation or isomerization may lead to instability or heterogeneity of the molecule. To reduce sequence burden, PTM motifs can be removed by mutation. VH or VL sequences were scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). Next, "hotspot" residues (e.g., D or G in the DG motif) were mutated to their germline counterparts or other residues with similar biophysical properties. Antibodies consisting of sequence variants after PTM removal were then recombinantly produced using established molecular biology techniques.

[0239] The designed mutants obtained by removing PTMs from PR300159 and PR300186 are shown in Table 7. The amino acid sequences of these anti-MSLN antibodies are shown in Tables 1 to 4. PR300159-1, PR300159-3, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8, and PR300159-9 are PTM-removed antibodies derived from PR300159. PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10 are PTM-removed antibodies derived from PR300186.

[0240] [Table 7]

[0241] Example 6. Binding activity of antibodies to human and cynomolgus monkey MSLN proteins Human MSLN (Acro Biosystems, catalog number MSN-H5223) or cynomolgus monkey MSLN protein (Harbourbiomed, lot number 2019072202) was diluted with PBS to a concentration of 1 μg / mL. 100 μL of the diluted human MSLN or cynomolgus monkey MSLN was added per well of an ELISA microplate, and the plate was incubated overnight at 4°C. The plates were blocked with ELISA blocking solution (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37°C for 1 hour. Then, the plates were washed and incubated with anti-MSLN antibodies (PR300186, PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10) diluted to 15 μg / mL (100 nM, 10-point dilutions) at 37°C for 1 hour. The plate was then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-035-088) for 1 hour at 37°C. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biopanda, catalog number: TMB-S-003) was added, and the plate was incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μL of ELISA stop solution (Solarbio, catalog number: C1058), and the optical density at 450 nm (OD450nm) was measured using an ELISA plate reader (Molecular Devices, Spectramax384plus).

[0242] The results for PR300186 and PR300186 PTM-depleted antibodies (PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9, and PR300186-10) are shown in Figures 6, 7, and Table 8. The results show that all PTM-depleted antibodies derived from PR300186 exhibited good binding activity to both human and cynomolgus monkey MSLN proteins, with EC50 values comparable to that of PR300186.

[0243] [Table 8]

[0244] Example 7. Binding activity of antibodies to MSLN-expressing cells The binding of recombinant anti-MSLN antibodies to the COV644 cell line (ECACC, catalog: 07071908) was tested by flow cytometry. Briefly, anti-MSLN antibodies were serially diluted in staining buffer (PBS containing 2% FBS) at 1–2 × 10 5 50 μL of diluted antibody solution was added to 50 μL of cell suspension containing 100 cells and incubated at 4°C for 1 hour. The cells were washed twice with staining buffer (PBS containing 2% FBS), and 100 μL of a 1:1000 diluted florescent-labeled anti-human IgG antibody (AlexaFluor® 488 AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, catalog 109-545-088) was added to each well. After incubation at 4°C for 1 hour, the cells were washed twice with staining buffer and subjected to flow cytometry.

[0245] The results for the PR300159 PTM-depleted antibodies are shown below in Figure 8 and Table 9. The results suggest that the PTM-depleted antibodies PR300159-1, PR300159-3, PR300159-5, PR300159-6, and PR300159-8 exhibited equivalent binding activity to COV644 cells compared to PR300159.

[0246] [Table 9]

[0247] The results for the PTM-depleted antibodies derived from PR300186 are shown in Figure 9. The results suggest that the PTM-depleted antibodies PR300186-3, PR300186-9, and PR300186-10 showed superior binding activity to COV644 cells compared to PR300186.

[0248] Example 8. Antibody internalization by MSLN-expressing cells In this example, pHAb amine-reactive dye (Promega, catalog number G9841) was used to determine antigen-based internalization of anti-MSLN antibodies into COV644 cells. pHAb dyes are pH-sensing dyes that exhibit very low fluorescence above pH 7 and dramatically increase fluorescence when the solution becomes acidic. When pHAb dye-labeled antibodies bind to the outside of the cell membrane at neutral pH, no or very weak fluorescence is detected. After internalization, fluorescence increases in the low pH environment of endosomes and lysosomes.

[0249] The antibody was labeled with pHAb dye, and the DAR was calculated according to the kit's instructions. The labeled antibody was then incubated with COV644 for 24 hours at 4°C (used as a background control, as internalization activity at this temperature is very low) or 37°C. Fluorescence was then detected with an excitation maximum (Ex) of 532 nm and an emission maximum (Em) of 560 nm. The final normalized result was calculated by subtracting the background fluorescence intensity at 4°C from the fluorescence intensity at 37°C, and dividing the result by the DAR of the antibody's pHAbDye. A higher value indicates higher internalization activity.

[0250] The results of the internalization rates of PR300159 and PR300159-derived PTM-depleted antibodies are shown in Figure 10 and Table 10. The results suggest that the PR300159-1, PR300159-4, PR300159-5, PR300159-6, PR300159-7, and PR300159-8 antibodies exhibit comparable internalization by COV644 cells compared to PR300159.

[0251] The results of the internalization rates of PR300186 and PR300186-derived PTM-depleted antibodies are shown in Figure 11 and Table 10. The results suggest that PR300186-3, PR300186-9, and PR300186-10 exhibit better internalization by COV644 cells compared to PR300186.

[0252] [Table 10]

[0253] Example 9. Binding activity of antibodies to soluble MSLN protein by BLI method In this example, the binding kinetics of anti-MSLN antibodies to soluble MSLN were analyzed using Octet Red 384 (Fortebio). For biolayer interferometry (BLI) analysis, recombinant human MSLN-His tag (AcroBiosystems, catalog no. MSN-H5223) was serially diluted in 1x kinetic buffer (Fortebio). The anti-MSLN antibody was diluted to 5 μg / mL. The diluted antibody, antigen, and regeneration buffer (10 mM glycine, pH 1.75) were then added to a 96-well plate (Greiner). The association and dissociation rate constants were measured using an AHC sensor (Fortebio). After each binding experiment, the sensor surface was regenerated using regeneration buffer. Traces were processed using Octet Data Analysis Software (version 11.0, Pall ForteBio, CA, USA). The K of antibody binding to soluble human MSLN was calculated. D The values are summarized in Table 11.

[0254] As shown in Table 11, in Octet analysis, the PTM-depleted antibodies derived from PR300159 showed comparable binding affinity to soluble MSLN compared to PR300159.

[0255] PR300186 and PR300186-10 have low binding affinity for soluble MSLN, which is advantageous because when used as therapeutic antibodies, it is desirable to bind to cell surface MSLN on tumor cells rather than soluble MSLN in the circulatory system.

[0256] [Table 11]

[0257] Example 10. Soluble MSLN interference assay Antibodies that bind to the membrane-bound form of MSLN are advantageous for diagnostic and therapeutic purposes: they do not bind to soluble MSLN, resulting in lower background in diagnostic imaging applications and allowing the use of lower doses in therapeutic applications.

[0258] Antibodies PR300159 and PR300186, and PTM-depleted antibodies derived from PR300159-8 or PR300186-10, were tested for binding to mesothelin-expressing COV644 cells in the presence or absence of 90 nM soluble MSLN (sMSLN). The experiment was performed as described in Example 4, except that 90 nM soluble MSLN was added to the serially diluted antibody in the +sMSLN group.

[0259] As shown in Figure 12, the binding of PR300159 to COV644 cells was reduced in the presence of 90 nM soluble MSLN compared to its absence. As shown in Figure 13, PR300186 showed less change, suggesting that soluble MSLN interferes less with the binding of PR300186 to membrane-bound MSLN.

[0260] As shown in Figures 14 and 15, in the presence of 90 nM soluble MSLN, the binding of PR300159-8 and PR300186-10 to COV644 cells was reduced compared to that in the absence of soluble MSLN, but the binding of PR300186-10 was less altered, suggesting that soluble MSLN interfered less with the binding of these antibodies to membrane-bound MSLN.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to mesothelin, the antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), (1) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (2) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 28; (3) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 26, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 27; (4) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (5) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 30; (6) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 31; (7) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 32; (8) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 33; (9) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 25, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 29; (10) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (11) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (12) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (13) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (14) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 68; (15) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 67; (16) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 64, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (17) The VH comprises HCDR1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 66, and the VL comprises LCDR1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; (18) The VH comprises HCDR1 to HCDR3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and the VL comprises LCDR1 to HCDR3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; or (19) An antibody or antigen-binding fragment thereof that specifically binds to mesothelin, wherein the VH comprises HCDR1 to HCDR3 of a VH having the amino acid sequence set forth in SEQ ID NO: 65, and the VL comprises LCDR1 to LCDR3 of a VL having the amino acid sequence set forth in SEQ ID NO:

69.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, (1) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (2) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 18, respectively; (3) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 17, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively; (4) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (5) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 20, respectively; (6) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 21, respectively; (7) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 22, respectively; (8) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 23, respectively; (9) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 19, respectively; (10) The VH comprises HCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and the VL comprises LCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (11) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (12) The VH comprises HCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and the VL comprises LCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (13) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (14) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 61, 54, and 56, respectively; (15) The VH comprises HCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and the VL comprises LCDR1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 54, and 56, respectively; (16) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 47, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (17) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 60, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; (18) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 47, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively; or (19) The VH comprises HCDR1 to HCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 49, respectively, and the VL comprises LCDR1 to LCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 62, 54, and 56, respectively. An antibody or an antigen-binding fragment thereof.

3. 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; (9) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; (10) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (11) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (12) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (13) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (14) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; (15) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67; (16) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (17) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; (18) The VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69; or (19) An antibody or antigen-binding fragment thereof, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 65, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

69.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain (HC) and a light chain (LC); (1) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:37; (2) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38; (3) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37; (4) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (5) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40; (6) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41; (7) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42; (8) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43; (9) the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39; (10) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (11) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (12) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (13) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (14) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75; (15) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; (16) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (17) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; (18) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (19) The HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 72, and the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

76. An antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof specifically binds to membrane-bound mesothelin.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof binds to membrane-bound mesothelin with higher affinity than its binding affinity to soluble mesothelin.

7. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof binds to membrane-bound mesothelin with an affinity that is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold greater than the binding affinity to soluble mesothelin.

8. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof does not bind to soluble MSLN.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

12. The antigen-binding fragment may be Fab, Fab', F(ab') ２ 12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which is selected from the group consisting of Fd, Fd', Fv, scFv, ds-scFv, and dAb.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody is monovalent, bivalent, or multivalent.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is conjugated to a fluorescent label, a radioactive label, or a cytotoxic agent.

16. A bispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen, or an immune checkpoint molecule.

17. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, or the bispecific antibody of claim 16.

18. A vector comprising the nucleic acid of claim 17.

19. A host cell comprising a nucleic acid according to claim 17 or a vector according to claim 18.

20. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the bispecific antibody according to claim 16.

21. 21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the bispecific antibody according to claim 16, or the nucleic acid according to claim 17, or the vector according to claim 18, or the host cell according to claim 19, or the antibody-drug conjugate according to claim 20, and optionally a pharmaceutically acceptable carrier or excipient.

22. 22. The pharmaceutical composition of claim 21, further comprising a second therapeutic agent selected from the group consisting of an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

23. 20. A method for treating cancer in a subject, comprising the step of administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the bispecific antibody according to claim 16, or the nucleic acid according to claim 17, or the vector according to claim 18, or the host cell according to claim 19, or the antibody-drug conjugate according to claim 20, or the pharmaceutical composition according to claim 21 or 22.

24. 24. The method of claim 23, wherein the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

25. 25. The method of claim 23 or 24, further comprising administering to the subject a second therapeutic agent.

26. 26. The method of claim 25, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

27. 20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, or the bispecific antibody of claim 16, or the nucleic acid of claim 17, or the vector of claim 18, or the host cell of claim 19, the antibody-drug conjugate of claim 20, or the pharmaceutical composition of claim 21 or 22, in the manufacture of a medicament for treating cancer in a subject.

28. 28. The use of claim 27, wherein the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

29. 29. The use of claim 27 or 28, wherein the medicament further comprises a second therapeutic agent, optionally wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

30. 29. The use of claim 27 or 28, wherein the agent is administered in combination with a second therapeutic agent, optionally wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

31. 21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the bispecific antibody according to claim 16, or the nucleic acid according to claim 17, or the vector according to claim 18, or the host cell according to claim 19, the antibody-drug conjugate according to claim 20, or the pharmaceutical composition according to claim 21 or 22, for use in treating cancer in a subject.

32. 32. The antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition for use according to claim 31 , wherein the cancer is selected from the group consisting of mesothelioma, prostate cancer, lung cancer, abdominal cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (e.g., triple-negative breast cancer), and ovarian cancer.

33. 33. The antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate, or pharmaceutical composition for use according to claim 31 or 32, further comprising the step of administering to the subject a second therapeutic agent, optionally wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

34. (a) obtaining a biological sample from a subject; (b) contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 15; (c) detecting binding of the antibody to the sample; wherein increased binding of the antibody or antigen-binding fragment thereof to the sample compared to binding of the antibody or antigen-binding fragment thereof to a control sample identifies the subject as having a mesothelin-positive cancer.

35. (a) administering to a subject the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 conjugated to a detectable marker; (b) detecting the presence of the marker; 1. A method for imaging mesothelin-positive cancer in a subject, comprising:

36. (a) the detectable marker １１１ In, preferably wherein the detection of the marker is single photon emission computed tomography; (b) the detectable marker is ８９ 35. The method of claim 34, wherein the marker is Zr, preferably wherein detection of the marker is by positron emission tomography.

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