Anti-mesothelin antibody

Abstract

Provided are antibodies and antigen-binding fragments thereof that bind to mesothelin, as well as 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. Also provided are pharmaceutical compositions and conjugates comprising the antibodies, as well as methods of treatment by administering the antibodies.

Description

Technical Field

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

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

[0003] Mesothelin (MSLN) is a cell surface molecule expressed as a 71 kD precursor protein that is further processed into a 40 kD glycoprotein that is anchored on the cell surface by glycosylphosphatidylinositol (GPI). MSLN shows very limited and low expression in normal tissues. It is expressed in mesothelial cells that form the pleura, pericardium, and peritoneum, where it appears to play a role in cell adhesion (Chang et al. (1996), PNAS, Vol. 93: pp. 136-140). It has also been suggested that soluble cleaved MSLN plays a role in megakaryocyte stimulation, but no defects in development were shown after knockout in mice, so its biological role is unclear (Yamaguchi et al. (1994), Vol. 269, No. 2: pp. 805-808; Bera et al. (2000), Vol. 20, No. 8: pp. 2902-2906).

[0004] In contrast, MSLN is highly expressed in several human cancers, including nearly all mesotheliomas and pancreatic cancers and approximately 70% of ovarian cancers and 50% of lung adenocarcinomas (Hassan and Ho (2008), Eur. J. Cancer, Vol. 44: pp. 46-53; Miettinen and Sarlomo-Rikala (2003), Am. J. Surg. Pathol., Vol. 27: pp. 150-158; Ordonez (2003), Am. J. Surg. Pathol., Vol. 27: pp. 1418-1428; Ho et al. (2007), Clin. Cancer Res., Vol. 13: pp. 1571-1575). Its high expression level makes MSLN an attractive candidate for targeted therapy because it promotes proliferation and invasion, playing an important role in tumor progression (Servais et al. (2012), Clin. Cancer Res., Vol. 18, No. 9: pp. 2478-2489).

[0005] Cell adhesion mediated by the interaction of MSLN and MUC16 plays an important role in the peritoneal implantation of ovarian cancer cells and increases the mobility and invasiveness of pancreatic cancer cells (Rump et al. (2004), J Biol Chem., Vol. 279, No. 10: pp. 9190-9198; Gubbels et al. (2006), Mol Cancer, Vol. 5, No. 1: p. 50; Coehlo et al., Expert Rev Anticancer Ther., Vol. 18, No. 2: pp. 177-186; Chen et al. (2013), Sci Rep., Vol. 3: p. 1870). In particular, pancreatic tumors often progress to the middle and late stages before patients feel any symptoms of the disease, and the average survival time is short (usually less than one year). This prognosis is poor because the tumors are usually not (completely) removable by surgery and have already metastasized (usually undetectable). Chemotherapy also fails to significantly prolong survival time or cure the disease. Despite several attempts, there has been no successful immunotherapy to date. Even the most famous anti-mesothelin monoclonal antibody (Amatuximab) that binds to a soluble form of MSLN has not been particularly successful (Baldo and Cecco (2017), Onco. Targets Ther., Vol. 10: pp. 5337-5353; Nicolaides et al. (2018), Cancer Biology & Therapy, Vol. 19, No. 7: pp. 622-630). Unfortunately, tumor-induced mesothelin shedding creates many problems, although it can be used as a biomarker for disease (Hassan et al. (2006), Clin. Cancer Res., Vol. 12: pp. 447-453). Shedding poses problems for tumor imaging and potential radiotherapy. MSLN shed into the bloodstream results in high background for imaging based on radiolabeled antibodies and is toxic because the radiolabeled antibodies bind to shed mesothelin throughout the body. This requires the administration of high and therefore toxic doses of anti-mesothelin antibodies. Summary of the invention

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

[0007] In a first aspect, the present invention provides an antibody or an 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), and wherein

[0008] (1) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 27;

[0009] (2) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:25, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:28;

[0010] (3) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:26, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:27;

[0011] (4) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:29;

[0012] (5) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:30;

[0013] (6) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:31;

[0014] (7) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:32;

[0015] (8) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:33;

[0016] (9) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:25, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:29;

[0017] (10) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0018] (11) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:64, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0019] (12) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0020] (13) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:68;

[0021] (14) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:68;

[0022] (15) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:66, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0023] (16) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:64, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:69;

[0024] (17) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:66, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:69;

[0025] (18) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; or

[0026] (19) VH comprises HCDRs 1-3 of VH having the amino acid sequence shown in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence shown in SEQ ID NO:69.

[0027] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the Kabat and Chothia combined numbering system.

[0028] In some embodiments, 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), and wherein

[0029] (1) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 13, respectively;

[0030] (2) VH comprises HCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 18, respectively;

[0031] (3) VH comprises HCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 17, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively;

[0032] (4) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 19, respectively;

[0033] (5) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 20, respectively;

[0034] (6) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 21, respectively;

[0035] (7) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 22, respectively;

[0036] (8) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 23, respectively;

[0037] (9) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 9, 11, and 19, respectively;

[0038] (10) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 52, 54, and 56, respectively;

[0039] (11) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 52, 54, and 56, respectively;

[0040] (12) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 54, and 56, respectively;

[0041] (13) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 61, 54, and 56, respectively;

[0042] (14) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 61, 54, and 56, respectively;

[0043] (15) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 54, and 56, respectively;

[0044] (16) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively;

[0045] (17) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively;

[0046] (18) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively; or

[0047] (19) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 62, 54, and 56, respectively.

[0048] In some embodiments, 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), and wherein:

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (5) 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 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;

[0054] (6) 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 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;

[0055] (7) 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 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;

[0056] (8) 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 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;

[0057] (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;

[0058] (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;

[0059] (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;

[0060] (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;

[0061] (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;

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] (18) VH comprises an amino acid sequence that has 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 that has 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

[0067] (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.

[0068] In some embodiments, the present 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), and wherein:

[0069] (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;

[0070] (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;

[0071] (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;

[0072] (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;

[0073] (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;

[0074] (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;

[0075] (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;

[0076] (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;

[0077] (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;

[0078] (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;

[0079] (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;

[0080] (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;

[0081] (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;

[0082] (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;

[0083] (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;

[0084] (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;

[0085] (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;

[0086] (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

[0087] (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.

[0088] 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 a higher affinity than it binds to soluble mesothelin. In some preferred embodiments, the antibody or antigen binding fragment thereof binds to membrane-bound mesothelin with an affinity at least two times, at least three times, at least five times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, or at least 100 times greater than its affinity for binding 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.

[0089] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0090] In some embodiments, the antibody is an isotype selected from IgG, IgA, IgM, IgE and IgD. In preferred embodiments, the antibody is a subtype selected from IgG1, IgG2, IgG3 and IgG4.

[0091] In some embodiments, the antigen binding fragment is selected from Fab, Fab', F(ab') 2 , Fd, Fd', Fv, scFv, ds-scFv and dAb.

[0092] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0093] In some embodiments, the antibodies are monovalent, bivalent, or multivalent.

[0094] In some embodiments, the antibody or antigen-binding fragment is linked to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0095] 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 present invention and a second antigen-binding region that specifically binds to a tumor-associated antigen, an immune cell antigen or an immune checkpoint molecule.

[0096] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention.

[0097] In a fourth aspect, the present invention provides a vector comprising the nucleic acid of the third aspect of the present invention.

[0098] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid of the third aspect of the present invention or the vector of the fourth aspect of the present invention.

[0099] In a sixth aspect, the present invention provides an antibody drug conjugate (ADC), wherein the ADC comprises the antibody or antigen-binding fragment thereof of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention.

[0100] In the seventh aspect, the present invention provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the bispecific antibody of the second aspect of the present invention, or the nucleic acid of the third aspect of the present invention, or the vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the antibody-drug conjugate of the sixth aspect of the present invention; and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0101] In some embodiments, the composition further comprises a second therapeutic agent selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0102] In an eighth aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody drug conjugate or pharmaceutical composition of the present invention.

[0103] In some specific embodiments, the cancer is selected from 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.

[0104] 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, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0105] In a ninth aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an antibody-drug conjugate or a pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer in a subject.

[0106] In some embodiments, the cancer is selected from mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (eg, triple negative breast cancer), and ovarian cancer.

[0107] In some embodiments, the medicament further comprises a second therapeutic agent, optionally, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0108] In some embodiments, the medicament is administered in combination with a second therapeutic agent, optionally selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

[0109] In a tenth aspect, the present invention provides an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an antibody drug conjugate or a pharmaceutical composition of the present invention for use in a method of treating cancer in a subject.

[0110] In some embodiments, the cancer is selected from mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (eg, triple negative breast cancer), and ovarian cancer.

[0111] 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.

[0112] In an eleventh aspect, the present invention provides a method for diagnosing mesothelin-positive cancer in a subject, the method comprising:

[0113] (a) obtaining a biological sample from a subject,

[0114] (b) contacting the sample with the antibody or antigen-binding fragment thereof of the present invention; and

[0115] (c) detecting binding of the antibody to the sample,

[0116] 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 mesothelin-positive cancer.

[0117] In a twelfth aspect, the present invention provides a method for imaging a mesothelin-positive cancer in a subject, the method comprising:

[0118] (a) administering to the subject an antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is conjugated to a detectable marker; and

[0119] (b) detecting the presence of the marker.

[0120] In some preferred embodiments, (a) the detectable marker is 111 In, and preferably the detection of the marker is performed by single photon emission computed tomography, or

[0121] (b) The detectable marker is 89 Zr, and preferably the detection of this marker is performed by positron emission tomography. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Protocol for generating anti-mesothelin antibodies. HarbourH2L2 transgenic mice were immunized by repeated injections with recombinant mesothelin protein, followed by hybridoma generation and clonal screening to identify MSLN-specific tetrameric (H2L2) antibodies.

[0123] Figure 2 .Workflow of screening strategies and methods for single B cell clone screening.

[0124] Figure 3 . Binding of antibodies PR300159 and PR300186 to membrane-bound human MSLN on the surface of CHOK1-human MSLN cells.

[0125] Figure 4 . Binding of antibodies PR300159 and PR300186 to membrane-bound cynomolgus monkey MSLN on the surface of CHOK1-cynomolgus monkey MSLN cells.

[0126] Figure 5 .Binding of antibodies PR300159 and PR300186 to membrane-bound human MSLN endogenously expressed on the COV644 cell line.

[0127] Figure 6 .PR300186 antibody and PR300186PTM removal antibody bind to human MSLN protein.

[0128] Figure 7 .Binding of PR300186 antibody and PR300186PTM removal antibody to cynomolgus monkey MSLN protein.

[0129] Figure 8 Binding of PR300159 antibody and PR300159PTM removal antibody to COV 644 cells.

[0130] Fig. 9 Binding of PR300186 antibody and PR300186PTM removal antibody to COV 644 cells.

[0131] Fig.10 Internalization of PR300159 antibody and PR300159PTM removal antibody on COV644 cells.

[0132] Fig.11 Internalization of PR300186 antibody and PR300186PTM removal antibody on COV644 cells.

[0133] Fig.12 Binding of antibody PR300159 to COV644 cells in the presence or absence of soluble MSLN (sMSLN): (A) in the absence of sMSLN; (B) in the presence of 90 nM sMSLN.

[0134] Fig.13 Binding of antibody PR300186 to COV644 cells in the presence or absence of soluble MSLN (sMSLN): (A) in the absence of sMSLN; (B) in the presence of 90 nM sMSLN.

[0135] Fig.14 .Binding of antibody PR300159-8 to COV644 cells in the presence or absence of soluble MSLN (sMSLN).

[0136] Fig.15 Binding of antibody PR300186-10 to COV644 cells in the presence or absence of soluble MSLN (sMSLN).

[0137] sequence

[0138] The amino acid sequences of the light chain, heavy chain, light chain variable region (VL), heavy chain variable region (VH), CDRs and FWRs of the light chain and heavy chain are shown in Tables 1 to 4 below.

[0139] Table 1. Sequences of heavy and light chains of antibodies

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] Table 2. Sequences of the heavy chain variable region and light chain variable region of antibodies

[0148]

[0149]

[0150]

[0151] Table 3. Sequences of HCDR1-3 and HFWR1-4 of antibodies (according to Kabat and Chothia combination system)

[0152]

[0153]

[0154] Table 4. Sequences of LCDR1-3 and LFWR1-4 of antibodies (according to Kabat and Chothia combinatorial systems)

[0155]

[0156] DETAILED DESCRIPTION

[0157] The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of the embodiments in conjunction with the accompanying drawings.

[0158] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and are used to generally understand the present invention. These embodiments should not be construed as limiting the scope of the present invention. Throughout the specification, identical or similar elements and elements with identical or similar functions are represented by the same reference numerals.

[0159] Unless otherwise noted or defined, all terms used have their ordinary meanings in the art, which are clear to those skilled in the art. For example, reference is made to standard manuals such as Leuenberger, HGW, Nagel, B. and KlbI, 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 to 3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., Current protocols inmolecular 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 York (2005), and the general background art cited above.

[0160] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies.

[0161] Unless otherwise stated or defined, the term "comprise" and variations thereof such as "include" and "comprising" should be understood to imply the inclusion of the stated elements or steps or groups of elements or steps, but not the exclusion of any other elements or steps or groups of elements or steps. The term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone, or may include some additional substances, such as X+Y.

[0162] The term "about" in relation to a numerical value x is optional and means, for example, x±10% or x±5%.

[0163] As used herein, the term "antibody" refers to an immunoglobulin molecule with the ability to specifically bind to a specific antigen. Antibodies typically include a variable region and a constant region in each of a heavy chain and a light chain. The variable regions of the heavy and light chains of an antibody include a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, which includes various cells of the immune system (such as effector cells) and components of the complement system, such as the first component CIq in the classical pathway of complement activation. Most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antigen-binding portion of an antibody.

[0164] 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 are used to align the CDRs to specifically bind to the epitope of the antigen. The CDRs contain the amino acid residues of the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL domain and the VH domain contain the following framework regions (FRs) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively, and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0165] The amino acid assignments for each VL and VH domain are consistent with any conventional definition of CDRs. Conventional 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 and Lesk, J. Mol. Biol., Vol. 196: pp. 901-917, 1987; Chothia et al., Nature, Vol. 342: pp. 878-883, 1989); the combination of Chothia Kabat CDRs (also known as Chothia and Kabat combined CDRs), in which each CDR is a combination of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the contact definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number.

[0166] Although the present disclosure relates to CDRs defined according to either of these numbering systems, preferred embodiments relate to CDRs defined according to a combination of Chothia and Kabat.

[0167] Table 5. Definition of CDRs for antibodies (see http: / / bioinf.org.uk / abs / )

[0168] Kabat Chothia combination LCDR1 L24--L34 L24--L34 L24-L34 LCDR2 L50--L56 L50--L56 L50-L56 LCDR3 L89--L97 L89--L97 L89-L97 HCDR1 H31--H35 H26--H32 H26-H35 HCDR2 H50--H65 H52--H56 H50-H65 HCDR3 H95--H102 H95--H102 H95-H102

[0169] In Table 5, Laa-Lbb may refer to an 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 an 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 an amino acid sequence from position 24 to position 34 (according to the Chothia numbering system) starting from the N-terminus of the antibody light chain; H26-H32 may refer to an amino acid sequence from position 26 to position 32 (according to the Chothia numbering system) starting from the N-terminus of the antibody heavy chain.

[0170] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0171] 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 (eg, MSLN). Studies have shown that the antigen-binding function of an antibody can be achieved by a fragment of a full-length antibody.

[0172] Examples of antigen-binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (ed. by Paul, Supplementary Version 3, 1993)); (iv) a F(ab')2 fragment, i.e., a monovalent fragment consisting of a VH domain and a CH1 domain. (v) an Fd fragment consisting of a VH domain and a CH1 domain and one or more cysteine ​​residues located at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; (vii) a dAb fragment (Ward et al., Nature (1989), Vol. 341: pp. 544-546), which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) nanobodies, i.e., heavy chain variable regions comprising a single variable domain and two constant domains. In addition, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, these domains can still be connected by synthetic linkers using recombinant methods so that these domains can be made into a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988), Science, Vol. 242: pp. 423-426; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85: pp. 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibodies. In addition, the term also includes "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) and a modified form of any of the aforementioned fragments, which form an antigen-binding region together with a complementary light chain polypeptide, and the modified forms of these fragments retain antigen-binding activity.

[0173] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments screened for utility in the same manner as intact antibodies.

[0174] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as a non-random binding reaction 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 antigen-antibody dissociation, is a measure of the strength of binding between an antigenic determinant (epitope) and an antigen binding site on an antibody: the smaller the KD value, the stronger the binding strength between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0175] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to both the affinity between an antigenic determinant (epitope) and its antigen binding site on an antibody and the number of associated binding sites present on the antibody. -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 (such as at least 10 12 M -1 ) binding affinity. Any K D Value greater than 10 -4 M is generally considered to indicate non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biomembrane interferometry (BLI) assays and sandwich competition assays, as well as different variations of these methods known per se in the art.

[0176] The term "epitope" refers to a site on an antigen that binds to an antibody. An epitope can be formed by continuous amino acids, or by non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are usually lost when treated with denaturing solvents. An epitope typically includes at least 3, more typically at least 5 or 8 to 10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore a specific target of an antibody or its antigen-binding fragment.

[0177] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at the same position in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y, and is detailed as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs 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).

[0178] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is substituted with another amino acid residue having a similar chemical structure and which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB-A-2 357768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferred) types and / or combinations of such substitutions may be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0179] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0180] Particularly preferred conservative substitutions are as follows: 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.

[0181] Any amino acid substitution applied to the polypeptides described herein may also be based on the following analyses: analysis of the frequency of amino acid variation between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978; analysis of structure forming potentials developed by Chou and Fasman, Biochemistry, Vol. 13: p. 211, 1974 and Adv. Enzymol., Vol. 47: p. 45-149, 1978; and analysis of structure forming potentials developed by Eisenberg et al., Proc. Nat. Acad Sci. USA, Vol. 81: p. 140-144, 1984; Kyte and Doolittle, J Mol. Biol., Vol. 157: p. 105-132, 198 1 year; and Goldman et al., Ann. Rev. Biophys. Chem., Vol. 15: pp. 321-353, 1986, the entire contents of which are incorporated herein by reference.

[0182] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibody constituting the population is identical except for a small amount of possible naturally occurring mutations. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology in this article, and should not be construed as requiring the production of antibodies by any particular method.

[0183] The term "bispecific antibody" in the context of the present invention is understood to mean an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as different target binding, but also includes binding to different epitopes in one target.

[0184] 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.

[0185] 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.

[0186] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0187] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and is substantially not deleterious to the recipient thereof, and / or such carrier or adjuvant is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0188] As used herein, the terms "treatment" and "treating" refer to the administration of an agent or the performance of a procedure in order to obtain an effect. The effect may be a preventive effect that completely or partially prevents a disease or its symptoms, and / or may be a therapeutic effect that partially or completely cures a disease and / or disease symptoms. As used herein, "treatment" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by a primary disease); (b) inhibiting the disease, i.e., preventing the disease from developing; and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any marker of success in the treatment or improvement or prevention of cancer, including any objective or subjective parameter, such as mitigation; relief; reducing symptoms or making the patient more tolerant to the disease condition; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate, stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to reducing, eliminating or preventing a disease, disease symptoms, or side effects of a disease in a subject.

[0189] As used herein, the term "effective amount" refers to an amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for the disease.

[0190] As used herein, the term "subject" refers to any mammalian subject in need of diagnosis, treatment or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sports animals or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0191] The terms "cyno / cynomolgus / Cynomolgus macaques" are used interchangeably herein and refer to the cynomolgus MSLN. The term includes any MSLN variants, isoforms, and species homologs that are naturally expressed by cynomolgus cells or expressed on cells transfected with a gene or cDNA encoding cynomolgus MSLN that is naturally expressed on cynomolgus cells.

[0192] Anti-MSLN antibody

[0193] The present invention provides anti-mesothelin antibodies.

[0194] In a first aspect, the present invention provides an antibody or an 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), and wherein

[0195] (1) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 27;

[0196] (2) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:25, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:28;

[0197] (3) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:26, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:27;

[0198] (4) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:29;

[0199] (5) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:30;

[0200] (6) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:31;

[0201] (7) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:32;

[0202] (8) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:24, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:33;

[0203] (9) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:25, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:29;

[0204] (10) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0205] (11) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:64, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0206] (12) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0207] (13) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:68;

[0208] (14) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:68;

[0209] (15) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:66, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:67;

[0210] (16) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:64, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:69;

[0211] (17) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO:66, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO:69;

[0212] (18) VH comprises HCDRs 1-3 of VH having the amino acid sequence set forth in SEQ ID NO: 63, and VL comprises LCDRs 1-3 of VL having the amino acid sequence set forth in SEQ ID NO: 69; or

[0213] (19) VH comprises HCDRs 1-3 of VH having the amino acid sequence shown in SEQ ID NO:65, and VL comprises LCDRs 1-3 of VL having the amino acid sequence shown in SEQ ID NO:69.

[0214] In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the EU Kabat definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the Chothia definition / numbering system. In some embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the AbM definition / numbering system. In some preferred embodiments, LCDRs 1-3 and HCDRs 1-3 are defined by the Kabat and Chothia combined numbering system.

[0215] In some embodiments, 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), and wherein

[0216] (1) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 13, respectively;

[0217] (2) VH comprises HCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 18, respectively;

[0218] (3) VH comprises HCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 17, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 11, and 13, respectively;

[0219] (4) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 19, respectively;

[0220] (5) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 20, respectively;

[0221] (6) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 21, respectively;

[0222] (7) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 22, respectively;

[0223] (8) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2, 4, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 9, 11, and 23, respectively;

[0224] (9) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 15, 16, and 6, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 9, 11, and 19, respectively;

[0225] (10) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 52, 54, and 56, respectively;

[0226] (11) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 52, 54, and 56, respectively;

[0227] (12) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 54, and 56, respectively;

[0228] (13) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 61, 54, and 56, respectively;

[0229] (14) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 61, 54, and 56, respectively;

[0230] (15) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 52, 54, and 56, respectively;

[0231] (16) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively;

[0232] (17) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 58, 60, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively;

[0233] (18) VH comprises HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 45, 47, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 62, 54, and 56, respectively; or

[0234] (19) VH comprises HCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 58, 59, and 49, respectively, and VL comprises LCDRs 1-3 comprising the amino acid sequences shown in SEQ ID NOs: 62, 54, and 56, respectively.

[0235] In some embodiments, the CDRs are defined by the Kabat Chothia combinatorial system.

[0236] In some embodiments, 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), and wherein

[0237] (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;

[0238] (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;

[0239] (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;

[0240] (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;

[0241] (5) 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 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;

[0242] (6) 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 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;

[0243] (7) 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 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;

[0244] (8) 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 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;

[0245] (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;

[0246] (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;

[0247] (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;

[0248] (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;

[0249] (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;

[0250] (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;

[0251] (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;

[0252] (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;

[0253] (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;

[0254] (18) VH comprises an amino acid sequence that has 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 that has 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

[0255] (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.

[0256] In some embodiments, VL comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 27 to 33 and 67 to 69, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the antibody of the VL comprising the functional variant retains the ability to bind to MSLN. In some embodiments, VH comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 24 to 26 and 63 to 66, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the antibody of the VH comprising the functional variant retains the ability to bind to MSLN.

[0257] The 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.

[0258] In some embodiments, 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 and is formed by insertion, deletion and / or substitution of one or more amino acids in any one of SEQ ID NOs: 27 to 33 and 67 to 69. In some embodiments, 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 and is formed by insertion, deletion and / or substitution of one or more amino acids in any one of SEQ ID NOs: 24 to 26 and 63 to 66.

[0259] In the case of functional variants, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, 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, still 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.

[0260] In some embodiments, insertions, deletions and / or substitutions may be made at the framework (FR) regions, such as at FR1, FR2, FR3 and / or FR4.

[0261] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0262] Particularly preferred conservative substitutions are as follows: 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.

[0263] In a preferred embodiment, VL comprises the amino acid sequence as shown in SEQ ID NO:27, and VH comprises the amino acid sequence as shown in SEQ ID NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:28, and VH comprises the amino acid sequence as shown in SEQ ID NO:25; or VL comprises the amino acid sequence as shown in SEQ ID NO:27, and VH comprises the amino acid sequence as shown in SEQ ID NO:26; or VL comprises the amino acid sequence as shown in SEQ ID NO:29, and VH comprises the amino acid sequence as shown in SEQ ID NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:30, and VH comprises the amino acid sequence as shown in SEQ ID NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:31, and VH comprises the amino acid sequence as shown in SEQ ID NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:32, and VH comprises the amino acid sequence as shown in SEQ ID NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:33, and VH comprises the amino acid sequence as shown in SEQ ID NO:34. NO:24; or VL comprises the amino acid sequence as shown in SEQ ID NO:29, and VH comprises the amino acid sequence as shown in SEQ ID NO:25; or VL comprises the amino acid sequence as shown in SEQ ID NO:67, and VH comprises the amino acid sequence as shown in SEQ ID NO:63; or VL comprises the amino acid sequence as shown in SEQ ID NO:67, and VH comprises the amino acid sequence as shown in SEQ ID NO:64; or VL comprises the amino acid sequence as shown in SEQ ID NO:67, and VH comprises the amino acid sequence as shown in SEQ ID NO:65; or VL comprises the amino acid sequence as shown in SEQ ID NO:68, and VH comprises the amino acid sequence as shown in SEQ ID NO:65; or VL comprises the amino acid sequence as shown in SEQ ID NO:68, and VH comprises the amino acid sequence as shown in SEQ ID NO:63; or VL comprises the amino acid sequence as shown in SEQ ID NO:67, and VH comprises the amino acid sequence as shown in SEQ ID NO:66; or VL comprises the amino acid sequence as shown in SEQ ID NO:69, and VH comprises the amino acid sequence as shown in SEQ ID NO: NO:64; or VL comprises the amino acid sequence as shown in SEQ ID NO:69, and VH comprises the amino acid sequence as shown in SEQ ID NO:66;Or VL comprises the amino acid sequence shown in SEQ ID NO: 69, and VH comprises the amino acid sequence shown in SEQ ID NO: 63; or VL comprises the amino acid sequence shown in SEQ ID NO: 69, and VH comprises the amino acid sequence shown in SEQ ID NO: 65. ;

[0264] Based on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be classified as lambda (λ) chain or kappa (κ) chain. The antibodies disclosed herein can be any of the above types or subtypes.

[0265] In some embodiments, the antibody can be an isotype selected from IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody can be a subtype selected from IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

[0266] The antibodies disclosed herein may be complete 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 a modified form of any of the aforementioned fragments, wherein the modified forms of these fragments retain antigen-binding activity.

[0267] In some embodiments, the antigen binding fragment can be selected from Fab, Fab', F(ab') 2 , Fv, scFv and ds-scFv. In a preferred embodiment, the antigen binding fragment is Fab or scFv.

[0268] In some embodiments, the present 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), and wherein:

[0269] (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;

[0270] (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;

[0271] (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;

[0272] (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;

[0273] (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;

[0274] (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;

[0275] (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;

[0276] (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;

[0277] (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;

[0278] (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;

[0279] (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;

[0280] (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;

[0281] (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;

[0282] (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;

[0283] (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;

[0284] (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;

[0285] (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;

[0286] (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

[0287] (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.

[0288] In some embodiments, the light chain comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 37 to 43 and 74 to 76, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the antibody comprising the light chain of the functional variant retains the ability to bind to MSLN. In some embodiments, the heavy chain comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 34 to 36 and 70 to 73, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the antibody comprising the heavy chain of the functional variant retains the ability to bind to MSLN.

[0289] 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.

[0290] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, 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.

[0291] In some embodiments, insertions, deletions and / or substitutions may be made at the framework (FR) regions, such as FR1, FR2, FR3 and / or FR4; and / or at the constant regions, such as CL, CH1, CH2 and / or CH3.

[0292] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0293] In a preferred embodiment, the light chain comprises the amino acid sequence as shown in SEQ ID NO:37, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:38, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:35; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:37, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:36; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:39, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:40, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:41, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:42, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:43, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:44. NO:34; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:39, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:35; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:74, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:70; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:74, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:71; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:74, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:72; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:75, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:72; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:75, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:70; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:74, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:73; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:76, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:77. NO:71; or the light chain comprises the amino acid sequence as shown in SEQ ID NO:76, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO:73;Or the light chain comprises the amino acid sequence shown in SEQ ID NO: 76, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 70; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 76, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72. ;

[0294] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0295] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described in, for example, Dall'Acqua WF et al., J Immunol. Vol. 177 No. 2: pp. 1129-1138 (2006) and Hezareh M, J Virol., Vol. 75 No. 24: pp. 12161-12168 (2001). In some embodiments, the Fc region of the antibody comprises a wild-type IgG1 Fc with L234A, L235A and G237A mutations.

[0296] In some embodiments, the antibody is mutated at one or more post-translational modification sites. In one embodiment, the Fc region comprises a mutation that removes an acceptor site for Asn-linked glycosylation, or the Fc region is manipulated to eliminate the effector function of the antibody.

[0297] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. In addition to conserved PTM sites in antibodies, such as conserved N-glycosylation sites on the CH2 domain of IgG1 antibodies, other PTM sites (i.e., CDR regions) that occur in the antigen binding site of antibodies may also reduce antigen binding activity or reduce chemical stability. For example, deamidation or isomerization may make the molecule unstable and heterogeneous. In order to reduce sequence susceptibility, PTM motifs can be removed by mutation. Scan VH sequences or VL sequences to observe the presence of PTM motifs such as isomerization motifs (e.g., DG). Then mutate the "hot spot" residues (e.g., D or G in the DG motif) to the corresponding residues in the germline sequence or other residues with similar biophysical properties.

[0298] Bispecific Antibodies

[0299] In a second aspect, the present application provides a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0300] Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate a significant tumor-specific immune response. 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 usually silent (i.e., not expressed) in normal cells, those that are only expressed at certain stages of differentiation, and those that are expressed over time, such as embryonic antigens and fetal antigens. Other tumor antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations. Other tumor antigens can be encoded by viral genes, such as those carried by RNA tumor viruses and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be produced by those skilled in the art.

[0301] 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 gpl20, melanoma-associated antigen, CD200R1, MUC-1, mutant p53, mutant Ras, ROR1, VEGFR2, and combinations thereof.

[0302] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen can be selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D, or any combination thereof.

[0303] In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule can be selected from PD-1, PD-L1, CTLA-4, etc.

[0304] In some embodiments, the bispecific antibody comprises a single polypeptide chain comprising a first antigen binding region and a second antigen binding region, and optionally an Fc region.

[0305] The Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0306] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. Vol. 177 No. 2: pp. 1129-1138 (2006) and Hezareh M, J Virol., Vol. 75 No. 24: pp. 12161-12168 (2001).

[0307] In one embodiment, the Fc region comprises a mutation that removes an Asn-linked glycosylated acceptor site, or the Fc region is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation.

[0308] In another embodiment, the Fc region is glycoengineered to reduce fucose, thereby enhancing ADCC, for example, by adding compounds to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010), Biotechnol. Bioeng., Vol. 105: p. 350, or by using FUT8 knockout cells, for example, as described in Yamane-Ohnuki et al. (2004), Biotechnol. Bioeng, Vol. 87: p. 614. ADCC may alternatively be used et al. (1999), Nature Biotech, Vol. 17: 176. In another embodiment, the Fc region has been engineered to enhance complement activation, for example, as described in Natsume et al. (2009), Cancer Sci., Vol. 100: 2411.

[0309] Nucleic Acids

[0310] In a third aspect, the present invention provides a nucleic acid 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.

[0311] The term "polynucleotide" or "nucleic acid" includes both single-stranded nucleotide polymers and double-stranded nucleotide polymers. The nucleotides comprising nucleic acids can be modified forms of ribonucleotides or deoxyribonucleotides or any type of nucleotides. The modifications include base modifications (such as bromouridine and inosine derivatives), ribose modifications (such as 2', 3'-dideoxyribose) and internucleotide linkage modifications (such as thiophosphates, dithiophosphates, selenophosphates, diselenphosphates, anilinothiophosphates (phosphoroanilothioate), anilinophosphates (phoshoraniladate) and phosphoramidates.

[0312] For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present 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 of the heavy chain variable region sequences disclosed herein.

[0313] For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present 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 of the light chain variable region sequences disclosed herein.

[0314] For example, the present invention provides nucleic acid molecules encoding the following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98% or at least 99% identical to nucleic acids encoding the following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein.

[0315] For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any one of the heavy chain variable region sequences disclosed herein.

[0316] In some embodiments, the invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein.

[0317] The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

[0318] In some embodiments, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence, which heavy chain variable region sequence comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0319] For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence, which comprises the CDR sequence of any one of the light chain variable region sequences disclosed herein.

[0320] In some embodiments, the invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein.

[0321] The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

[0322] In some embodiments, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence, which light chain variable region sequence comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0323] For example, the present invention provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein; and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence comprising any of the groups of three CDR sequences disclosed herein; and (ii) a light chain variable region sequence comprising any of the groups of three CDR sequences disclosed herein. The present invention also provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein; and (ii) a light chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence 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, which comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively; and (ii) a light chain variable region sequence, which comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0324] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present 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 present 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.

[0325] Thus, 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 is used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from 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.

[0326] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0327] 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 can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from 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.

[0328] In some embodiments, the RNA can be introduced into human cells in vivo. In some embodiments, the RNA of the invention is contained in a carrier or delivery agent.

[0329] In some specific 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 (such as a liposome). In some embodiments, the mRNA can be introduced into human cells in vivo via a vector or delivery system (such as a liposome) and express the MLSN antibody of the present invention in vivo.

[0330] The mRNA of the present invention can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from 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.

[0331] Carrier

[0332] In a fourth aspect, the present invention also provides a vector comprising a nucleic acid 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.

[0333] In some embodiments, the vector is a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-MSLN antibody. For example, the present invention provides a recombinant expression vector comprising any of the above nucleic acid molecules.

[0334] Any vector may be suitable for use in 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, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papillomatosis virus 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-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP and pLXIN-Luc.

[0335] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. For example, vectors can be selected from 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.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can 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).

[0336] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, which are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, 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 can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0337] Therefore, the vector can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from 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. The vector of the present invention can be introduced into a cell. In some embodiments, the vector of the present invention can be introduced into a cell in vitro or in vitro. Optionally, the cell introduced with the vector can then be administered to a subject. In some embodiments, the vector of the present invention can be introduced into a cell in vivo.

[0338] For example, the vector can 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 can be administered to a subject and then enter the subject's cells in vivo, thereby integrating 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 into the genome of the cell, and then the cell expresses the anti-MSLN antibody or antigen-binding fragment thereof disclosed herein, so as to treat the diseases disclosed herein.

[0339] Host cells

[0340] In a fifth aspect, the present invention also provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

[0341] Any cell can be used as a host cell for nucleic acids or vectors disclosed herein. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example Salmonella typhimurium; Serratia, for example Serratia marcescens. marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. 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.

[0342] The host cells of the present invention are prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the anti-MSLN antibody or antigen-binding fragment thereof disclosed herein in vivo to treat the diseases disclosed herein.

[0343] The present invention also provides a host cell into which any of the above vectors has been introduced. The present invention also provides a method for producing the antibodies and antibody fragments of the present invention by culturing the host cell under conditions that allow the production of the antibodies or antibody fragments and recovering the antibodies and antibody fragments thus produced.

[0344] Antibody Drug Conjugates

[0345] In a sixth aspect, the present invention provides an antibody drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0346] In the context of the present disclosure, "conjugate" refers to 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 marker, 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 linked 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 (such as bispecific or trispecific) antibodies.

[0347] In some embodiments, the effector molecule can be a detectable marker or an immunotoxin. Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38 and PE40), diphtheria toxin (DT), botulinum toxin or its modified toxins, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds, usually causing death by hepatotoxicity. However, PE and DT can be modified to be used as an immunotoxin by removing the natural targeting components of the toxin (such as the domain 1a of PE and the B chain of DT) and replacing them with different targeting parts (such as antibodies). The term "conjugated" or "connected" can refer to making two polypeptides into a continuous polypeptide molecule. In one embodiment, an antibody is connected to an effector molecule. In another embodiment, the antibody connected to the effector molecule is further connected to a lipid or other molecule, to a protein or peptide, to increase its half-life in vivo. The connection can be carried out by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein the reaction between the antibody portion and the effector molecule has resulted in a covalent bond formed between the two molecules, thereby forming one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule.

[0348] The present invention provides immunoconjugates comprising a monoclonal antibody or antigen binding fragment 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.

[0349] The disclosed monoclonal antibodies may be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which a therapeutic agent is covalently linked to an antibody. A therapeutic agent is an agent having a specific biological activity against a specific target molecule or a cell carrying a target molecule. Those skilled in the art will appreciate that therapeutic agents may include various drugs such as vinblastine, daunomycin, etc.; cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin; encapsulating agents (such as liposomes) containing pharmacological compositions; radioactive agents such as 125 I. 32 P. 14 C. 3 H and 35 S and other labels; target moieties; and ligands.

[0350] The choice of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent may be a cytotoxin for causing death of a particular target cell, such as a tumor cell. Conversely, when it is desired to cause a non-lethal biological response, the therapeutic agent may be conjugated to a non-lethal pharmaceutical agent or a liposome containing a non-lethal pharmaceutical agent.

[0351] Using the therapeutic agents and antibodies described herein, a skilled artisan can easily construct multiple clones containing functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same effector portion or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0352] The effector molecule can be attached to the antibody of interest using any number of means known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The method of attaching the effector molecule to the antibody varies depending on the chemical structure of the effector. Polypeptides generally contain a variety of functional groups; such as carboxylic acids (COOH), free amines (-NH 2 ) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or connect additional reactive functional groups. Derivatization can include connecting any of many known linker molecules. The linker can be any molecule for connecting the antibody to the effector molecule. The linker can form a covalent bond with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art, and include but are not limited to straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody and effector molecule are polypeptides, the linker can be connected to these constituent amino acids through the side groups of the constituent amino acids (such as through a disulfide bond with cysteine), or to the alpha carbon amino group and carboxyl group of the terminal amino acid.

[0353] In some cases, when the immunoconjugate has reached its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site.

[0354] Cleavage of the linker can be facilitated by enzymatic activity or by conditions to which the immunoconjugate is subjected within the target cell or near the target site to release the effector molecule from the antibody.

[0355] In view of the numerous methods reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for attaching a given agent to an antibody or other polypeptide.

[0356] The antibodies disclosed herein may be derivatized or linked to another molecule (such as another peptide or protein). Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, the antibody functionality may be linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0357] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to produce bispecific antibodies). Suitable cross-linking agents include those with heterobifunctional groups having two distinct reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those with homobifunctional groups (such as disuccinimidyl suberate). Such linkers are commercially available.

[0358] The antibody can be conjugated to a detectable marker; for example, the detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, 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-l-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). The antibody or antigen binding fragment can also be conjugated to an enzyme for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, detection can be performed by adding additional reagents that use the additional reagents to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The antibody or antigen binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin protein binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0359] The antibody can be fused to a self-labeled protein tag (e.g., HaloTag). For example, a protein tag can be cloned at the end of a constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) that is designed to be covalently bound to a synthetic ligand. In some cases, the synthetic ligand comprises a chloroalkane linker (Los et al. (2008), ACS Chem Biol., Vol. 3, No. 6: pp. 373-82) connected to a fluorophore such as a near-infrared fluorophore.

[0360] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0361] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter gene (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). In some embodiments, the labels are connected by spacer arms of various lengths to reduce potential steric hindrance.

[0362] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of the target antigen 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.

[0363] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life or increasing tissue binding.

[0364] Toxins can be used with monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxins and their subunits, and botulinum toxins A to F. These toxins can be easily obtained from commercial sources (Sigma Chemical Company, St. Louis, MO). The toxins considered also include variants of toxins described herein (see, for example, see U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length natural (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to: elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions, and addition of one or more sequences at the carboxyl terminus (see, for example, Siegall et al., Biol. Chem. 264: 14256-14261, 1989).

[0365] The PE used in the monoclonal antibodies described herein may include native sequences, cytotoxic fragments of native sequences, and conservatively modified variants of native PE and its cytotoxic fragments. The cytotoxic fragments of PE include cytotoxic fragments with or without subsequent proteolysis or other treatments in target cells. The cytotoxic fragments of PE include PE40, PE38, and PE35. For additional descriptions of PE and its variants, see, e.g., U.S. Patent 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; PCT Publication Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA, Vol. 88: pp. 3358-3362, 1991; Kondo et al., J. Biol. Chem., Vol. 263: pp. 9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta, Vol. 1333: pp. C1-C6, 1997.

[0366] Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, 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, Vol. 113, No. 16: pp. 3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA, Vol. 105, No. 32: pp. 11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, which are incorporated herein by reference).

[0367] In some examples, PE is a variant resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood, Vol. 113 No. 16: pp. 3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other examples, PE is a variant designated as PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other examples, PE variants are PE with reduced immunogenicity. In other examples, PE is a variant designated as PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0368] Modification of PE may occur in any of the aforementioned variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE may include any substitution, such as substitution of one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or deletion of one or more T cell epitopes and / or B cell epitopes (see, e.g., U.S. Patent Application Publication No. 2015 / 0099707). The PE forms considered also include deimmunized forms of PE, such as forms with domain II deletion (e.g., PE24). Deimmunized forms of PE are described, for example, in PCT publications 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.

[0369] 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 the surface. Therefore, the antibodies disclosed herein can be directly or via a joint connected to a drug that will be delivered directly to cells expressing cell surface antigens. This can be used for treatment, diagnosis or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates or recombinant viruses. Nucleic acid therapy and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA, and oligonucleotides that form triplexes.

[0370] Alternatively, the molecule attached to the antibody can be an encapsulation system, such as a nanoparticle, a liposome or a micelle, which contains a therapeutic composition, such as a drug, a nucleic acid (e.g., an antisense nucleic acid) or another therapeutic moiety that is preferably protected from direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther., Vol. 28: pp. 341-365, 1985).

[0371] The antibodies described herein may also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such uses 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 or32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0372] The means for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using photodetectors to detect emitted illumination. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

[0373] Pharmaceutical composition

[0374] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the bispecific antibody of the second aspect of the present invention, or the nucleic acid of the third aspect of the present invention, or the vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the ADC of the sixth aspect of the present invention; and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0375] The present invention provides pharmaceutical compositions comprising antibodies 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 solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. 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, the composition for intravenous administration is typically a sterile isotonic buffered aqueous solution.

[0376] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise an 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 agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of "Remington's Pharmaceutical Sciences", which is a standard reference text in this field, and is 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 solvents 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. Unless any conventional media or agents are incompatible with the active compound, these media or agents are considered to be used in the composition. Supplementary active compounds may also be incorporated into the composition.

[0377] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. The pH value can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0378] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of preparation and storage; and it must be preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity. The preventive effect on microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. The sustained absorption of the injectable composition can be achieved by including an agent that delays absorption (for example, aluminum monostearate and gelatin) in the composition.

[0379] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into an appropriate solvent with one or more of the components listed above, as required, followed by sterilization filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from the components listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

[0380] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is orally administered and spit out or swallowed after rinsing the mouth. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants such as colloidal dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or orange flavoring.

[0381] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0382] 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 penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream well known in the art.

[0383] The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0384] In one embodiment, the active compound is prepared with carriers that protect the compound from rapid discharge 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 can be used. Methods for preparing such preparations are obvious to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0385] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect when associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compounds for the treatment of individuals, and are directly dependent on these factors.

[0386] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0387] The present invention provides therapeutic compositions comprising the anti-MSLN antibodies or antigen-binding fragments thereof of the present invention. The therapeutic compositions according to the present invention will be administered with suitable carriers, excipients and other agents, which are incorporated into formulations to improve transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998), JPharm Sci Technol, Vol. 52: pp. 238-311.

[0388] Generation method

[0389] Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler and Milstein, Nature, Vol. 256: p. 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0390] Immunizing agents generally include protein antigens, fragments thereof, or fusion proteins thereof. Typically, if human cells are needed, peripheral blood lymphocytes are used, or if non-human mammalian sources are needed, spleen cells or lymph node cells are used. Lymphocytes are then fused to immortalized cell lines using suitable fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells from rodents, cattle, and humans. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will generally contain hypoxanthine, aminopterin, and thymidine ("HAT culture medium"), which prevent the growth of HGPRT-deficient cells.

[0391] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibodies by the selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. More preferred immortalized cell lines are murine myeloma cell 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 interspecies myeloma cell lines for the production of human monoclonal antibodies have also been described. (See Kozbor, J. Immunol., Vol. 133: p. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63)).

[0392] The presence of monoclonal antibodies against the antigen in the culture medium of the hybridoma cells can then be determined. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). These techniques and assays are known in the art. The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., Vol. 107: p. 220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0393] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites.

[0394] 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.

[0395] Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). The hybridoma cells of the present invention are used as a preferred source of such DNA. After isolation, the DNA can be placed in expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to achieve the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences for human heavy and light chain constant regions (see U.S. Pat. No. 4,816,567; Morrison, Nature, Vol. 368: pp. 812-813 (1994)) or by covalently linking immunoglobulin coding sequences to all or part of the coding sequences for non-immunoglobulin polypeptides. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0396] A fully human antibody is an antibody molecule in which the entire sequence of both the light chain and the heavy chain (including CDR) is derived 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 tri-hybridoma technology; human B cell hybridoma technology (see Kozbor et al., 1983, Immunol Today, Vol. 4: p. 72); and EBV hybridoma technology to produce human monoclonal antibodies (see Cole et al., 1985, in: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983, Proc Natl Acad Sci USA, Vol. 80: pp. 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).

[0397] In addition, humanized antibodies can be produced in transgenic plants as a low-cost production 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 transformation of plants can be achieved by using Agrobacterium tumefacien or particle bombardment. For example, by transformation, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed in a bacterial culture, i.e., Agrobacterium tumefaciens strain BLA4404. Infiltration of plants can be accomplished by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Separation and purification of antibodies can be easily performed by many methods known to those skilled in the art. Other methods for producing antibodies in plants are described in, for example, Fischer et al., Vaccine, 2003, Vol. 21: 820-825; and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, 55-78. Therefore, the present invention also provides any cell or plant comprising a vector encoding an antibody of the present invention or producing an antibody of the present invention.

[0398] Furthermore, the (human) antibodies of interest may be produced in fungi. For example, the fungus may be Myceliophthora thermophila (e.g., Myceliophthora thermophila strain C1; Visser et al. (2011), Industrial Biotechnology, Vol. 7, No. 3: pp. 214-223). Other examples include Aspergillus species (e.g., A. oryzae (Huynh et al. (2020), Fungal Biology and Biotechnology, Vol. 7: p. 7), A. niger (Ward et al. (2004), Environ. Microbiol., Vol. 70: pp. 2567-2576), or A. awamori (Joosten et al. (2003), Microb. Cell Fact, Vol. 2: p. 1)) and Trichoderma species (e.g., T. reesei ( et al. (1993), Biotechnology, Vol. 11: pp. 591-595). In other cases, the fungus can be a yeast, such as Saccharomyces cerevisiae, Candida boidinii, Hansenula polymorpha, Pichia methanolica, Pichia pastoris, Yarrowia lipolytica, Kluyveromyces lactis, or Ogataeaminuta (Joosten et al. (2003); Suzuki et al. (2017), J Biosci Bioeng., Vol. 124: pp. 156-163).

[0399] In addition, human antibodies can also be produced using other technologies, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., Vol. 227: p. 381 (1991); Marks et al., J. Mol. Biol., Vol. 222: p. 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar to that observed in humans in all aspects including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in WO 2006 / 008548, WO 2007 / 096779, WO 2010 / 109165, WO 2010 / 070263, WO 2014 / 141189 and WO 2014 / 141192.

[0400] A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a 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 a heavy chain and a light chain.

[0401] In a further improvement on this procedure, PCT Publication WO 99 / 53049 discloses methods for identifying clinically relevant epitopes on immunogens and associated methods for selecting antibodies that immunospecifically bind to the relevant epitopes with high affinity.

[0402] The antibody can be expressed using a vector containing a DNA fragment encoding the above-mentioned single-chain antibody.

[0403] 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 portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins, such as those described in PCT / US 95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting portion (e.g., an antibody specific for a target cell) and a nucleic acid binding portion (e.g., protamine); plasmids; phages, etc. Vectors may be chromosomal, non-chromosomal or synthetic.

[0404] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. Preferred are DNA viral vectors. These vectors include poxvirus vectors such as orthopoxvirus or fowlpoxvirus vectors, herpesvirus vectors such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, Vol. 64: p. 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, Vol. 90: p. 7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA, Vol. 87: p. 1149 (1990), adenovirus vectors (see LeGal ... LaSalle et al., Science, Vol. 259: p. 988 (1993); Davidson et al., Nat. Genet, Vol. 3: p. 219 (1993); Yang et al., J. Virol., Vol. 69: p. 2004 (1995) and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet., Vol. 8: p. 148 (1994).

[0405] Poxvirus vectors introduce genes into the cytoplasm. Fowlpoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferably used to introduce nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be carried out by standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0406] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. In addition, micropump infusion systems such as the SynchroMed infusion system can be used to deliver particles by intracerebroventricular (icv) infusion. Bulk flow-based methods known as convection have also been shown to be effective in delivering macromolecules to extended areas of the brain and can be used to deliver vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA, Vol. 91: pp. 2076-2080 (1994); Morrison et al., Am. J. Physiol., Vol. 266: pp. 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injections, as well as oral or other known routes of administration.

[0407] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of MSLN in a sample. The antibodies can also be used to attempt to bind to MSLN and disrupt the interaction between MSLN and MUC16.

[0408] In a preferred embodiment, the antibodies of the invention are full length antibodies, containing an Fc region similar to a wild-type Fc region that binds an Fc receptor.

[0409] Treatment

[0410] The antibodies provided herein can be administered to slow down 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 the signs or symptoms of cancer. Suitable subjects may include those diagnosed with cancers expressing 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.

[0411] Provided herein is a method for treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody as described herein. Also provided herein is a method for inhibiting the metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of an antibody as described herein. In some embodiments, the mesothelin-positive cancer is 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.

[0412] The administration of the antibodies disclosed herein may also be accompanied by the administration of other anticancer agents or therapeutic treatments (such as surgical resection of tumors). Any suitable anticancer agent may be administered in combination with the antibodies disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, anti-hormones (e.g., anti-androgens), and anti-angiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0413] Another common treatment for some types of cancer is surgery, such as surgical removal of metastatic tumors. Another example of treatment is radiation therapy, such as administering radioactive material or energy (such as external beam radiation therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical removal.

[0414] Diagnosis and testing methods

[0415] 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 a blood sample, tissue from a biopsy, autopsy, and pathological specimens. Biological samples also include tissue sections, e.g., frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0416] 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. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having a mesothelin-positive cancer.

[0417] In another embodiment, a method is provided for confirming a diagnosis of mesothelin-positive cancer in a subject by contacting a sample from a subject diagnosed with mesothelin-positive cancer with a mesothelin-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. Increased binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms a diagnosis of mesothelin-positive cancer in the subject.

[0418] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0419] In other examples, the method further comprises 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.

[0420] In some instances, the cancer is 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.

[0421] In some examples, the control sample is a sample from a subject without cancer. In certain examples, the sample is a blood or tissue sample.

[0422] In some embodiments of the diagnostic and detection methods, the anti-MSLN antibody is directly labeled with a detectable label. In another embodiment, the anti-MSLN antibody (first antibody) is unlabeled, and the second antibody or other molecule that can bind to the first antibody is labeled. As is well known to those skilled in the art, a secondary antibody is selected that is capable of specifically binding to a particular type and class of the first antibody. For example, if the first antibody is human IgG, the secondary antibody can be 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.

[0423] 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 exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or3 H.

[0424] In another embodiment, mesothelin in a biological sample can be measured by a competitive immunoassay using a mesothelin protein standard labeled with a detectable substance and an unlabeled anti-MSLN antibody. In this assay, the biological sample, the labeled MSLN protein standard, and the anti-MSLN antibody are combined, and the amount of the 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 the labeled MSLN protein standard bound to the anti-MSLN antibody.

[0425] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, an anti-MSLN antibody can be used to detect the production of MSLN 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 or a blood or serum sample). In some examples, the MSLN is a 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).

[0426] In one embodiment, a kit for detecting MSLN in a biological sample (such as a blood sample or a tissue sample) is provided. For example, in order to verify a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. The kit for detecting the polypeptide will generally include a monoclonal anti-MSLN antibody, such as any of the monoclonal antibodies disclosed herein. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme label).

[0427] In one embodiment, the kit includes instructional materials that disclose methods of using the anti-MSLN antibody. The instructional materials may be written in electronic form (such as a computer disk or CD), or may be visual (such as a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally contain means for detecting the label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label (such as a secondary antibody), etc.). The kit may additionally include buffers and other reagents routinely used to implement a particular method. These kits and appropriate contents are well known to those skilled in the art.

[0428] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassay may vary depending on the particular format used, the method of detecting MSLN in a biological sample generally includes the step of contacting the biological sample with an anti-MSLN antibody. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0429] The antibodies disclosed herein can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. The antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low angle and obtuse angle light scatter detection channels, and impedance channels, as well as other more complex detection levels, to separate or sort cells (see U.S. Patent No. 5,061,620). As disclosed herein, any monoclonal antibody that binds to mesothelin can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation.

[0430] Example

[0431] Example 1. Animal immunization protocol

[0432] To obtain MSLN-specific antibodies, Harbour H2L2 transgenic mice (https: / / harbourantibodies.com / ) were immunized by different methods. These immunizations produced a number of antibodies that bound to the MSLN extracellular (ECD) protein and cells expressing MSLN such as COV644 cells (an ovarian epithelial-mucinous carcinoma cell line expressing MSLN).

[0433] Specifically, recombinant human MSLN ECD His-tag protein (Acro Biosystem, catalog number MSN-H5223) or recombinant cynomolgus monkey MSLN ECD His-tag protein (having the sequence listed below, SEQ ID NO: 77) was used as an immunogen to immunize Harbour H2L2 transgenic mice.

[0434] Cynomolgus monkey MSLN ECD His-tag protein (SEQ ID NO: 77)

[0435] DVERTTCPPEKEVHEIDESLIFYKKRELEACVDAALLAAQMDRVDAIPFTYEQLDVLKHKLDELYPQGYPESVIRHLGHLFLKMSPEDIRKWNVTSLETLKALLKVSKGHEMSAQVATLIDRVVVGRGQLDKDTVDTLTAFCPGCLCSLSPERLSSVPPSVIGAV RPQDLDTCGPRQLDVLYPKARLAFQNMSGSEYFVKIRPFLGGAPTEDVKALSQQNVSMDLATFMKLRREAVLPLTVAEVQKLLGPHVEGLKVEEQHSPVRDWILKQRQDDLDTLGLGLQGGIPNGYLILDLSVREALSGTPCLLGPGPVLTVLALLLASHHHHHH

[0436] The protocol for generating anti-mesothelin antibodies by antigen immunization and hybridoma clone screening is shown in Figure 1 middle.

[0437] Examples of immunization protocols are listed in Table 6 below. Briefly, 50 μg of immunogen was administered subcutaneously or intraperitoneally to each mouse together with an adjuvant (Sigma, S6322) for the first booster immunization, and 25 μg of immunogen was administered together with an adjuvant for subsequent booster immunizations. Immunization was performed every two weeks for a total of 6 times. Final immunization was performed intraperitoneally with the immunogen diluted in PBS. Serum titers for recombinant human MSLN ECD His-tag protein (Acro Biosystem, catalog number MSN-H5223) were tested using ELISA and FACS.

[0438] Table 6. Immunization schedule

[0439]

[0440] Example 2. Screening of MSLN-specific antibodies

[0441] 2.1 Generation of hybridomas and screening of MSLN-specific antibodies

[0442] Monoclonal antibodies can be prepared using hybridoma methods such as those described in Kohler and Milstein, Nature, Vol. 256: p. 495 (1975). In the hybridoma method, lymphocytes are fused with immortalized cell lines to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodents. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains 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 assays (ELISA). After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution steps and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0443] 2.2 Single B cell screening of MSLN-specific antibodies

[0444] The Optofluidic system is used for single B cell screening. The system uses optoelectronic positioning (OEPTM) technology to move single cells and allows for simultaneous biological function testing, experimental analysis, positive clone selection, and other operations under cell culture conditions. The Beacon platform can perform these tasks in a massively parallel, automated manner for thousands of cells.

[0445] In this example, a plasma cell discovery workflow was used. In each experiment, up to 14,000 individual plasma cells were screened for the secretion of MSLN-specific antibodies. Plasma cells secreting antigen-specific antibodies were then transferred to 96-well plates for subsequent single B cell sequencing to identify the heavy and light chains of antibodies produced by a single B cell (monoclonal). Figure 2 The screening strategy and process are shown.

[0446] This embodiment uses a single B cell sequencing method to obtain the sequences of the heavy and light chains of antibodies from a single plasma cell. The general procedure includes extraction and purification of total RNA, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification, cloning and transfection of the DNA sequences encoding the heavy and light chains of antibodies, and Sanger sequencing. The obtained sequences are subjected to uniqueness and cluster analysis, and then the DNA sequences encoding the paired heavy and light chains of antibodies are synthesized.

[0447] Example 3. Antibody Production and Purification

[0448] The recombinant plasmid encoding the target antibody was transiently transfected into HEK293-6E cells or 293-F cells using PEI (Polyscience, 24885). After transfection, the cells were incubated at 37°C with 5% CO 2 Incubate at 40 °C and shake at 120 rpm. 6 to 7 days after transfection, harvest the cell culture supernatant containing the target antibody by centrifugation and filtration. Purify the monoclonal antibody using protein A magnetic beads (AmMag protein A magnetic beads, Genscript, L00695).

[0449] The purity of the antibodies was tested by SEC-HPLC (Agilent 1260 Infinity II HPLC system, Welch Xtimate SEC-300 column, 1× PBS at pH 7.4 as mobile phase) and SDS-PAGE (SurePAGE, Bis-Tris, 10×8, 4%-12%, 12-well, Genscript, M00653). The recombinant antibodies were successfully expressed and purified for further characterization.

[0450] Two antibodies PR300159 and PR300186 were obtained. The amino acid sequences of antibodies PR300159 and PR300186 are listed in Tables 1 to 4 above.

[0451] At the same time, the anti-MSLN antibody Amatuximab was also produced according to the above procedures using the 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.

[0452] Example 4. Antibody Binding Activity to Cells Expressing MSLN

[0453] The binding of recombinant anti-MSLN antibodies to cells expressing human or cynomolgus MSLN was tested by flow cytometry. In this embodiment, cell lines expressing MSLN include CHOK1 cell lines that have been transfected to express human MSLN (CHOK1-hu MSLN, supplier: Kyinno, catalog number: KC-1152) or cynomolgus MSLN (CHOK1-cyno MSLN, supplier: Kyinno, catalog number: KC-1174) on the surface, and also include COV644 cell lines (ECACC, catalog number: 07071908).

[0454] Briefly, anti-MSLN antibody was serially diluted in staining buffer (PBS buffer containing 2% FBS). 50 μL of diluted antibody solution was added to 50 μL of 1-2×10 5 The cells were washed twice with staining buffer (PBS buffer containing 2% FBS) and 100 μL of 1:1000 diluted fluorescently labeled anti-human IgG antibody (Alexa Fluor 488AffiniPure goat anti-human IgG (H+L), Jackson ImmunoResearch, catalog number 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 non-related IgG isotype control (Crownbio) were used as positive and negative controls, respectively.

[0455] The results are shown below Figures 3 to 5 The results showed that both PR300159 and PR300186 showed strong binding activity to cells expressing human MSLN and cells expressing cynomolgus monkey MSLN, with EC50 values ​​comparable to PR000685 (Amatuximab). These results indicate that the anti-MSLN antibodies PR300159 and PR300186 can bind to human MSLN and cynomolgus monkey MSLN on the cell membrane with high affinity.

[0456] Example 5. Antibody Engineering

[0457] The VH and VL sequences of the anti-MSLN antibodies PR300159 and PR300186 were further optimized by the PTM removal procedure.

[0458] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. In addition to the conserved PTM sites in antibodies, such as the conserved N-glycosylation sites on the CH2 domain of IgG1 antibodies, other PTM sites (i.e., CDR regions) that occur in the antigen binding site of antibodies may also reduce antigen binding activity or reduce chemical stability. For example, deamidation or isomerization may make the molecule unstable and heterogeneous. In order to reduce sequence susceptibility, PTM motifs can be removed by mutation. Scan the VH sequence or VL sequence to observe the presence of PTM motifs such as isomerization motifs (e.g., DG). Then mutate the "hot spot" residues (e.g., D or G in the DG motif) to the corresponding residues in the germline sequence or other residues with similar biophysical properties. Then, antibodies consisting of sequence variants after the removal of PTMs are recombined and produced by mature molecular biology techniques.

[0459] The designed variants 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-removing antibodies derived from PR300186.

[0460] Table 7. Designed PR300159 derivative variants and PR300186 derivative variants

[0461]

[0462]

[0463] Example 6. Antibody Binding Activity to Human MSLN Protein and Cynomolgus Monkey MSLN Protein

[0464] Human MSLN (Acro Biosystems, catalog number MSN-H5223) or cynomolgus monkey MSLN protein (Harbourbiomed, lot number: 2019072202) was diluted to a concentration of 1 μg / mL in PBS. 100 μL of diluted human MSLN or cynomolgus monkey MSLN was added to each well of the ELISA microplate, and the plate was incubated overnight at 4°C. The plates were blocked with ELISA blocking buffer (containing 2% weight / volume BSA, 0.05% (volume / volume) Tween-20, PBS buffer, pH 7.4) at 37°C for 1 hour, then 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 at 15 μg / mL (100 nM, 10-fold dilution, 8 points) 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) at 37°C for 1 hour. 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Biopanda, catalog number: TMB-S-003) was added, and the plate was incubated at room temperature for 15 minutes. 100 μL of ELISA stop solution (Solarbio, catalog number: C1058) was added to stop the reaction, and the optical density (OD450nm) at 450nm was measured by an ELISA plate reader (Molecular Devices, Spectra max 384plus).

[0465] The results of PR300186 and PR300186PTM removal antibodies (PR300186-2, PR300186-3, PR300186-4, PR300186-5, PR300186-6, PR300186-7, PR300186-8, PR300186-9 and PR300186-10) are shown in Figure 6 , Figure 7 and in the following Table 8. The results indicated that all PTM removal antibodies derived from PR300186 showed good binding activity to both human MSLN protein and cynomolgus monkey MSLN protein, with EC50 values ​​comparable to those of PR300186.

[0466] Table 8. Binding of PR300186PTM removal antibodies to human MSLN protein and cynomolgus monkey MSLN protein

[0467]

[0468]

[0469] Example 7. Antibody Binding Activity to Cells Expressing MSLN

[0470] The binding of recombinant anti-MSLN antibodies to the COV644 cell line (ECACC, catalog number: 07071908) was tested by flow cytometry. Briefly, the anti-MSLN antibody was serially diluted in staining buffer (PBS buffer containing 2% FBS). 50 μL of the diluted antibody solution was added to 50 μL of 1-2×10 5 The cells were washed twice with staining buffer (PBS buffer containing 2% FBS) and 100 μL of 1:1000 diluted fluorescently labeled anti-human IgG antibody (Alexa Fluor 488 AffiniPure goat anti-human IgG (H+L), Jackson ImmunoResearch, catalog number 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.

[0471] The results of PR300159PTM removal antibody are shown in Figure 8 and in Table 9 below. The results showed that PTM removal antibodies PR300159-1, PR300159-3, PR300159-5, PR300159-6 and PR300159-8 showed comparable binding activity to COV644 cells compared to PR300159.

[0472] Table 9. Binding of PR300159PTM removal antibodies to cell surface MSLN by FACS

[0473]

[0474] The results of the PTM removal antibody derived from PR300186 are shown in Fig. 9 The results showed that the PTM removal antibodies PR300186-3, PR300186-9 and PR300186-10 showed better binding activity to COV644 cells compared with PR300186.

[0475] Example 8 Internalization of Antibodies by Cells Expressing MSLN

[0476] In this example, pHAb amine-reactive dye (Promega, catalog number G9841) was used to measure antigen internalization of anti-MSLN antibody in COV644 cells. pHAb dye is a pH sensor dye that has very low fluorescence at pH>7 and a significant increase in fluorescence when the solution becomes acidic. When antibodies labeled with pHAb dye bind to the outside of the cell membrane at neutral pH, no or very low fluorescence can be monitored. After internalization, the fluorescence becomes stronger in the low pH environment of endosomes and lysosomes.

[0477] The antibody was labeled with pHAb dye and DAR was calculated according to the kit instructions. The labeled antibody was then incubated with COV644 at 4 ° C (the internalization activity at this temperature was very low, which was used as a background control) or 37 ° C for 24 hours. The fluorescence with an excitation maximum (Ex) at 532nm and an emission maximum (Em) at 560nm was then detected. The final normalized results are shown as the fluorescence intensity at 37 ° C minus the fluorescence intensity of the background at 4 ° C, and then divided by the DARs of the pHAb Dye of the antibody. The higher the value, the higher the internalization activity.

[0478] The internalization rate results of PR300159 and PTM removal antibodies derived from PR300159 are shown in Fig.10 and Table 10. The results showed that PR300159-1, PR300159-4, PR300159-5, PR300159-6, PR300159-7, PR300159-8 antibodies showed comparable internalization by COV644 cells compared to PR300159.

[0479] The internalization rate results of PR300186 and PTM removal antibodies derived from PR300186 are shown in Fig.11 and Table 10. The results showed that PR300186-3, PR300186-9 and PR300186-10 showed better internalization by COV644 cells compared with PR300186.

[0480] Table 10. Internalization of PR300159 and PR300186 PTM removal antibodies by cells expressing MSLN

[0481] Antibody Internalization FLU PR300159 22214 PR300159-1 13720 PR300159-3 9180 PR300159-4 20888 PR300159-5 20912 PR300159-6 17753 PR300159-7 22440 PR300159-8 21172 PR300159-9 8299 PR300186 6049 PR300186-3 7042 PR300186-9 8069 PR300186-10 8219

[0482] Example 9. Binding activity of antibodies to soluble MSLN protein by BLI method

[0483] In this example, the binding kinetics of anti-MSLN antibodies to soluble MSLN were analyzed by Octet Red384 (Fortebio). In the biomembrane interferometry (BLI) analysis, recombinant human MSLN-His tag (Acro Biosystems, catalog number: MSN-H5223) was serially diluted with 1× 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 rate constants of association and dissociation were measured using an AHC sensor (Fortebio). The sensor surface was regenerated with regeneration buffer after each binding experiment. The traces were processed using Octet data analysis software (version 11.0, Pall ForteBio, CA, USA). K of antibody binding to soluble human MSLN D The values ​​are summarized in Table 11.

[0484] As shown in Table 11, in the Octet analysis, the PTM removal antibodies derived from PR300159 showed comparable binding affinity to soluble MSLN compared to PR300159.

[0485] PR300186 and PR300186-10 showed low binding affinity to soluble MSLN, which is advantageous when applied as therapeutic antibodies because the antibodies bind better to cell surface MSLN on tumor cells rather than soluble MSLN in the circulation.

[0486] Table 11. Binding of PR300159 and PR300186 PTM removal antibodies to soluble human MSLN

[0487]

[0488] Example 10. Soluble MSLN Interference Assay

[0489] Antibodies that bind to the membrane-bound form of MSLN are advantageous for diagnostic and therapeutic purposes. Because they do not bind to soluble MSLN, they will have lower background for imaging applications, and they can be used at lower doses for therapeutic use.

[0490] The binding of antibodies PR300159 and PR300186 and PTM removal antibodies derived from PR300159-8 or PR300186-10 to mesothelin expressing COV644 was tested in the presence or absence of 90 nM soluble MSLN (sMSLN). The experiment was performed according to a similar procedure as shown in Example 4, except that 90 nM soluble MSLN was added to the serial dilutions of the +sMSLN group of antibodies.

[0491] like Fig.12 As shown, in the presence of 90 nM soluble MSLN, the binding of PR300159 to COV644 cells was reduced compared to the absence of soluble MSLN. Fig.13 As shown, PR300186 showed less change, suggesting that soluble MSLN interfered less with the binding of PR300186 to the membrane-bound form of MSLN.

[0492] like Fig.14 and Fig.15 As shown, in the presence of 90 nM soluble MSLN, PR300159-8 and PR300186-10 had reduced binding to COV644 cells compared to the absence of soluble MSLN. However, PR300186-10 showed less change, suggesting that soluble MSLN interfered less with the binding of these antibodies to the membrane-bound form of MSLN.

Claims

1. 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), and wherein (1) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 27; (2) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 25, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 28; (3) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 26, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 27; (4) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 29; (5) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 30; (6) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 31; (7) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 32; (8) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 24, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 33; (9) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 25, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 29; (10) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 63, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 67; (11) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 64, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 67; (12) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 65, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 67; (13) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 65, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 68; (14) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 68; (15) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 67; (16) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 64, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 69; (17) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 69; (18) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 63, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 69; or (19) The VH comprises HCDR 1-3 of VH having the amino acid sequence shown in SEQ ID NO:65, and the VL comprises LCDR 1-3 of VL having the amino acid sequence shown in SEQ ID NO:

69.

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

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: (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 that has 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 that has 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) The VH comprises an amino acid sequence that has 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 that has 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.

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), and wherein: (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 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:

76.

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 according to claim 5, wherein the antibody or antigen-binding fragment thereof binds to membrane-bound mesothelin with a higher affinity than it binds 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 two times, at least three times, at least five times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, or at least 100 times greater than its affinity for binding to soluble mesothelin.

8. The antibody or antigen-binding fragment thereof according to 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 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 IgG1, IgG2, IgG3 and IgG4.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, 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 according to any one of claims 1 to 13, wherein the antibody is monovalent, divalent or multivalent.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is linked 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 according to claim 1 or the bispecific antibody according to claim 16 .

18. A vector comprising the nucleic acid according to claim 17.

19. A host cell comprising the nucleic acid according to claim 17 or the 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. 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. The pharmaceutical composition of claim 21, wherein the composition further comprises a second therapeutic agent selected from the group consisting of an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

23. A method of treating cancer in a subject, the method comprising 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, the antibody drug conjugate according to claim 20, or the pharmaceutical composition according to claim 21 or 22.

24. The method of claim 23, wherein the cancer is selected from 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. The method of claim 23 or 24, further comprising administering to the subject a second therapeutic agent.

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

27. Use 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, the antibody-drug conjugate according to claim 20, or the pharmaceutical composition according to claim 21 or 22 in the preparation of a medicament for treating cancer in a subject.

28. The use according to claim 27, wherein the cancer is selected from 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. The use according to any one of claims 27 or 28, wherein the medicament further comprises a second therapeutic agent, optionally the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

30. The use according to any one of claims 27 or 28, wherein the medicament is administered in combination with a second therapeutic agent, optionally the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

31. 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, for treating cancer in a subject.

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 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.

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 administering to the subject a second therapeutic agent, optionally the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, siRNA, an antisense oligonucleotide, a polypeptide and a small molecule drug.

34. A method for diagnosing a mesothelin-positive cancer in a subject, the method comprising: (a) obtaining a biological sample from the subject, (b) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; and (c) detecting binding of the antibody to the sample, Wherein increased binding of the antibody or antigen-binding fragment thereof to the sample as 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 method for imaging a mesothelin-positive cancer in a subject, the method comprising: (a) administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody is conjugated to a detectable marker; as well as (b) detecting the presence of the marker.

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

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