Anti-mesothelin antibodies

Abstract

To provide antibody molecules that bind with higher affinity to immobilized mesothelin (MSLN) than to MSLN in solution.SOLUTION: The present invention provides an antibody molecule that binds mesothelin (MSLN), wherein the antibody molecule is a human IgG1 molecule, and an antigen-binding site of the antibody molecule comprises complementarity determining regions (CDRs) 1 to 6 of antibody: FS28-024-052; FS28-024-051; FS28-024-053; or FS28-024, where the CDR sequences are defined according to the ImMunoGeneTics (IMGT) numbering scheme, and / or the antigen-binding site of the antibody molecule comprises CDRs 1 to 6 of antibody: FS28-024-052; FS28-024-051; FS28-024-053; or FS28-024, where the CDR sequences are defined according to the Kabat numbering scheme.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibody molecules that bind to mesothelin (MSLN) and find use in the treatment and diagnosis of diseases and disorders, such as cancer. [Background technology]

[0002] 2. Background of the Invention MSLN is expressed at relatively low levels in mesothelial cells lining the pleura, peritoneum, and pericardium in healthy individuals (Hassan et al., 2005), but is highly expressed in several different cancers, including mesothelioma, squamous cell carcinoma, pancreatic cancer, lung cancer, gastric cancer, breast cancer, endometrial cancer, and ovarian cancer. The normal biological function of mesothelin is unclear. With respect to cancer, high expression levels of MSLN have been correlated with poor prognosis in ovarian cancer, cholangiocarcinoma, lung adenocarcinoma, and triple-negative breast cancer. Due to the limited expression of MSLN in normal cells compared to its high expression in tumor cells, MSLN is an attractive therapeutic target using monoclonal antibodies (Hassan et al., 2016).

[0003] MSLN is expressed as a 69-kDa precursor protein (628 amino acids). The precursor protein is then cleaved by the endoprotease furin to release a secreted N-terminal region called megakaryocyte potentiating factor (MPF), while the 40-kDa protein mature MSLN remains attached to the cell membrane via a glycosylphosphatidylinositol (GPI) linker. Human MSLN shares 60% and 87% amino acid identity with the mouse and cynomolgus monkey orthologues of MSLN, respectively.

[0004] Membrane-bound mature MSLN is shed from cells as a result of alternative splicing by generating variants lacking the membrane anchor sequence or protease cleavage by tumor necrosis factor alpha converting enzyme (TACE) (Sapede et al., 2008; Zhang et al., 2011). Soluble shed MSLN is found in the serum of patients and in the stroma of tumors, including malignant mesothelioma, ovarian cancer or highly metastatic cancer. Measuring soluble MSLN levels in the blood and exudates of mesothelioma patients is approved by the US FDA to monitor the patient's response to treatment and progression (Hollevoet et al., 2012; Creany et al., 2015).

[0005] Several antibody-based therapeutic approaches targeting MSLN have been developed and tested in clinical trials, mainly in mesothelioma, pancreatic cancer, and non-small cell lung cancer (Hassan et al., 2016). Approaches used include direct tumor cell killing through the use of anti-MSLN antibodies such as amatuximab with antibody-dependent cellular cytotoxicity (ADCC) activity, as well as antibody-drug conjugates (ADCs) such as SS1P-PE38 and anetumab ravtansine, which contain antibodies or antibody fragments conjugated to toxins. In addition, anti-MSLN-binding Fv fragments have been used in chimeric antigen receptor T-cell therapy, and bispecific antibodies such as ABBV-428 have moved from preclinical trials to clinical trials.

[0006] Amatuximab is a murine chimeric IgG1 κ mAb that blocks MSLN-MUC16 interaction and relies on ADCC function to eliminate tumor cells. Phase I trials have shown a good safety profile, but limited clinical efficacy when combined with chemotherapy (gemcitabine or pemetrexed / cisplatin), with minimal / no improvement in progression-free survival in malignant mesothelioma (NCT00738582, Hassan et al., 2014), and no overall response compared to the comparison arm in pancreatic cancer (NCT00570713). SS1-PE-38 is a recombinant immunotoxin containing the anti-MSLN scFv present in amatuximab bound to the protein synthesis inhibitor PE38. Despite the high antitumor activity observed in Phase I trials (77% partial response when combined with chemotherapy, NCT01445392), the clinical use of SS1P is limited by immunogenicity and dose-limiting vascular leak syndrome. LMB-100, an optimized form of SS1P-PE38 with reduced immunogenicity in vitro, is currently being tested alone and in combination with chemotherapy (NCT02798536, NCT02810418) in Phase I and II trials.

[0007] Anti-MSLN targeting is often used to deliver cytotoxic drugs to tumor cells. Anetumab ravtansine, a fully human IgG1 covalently linked to the antimitotic agent DM4, showed a 31% overall response rate (ORR) in a phase I study on ovarian cancer, primary peritoneal cancer, fallopian tube cancer, and advanced epithelioid type peritoneal predominant mesothelioma (NCT01439152). In mesothelioma, anetumab ravtansine showed a 50% ORR in combination with standard administration of chemotherapy (NCT02639091). Similar to another ADC antibody drug, RG7600, anetumab ravtansine showed a tolerable safety profile, although dose-limiting toxicities were observed in these studies consistent with those reported for the respective ADC moieties. To date, phase I data from BMS86148 are still not available.

[0008] In summary, unconjugated antibodies targeting MSLN have shown favorable safety profiles, but their therapeutic efficacy is limited, while ADCs have shown more potent antitumor activity, but are associated with dose-limiting toxicity. In terms of safety, ADCs may have a broader therapeutic window than immunotoxin therapy (Zhao et al., 2016). Several of these antibody-based MSLN therapies are undergoing Phase II clinical trials combining the therapy with either chemotherapy or immune checkpoint inhibitors such as PD-1 or PD-L1. Several bispecific molecules intended to engage the immune system are also in development, including ABBV-428 targeting MSLN and the costimulatory protein CD40, MSLN-CD3 bispecific T cell engager (BITE) and MSLN-CD47 bispecific molecules. Summary of the Invention [Problem to be solved by the invention]

[0009] Description of the Invention As explained above, mature MSLN, like other tumor-associated antigens (TAA), is known to be shed from the cell surface by enzymatic cleavage. The shed / soluble portion of MSLN is then removed from the tumor site. This is a challenge for therapeutics targeting MSLN, because the shed / soluble portion may act as a sink for therapeutics, removing them from the tumor site before they bind to the tumor. [Means for solving the problem]

[0010] The inventors undertook an extensive selection program to isolate antibody molecules that bind with higher affinity to immobilized MSLN than to MSLN in solution.

[0011] "Affinity" referred to herein may refer to the strength of binding interaction between an antibody molecule and its cognate antigen, as measured by KD.As will be readily apparent to those skilled in the art, if an antibody molecule can form multiple binding interactions with an antigen (e.g., an antibody molecule can bivalently bind to an antigen, optionally when the antigen is a dimer), the affinity measured by KD may also be affected by avidity, whereby avidity refers to the overall strength of the antibody-antigen complex.

[0012] Specifically, the antibody molecules of the present invention are believed to bind to MSLN with higher avidity and therefore bind to MSLN more strongly when the antibody is capable of binding to two MSLN molecules, such as when multiple copies of the antigen are immobilized on the surface, than when MSLN is in monomeric form, which is expected to be the case for MSLN in solution. Thus, without wishing to be bound by theory, it is believed that the antibody molecules of the present invention will not be rapidly removed from the tumor site because they do not remain bound to MSLN shed in solution in vivo due to the low affinity of the antibody for monomeric MSLN, and therefore will exert their therapeutic effect longer by binding MSLN to the surface of tumor cells. Preferential targeting of membrane-bound MSLN has been reported previously, but the molecules were isolated using a different approach than that used by the present inventors. Specifically, preferential targeting of membrane-bound MSLN has been reported by isolating molecules that target different regions of MSLN (Asgarov et al., 2017; Tang et al., 2013). For example, a single domain (VH domain only) antibody fused to a human Fc, SD1-Fc, targeting an epitope close to the cell membrane to promote CDC activity has been reported (Tang et al., 2013). However, it is unclear how such epitopes are exposed in various cancer contexts. In addition, MSLN-CD3 bispecific T cell engagers (BITEs) have also been reported to preferentially bind cell-bound MSLN. However, none of these molecules are full IgG molecules, and none of them have monovalent binding to their targets (via the VH domain in SD1-Fc or the scFv of BITE), and therefore are not capable of bivalently binding to MSLN.

[0013] The antibody molecules isolated by the present inventors bind to various epitopes / regions in MSLN. This is evident from the fact that some of the antibody molecules can block the binding of the ligand MUC16 to MSLN, while others cannot. Blocking MUC16 to MSLN is believed to be advantageous for inhibiting the metastasis of MUC16-expressing cancer cells to MSLN-expressing surfaces in the pleura and peritoneum (Chen et al., 2013). Other binding regions closer to the cell membrane may promote ADCC and CDC activity.

[0014] The anti-MSLN antibody molecule of the present invention has been shown to have ADCC activity and is therefore expected to be useful in cancer therapy. Specifically, the antibody molecule has been shown to be capable of targeting tumor cells that contain MSLN on their cell surface and mediating tumor cell killing via ADCC.

[0015] The antibody molecules of the present invention may also be useful for preparing ADCs comprising the antibody molecules of the present invention and a biologically active molecule, such as a toxin. Such molecules are also expected to be utilized in the treatment of cancers that contain MSLN on the cell surface by targeted delivery of the biologically active molecule to tumor cells.

[0016] The present inventors have realized that the anti-MSLN antibody of the present invention can be used to prepare a multispecific, e.g., bispecific, molecule that binds to a second antigen in addition to MSLN.Preferably, the multispecific molecule binds to the second antigen bivalently.In particular, the present inventors have prepared an anti-MSLN antibody molecule that comprises an additional antigen-binding site in each of the CH3 domains of the antibody molecule that can bivalently bind to the second antigen.

[0017] The second antigen bound by the antibody molecule can be an immune cell antigen, such as a member of the tumor necrosis factor receptor superfamily (TNFRSF). Tumor necrosis factor (TNF) receptors require clustering for activation. Specifically, initial ligation of a TNF receptor ligand to its receptor initiates a series of events that result in trimerization of the TNF receptor, followed by receptor clustering, activation of the NFkB intracellular signaling pathway, and subsequent immune cell activation. Thus, for a therapeutic agent to efficiently activate a TNFR receptor, several TNF receptor monomers need to be crosslinked together in a manner that mimics the trimeric ligand. Many anti-TNF receptor agonist antibodies require crosslinking by Fcγ receptors for their agonist activity or exhibit agonist activity in the absence of crosslinking. In either case, Fcγ receptors are found throughout the human body, so the agonist activity of the antibody is not restricted to a specific site.

[0018] It is expected that bispecific anti-MSLN antibody molecules containing constant domain binding sites for immune cell antigens will be able to conditionally activate immune cell antigens in the presence of MSLN without the need for, for example, Fcγ receptor-mediated crosslinking required by conventional antibody molecules. It is expected that binding of antibody molecules to MSLN will cause crosslinking of the antibody molecules at the sites of MSLN, which will therefore result in clustering and activation of immune cell antigens on the immune cell surface. It is therefore expected that the agonistic activity of the antibody molecules will be dependent on both the immune cell antigen and MSLN being present. In other words, the agonistic activity is expected to be conditional. Furthermore, it is expected that crosslinking of the antibody in the presence of MSLN will promote clustering of immune cell antigens bound via the constant domain antigen binding sites of the antibody molecules. It is therefore expected that when MSLN is a tumor antigen, the antibody molecules will be able to activate immune cells in a disease-dependent manner, for example, in the tumor microenvironment. This targeted activation of immune cells is expected to be beneficial in avoiding off-target side effects. The inventors have shown that a bispecific antibody molecule containing anti-MSLN and anti-CD137 binding sites was able to inhibit tumor growth and prolong survival in a mouse tumor model in the absence of Fcγ receptor binding.

[0019] An antibody molecule comprising an anti-MSLN Fab and a CH3 domain binding site specific for a second antigen binds bivalently to both MSLN and the second antigen. When the second antigen is an immune cell antigen, bivalent binding of both targets is expected to make the cross-linking between immune cells expressing the immune cell antigen and MSLN more stable, thereby extending the time during which the immune cells can localize to a specific site, such as the tumor microenvironment, and act on the disease, e.g., tumor. This differs from most conventional bispecific antibody formats, which are heterodimers and bind each target antigen monovalently via one Fab arm. Such monovalent interactions are expected to be not only more unstable, but also insufficient in many cases to induce clustering of immune cell antigens, such as TNF receptors, in the first place.

[0020] A further feature of the anti-MSLN antibody molecule of the present invention, which comprises a CH3 domain binding site specific for a second antigen, is that the two antigen binding sites for MSLN and the second antigen are both contained within the antibody structure itself. In particular, the antibody molecule does not require other proteins to be fused to it via linkers or other means to result in a molecule that bivalently binds to both of its targets. This has many advantages. In particular, the antibody molecule can be produced using methods similar to those used for the production of standard antibodies, since it does not contain additional fused moieties. This structure is also expected to result in improved antibody stability, since the linker can degrade over time, resulting in a heterogeneous population of antibody molecules. Those antibodies in a population with only one protein fused may not be able to induce conditional agonism of immune cell antigens, such as TNF receptors, as efficiently as those with two fused proteins. Cleavage or degradation of the linker can occur before or after administration of the therapeutic agent to the patient (e.g., by enzymatic cleavage or the patient's in vivo pH), thereby resulting in a decrease in its effectiveness in the patient's circulation. In the absence of a linker in an antibody molecule, the antibody molecule is expected to retain the same number of binding sites both before and after administration. Furthermore, the structure of the antibody molecule is also favorable in terms of the immunogenicity of the molecule, since the introduction of a fused protein or a linker, or both, may induce immunogenicity when the molecule is administered to a patient, resulting in a decrease in the efficacy of the therapeutic agent.

[0021] Thus, the present invention provides: [1] An antibody molecule that binds to mesothelin (MSLN), wherein the antigen-binding site of the antibody molecule is an antibody: (i) FS28-256-271, as set forth in SEQ ID NOs: 98, 73, 99, 20, 21 and 44, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 10, 11, 41, 20, 21 and 22, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 98, 73, 99, 20, 21, and 34, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 98, 73, 99, 20, 21, and 25, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 101, 73, 103, 20, 21, and 34, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 98, 73, 99, 20, 21, and 43, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 101, 73, 103, 20, 21, and 43, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 98, 73, 99, 20, 21, and 44, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 85, 73, 75, 20, 21, and 34, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 85, 73, 75, 20, 21, and 43, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 111, 73, 113, 20, 21, and 25, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 101, 73, 103, 20, 21, and 25, respectively; (xiii) FS28-256, as set forth in SEQ ID NOs: 71, 73, 75, 20, 21, and 25, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 10, 11, 32, 20, 21, and 22, respectively; (xv) FS28-024-053, as set forth in SEQ ID NOs: 10, 11, 51, 20, 21, and 22, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 10, 11, 12, 20, 21, and 22, respectively. Complementarity determining regions (CDRs) 1 to 6 of Here, the CDR sequences are defined according to the ImMunoGeneTics (IMGT) numbering scheme of the antibody molecule.

[0022] [2] An antibody molecule that binds to MSLN, the antigen-binding site of the molecule being an antibody: (i) FS28-256-271, as set forth in SEQ ID NOs: 97, 182, 100, 23, 24 and 44, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 13, 14, 42, 23, 24 and 22, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 97, 74, 100, 23, 24, and 34, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 97, 74, 100, 23, 24, and 25, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 102, 74, 104, 23, 24, and 34, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 97, 74, 100, 23, 24, and 43, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 102, 74, 104, 23, 24, and 43, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 97, 74, 100, 23, 24, and 44, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 86, 74, 76, 23, 24, and 34, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 86, 74, 76, 23, 24, and 43, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 112, 74, 114, 23, 24, and 25, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 102, 74, 104, 23, 24, and 25, respectively; (xiii) FS28-256, as set forth in SEQ ID NOs: 72, 74, 76, 23, 24, and 25, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 13, 14, 33, 23, 24, and 22, respectively; (xiv) FS28-024-053, as set forth in SEQ ID NOs: 13, 14, 52, 23, 24, and 22, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 13, 14, 15, 23, 24, and 22, respectively. comprising CDRs 1 to 6 of wherein the CDR sequences are defined according to the Kabat numbering scheme, for an antibody molecule.

[0023] [3] The antibody molecule according to [1] or [2], comprising a heavy chain variable (VH) domain and / or a light chain variable (VL) domain, preferably a VH domain and a VL domain.

[0024] [4] The antibody molecule according to any one of [1] to [3], comprising an immunoglobulin heavy chain and / or an immunoglobulin light chain, preferably an immunoglobulin heavy chain and an immunoglobulin light chain.

[0025] [5] Antibodies: (i) FS28-256-271, as set forth in SEQ ID NOs: 180 and 56, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 39 and 18, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 109 and 93, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 109 and 79, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 121 and 93, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 109 and 53, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 121 and 53, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 109 and 56, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 63 and 93, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 63 and 53, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 115 and 79, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 121 and 79, respectively; (xiii) FS28-256, as set forth in SEQ ID NOs: 69 and 79, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 30 and 18, respectively; (xv) FS28-024-053, as set forth in SEQ ID NOs: 49 and 18, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 8 and 18, respectively. An antibody molecule according to any one of [3] to [4], comprising a VH domain and / or a VL domain, preferably a VH domain and a VL domain.

[0026] [6] Antibodies: (i) FS28-256-271, as set forth in SEQ ID NOs: 176 and 95, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 35 and 16, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 105 and 83, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 105 and 77, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 123 and 83, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 105 and 90, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 123 and 90, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 105 and 95, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 81 and 83, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 87 and 90, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 117 and 77, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 123 and 77, respectively; (xiii) FS28-256, as set forth in SEQ ID NOs: 65 and 77, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 26 and 16, respectively; (xv) FS28-024-053, as set forth in SEQ ID NOs: 45 and 16, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 4 and 16, respectively. An antibody molecule described in any one of [1] to [5], comprising a heavy chain [not having LALA] and a light chain.

[0027] [7] Antibodies: (i) FS28-256-271, as set forth in SEQ ID NOs: 178 and 95, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 37 and 16, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 107 and 83, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 107 and 77, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 125 and 83, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 107 and 90, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 125 and 90, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 107 and 95, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 89 and 83, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 89 and 90, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 119 and 77, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 125 and 77, respectively; (xiii) FS28-256, as set forth in SEQ ID NOs: 67 and 77, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 28 and 16, respectively; (xv) FS28-024-053, as set forth in SEQ ID NOs: 47 and 16, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 5 and 16, respectively. An antibody molecule described in any one of [1] to [5], comprising a heavy chain [having LALA] and a light chain.

[0028] [8] The antibody molecule according to any one of [1] to [7], comprising CDRs 1 to 6, a VH domain, a VL domain, a light chain and / or a heavy chain of antibody FS28-256-271.

[0029] [9] The antibody molecule according to any one of [1] to [7], comprising CDRs 1 to 6, a VH domain, a VL domain, a light chain and / or a heavy chain of antibody FS28-024-052.

[0030]

[10] An antibody molecule described in any one of [1] to [9], wherein MSLN is MSLN bound to a cell surface.

[0031]

[11] An antibody molecule described in

[10] that binds to immobilized MSLN with higher affinity than MSLN in solution.

[0032]

[12] The antibody molecule described in

[11] , which binds to immobilized MSLN with an affinity (kD) of 8 nM or higher.

[0033]

[13] An antibody molecule described in

[11] or

[12] that binds to MSLN in solution with an affinity (kD) of 15 nM or lower.

[0034]

[14] An antibody molecule described in any one of [1] to

[13] , wherein MSLN is human MSLN.

[0035]

[15] MSLN is an antibody molecule described in

[14] , which consists of or comprises the sequence set forth in SEQ ID NO: 169.

[0036]

[16] The antibody molecule according to any one of [1] to

[13] , wherein MSLN is cynomolgus monkey MSLN.

[0037]

[17] MSLN is an antibody molecule described in

[16] , which consists of or comprises the sequence set forth in SEQ ID NO: 170.

[0038]

[18] The antibody molecule of any one of [1] to

[17] , comprising CDRs 1 to 6, a VH domain, a VL domain, a light chain and / or a heavy chain of antibody FS28-024-051, FS28-024-052, FS28-024-053 or FS28-024, wherein the antibody blocks binding of MUC16 to MSLN.

[0039]

[19] The antibody molecule of any one of [1] to

[17] , comprising CDRs 1 to 6, a VH domain, a VL domain, a light chain and / or a heavy chain of antibody FS28-256-271, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026 or FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018 or FS28-256, wherein the antibody does not block binding of MUC16 to MSLN.

[0040]

[20] The antibody molecule according to

[18] or

[19] , wherein MUC16 is human MUC16.

[0041]

[21] The antibody molecule of any one of [1] to

[20] , which is a multispecific antibody molecule and contains a second antigen-binding site that binds to a second antigen.

[0042]

[22] The antibody molecule described in

[21] , which is a bispecific, trispecific or tetraspecific antibody molecule.

[0043]

[23] The antibody molecule described in

[22] , which is a bispecific molecule.

[0044]

[24] The antibody molecule of any one of

[21] to

[23] , wherein the second antigen-binding site is located in a constant domain of the antibody molecule.

[0045]

[25] The antibody molecule according to any one of

[21] to

[24] , wherein the second antigen is an immune cell antigen.

[0046]

[26] The antibody molecule described in

[25] , wherein the immune cell antigen is a member of the tumor necrosis factor receptor superfamily (TNFRSF).

[0047]

[27] The antibody molecule described in

[26] , wherein the member of the TNFRSF is OX40 or CD137.

[0048]

[28] The antibody molecule according to any one of

[21] to

[27] , wherein the second antigen-binding site comprises a first sequence, a second sequence and / or a third sequence, wherein the first sequence, the second sequence and the third sequence are located in the AB structural loop, the CD structural loop and the EF structural loop of the constant domain, respectively.

[0049]

[29] An antibody molecule described in any one of

[24] to

[28] , wherein the constant domain is a CH3 domain.

[0050]

[30] An antibody molecule described in any one of

[26] to

[29] , which is capable of activating a TNFRSF member on an immune cell in the presence of MSLN bound to the tumor cell surface.

[0051]

[31] An antibody molecule described in any one of

[26] to

[30] , wherein binding of the antibody molecule to TNFRSF members and MSLN bound to the tumor cell surface causes clustering of TNFRSF members on the immune cell surface.

[0052]

[32] The antibody molecule according to

[30] or

[31] , wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell or a dendritic cell (DC).

[0053]

[33] The antibody molecule described in

[32] , wherein the immune cell is a T cell.

[0054]

[34] An antibody molecule described in any one of [1] to

[33] , which has antibody-dependent cellular cytotoxicity (ADCC) or is capable of inducing antibody-dependent cellular cytotoxicity (ADCC).

[0055]

[35] An antibody molecule described in any one of [1] to

[33] , which is modified to reduce or inhibit binding of the CH2 domain of the antibody molecule to one or more Fcγ receptors.

[0056]

[36] An antibody molecule according to any one of [1] to

[33] or

[35] , which does not bind to an Fcγ receptor.

[0057]

[37] The antibody molecule according to

[35] or

[36] , wherein the Fcγ receptor is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb and FcγRIII.

[0058]

[38] A conjugate comprising an antibody molecule according to any one of [1] to

[37] and a biologically active molecule.

[0059]

[39] A conjugate comprising an antibody molecule described in any one of [1] to

[37] and a detectable label.

[0060]

[40] One or more nucleic acid molecules encoding an antibody molecule described in any one of [1] to

[37] .

[0061]

[41] Antibodies: (i) FS28-256-271, as set forth in SEQ ID NOs: 181 and 57, respectively; (ii) FS28-024-052, as set forth in SEQ ID NOs: 40 and 19, respectively; (iii) FS28-256-021, as set forth in SEQ ID NOs: 110 and 94, respectively; (iv) FS28-256-012, as set forth in SEQ ID NOs: 110 and 80, respectively; (v) FS28-256-023, as set forth in SEQ ID NOs: 122 and 94, respectively; (vi) FS28-256-024, as set forth in SEQ ID NOs: 110 and 54, respectively; (vii) FS28-256-026, as set forth in SEQ ID NOs: 122 and 54, respectively; (viii) FS28-256-027, as set forth in SEQ ID NOs: 110 and 57, respectively; (ix) FS28-256-001, as set forth in SEQ ID NOs: 64 and 94, respectively; (x) FS28-256-005, as set forth in SEQ ID NOs: 64 and 54, respectively; (xi) FS28-256-014, as set forth in SEQ ID NOs: 116 and 80, respectively; (xii) FS28-256-018, as set forth in SEQ ID NOs: 122 and 80, respectively; (xiiii) FS28-256, as set forth in SEQ ID NOs: 70 and 80, respectively; (xiv) FS28-024-051, as set forth in SEQ ID NOs: 31 and 19, respectively; (xv) FS28-024-053, as set forth in SEQ ID NOs: 50 and 19, respectively; or (xvi) FS28-024, as set forth in SEQ ID NOs: 9 and 19, respectively. The nucleic acid molecule according to

[40] , comprising a VH domain and / or VL domain nucleic acid sequence.

[0062]

[42] (i) the heavy chain nucleic acid sequence of antibody FS28-256-271 set forth in SEQ ID NO: 179 or 177 and / or the light chain nucleic acid sequence of antibody FS28-256-271 set forth in SEQ ID NO: 96; (ii) the heavy chain nucleic acid sequence of antibody FS28-024-052 set forth in SEQ ID NO: 38 or 36 and / or the light chain nucleic acid sequence of antibody FS28-024-052 set forth in SEQ ID NO: 17; (iii) the heavy chain nucleic acid sequence of antibody FS28-256-021 set forth in SEQ ID NO: 108 or 106 and / or the light chain nucleic acid sequence of antibody FS28-256-021 set forth in SEQ ID NO: 92; (iv) the heavy chain nucleic acid sequence of antibody FS28-256-012 set forth in SEQ ID NO: 108 or 106 and / or the light chain nucleic acid sequence of antibody FS28-256-012 set forth in SEQ ID NO: 78; (v) the heavy chain nucleic acid sequence of antibody FS28-256-023 set forth in SEQ ID NO: 126 or 124 and / or the light chain nucleic acid sequence of antibody FS28-256-023 set forth in SEQ ID NO: 92; (vi) the heavy chain nucleic acid sequence of antibody FS28-256-024 set forth in SEQ ID NO: 108 or 106 and / or the light chain nucleic acid sequence of antibody FS28-256-024 set forth in SEQ ID NO: 91; (vii) the heavy chain nucleic acid sequence of antibody FS28-256-026 set forth in SEQ ID NO: 126 or 124 and / or the light chain nucleic acid sequence of antibody FS28-256-026 set forth in SEQ ID NO: 91; (viii) the heavy chain nucleic acid sequence of antibody FS28-256-027 set forth in SEQ ID NO: 108 or 106 and / or the light chain nucleic acid sequence of antibody FS28-256-027 set forth in SEQ ID NO: 96; (ix) the heavy chain nucleic acid sequence of antibody FS28-256-001 set forth in SEQ ID NO: 84 or 82 and / or the light chain nucleic acid sequence of antibody FS28-256-001 set forth in SEQ ID NO: 92; (x) the heavy chain nucleic acid sequence of antibody FS28-256-005 set forth in SEQ ID NO: 84 or 88 and / or the light chain nucleic acid sequence of antibody FS28-256-005 set forth in SEQ ID NO: 91; (xi) the heavy chain nucleic acid sequence of antibody FS28-256-014 set forth in SEQ ID NO: 120 or 118 and / or the light chain nucleic acid sequence of antibody FS28-256-014 set forth in SEQ ID NO: 78; (xii) the heavy chain nucleic acid sequence of antibody FS28-256-018 set forth in SEQ ID NO: 126 or 124 and / or the light chain nucleic acid sequence of antibody FS28-256-018 set forth in SEQ ID NO: 78; (xiii) the heavy chain nucleic acid sequence of antibody FS28-256 set forth in SEQ ID NO: 68 or 66 and / or the light chain nucleic acid sequence of antibody FS28-256 set forth in SEQ ID NO: 78; (xiv) the heavy chain nucleic acid sequence of antibody FS28-024-051 set forth in SEQ ID NO: 29 or 27 and / or the light chain nucleic acid sequence of antibody FS28-024-051 set forth in SEQ ID NO: 17; (xv) the heavy chain nucleic acid sequence of antibody FS28-024-053 set forth in SEQ ID NO: 48 or 46 and / or the light chain nucleic acid sequence of antibody FS28-024-053 set forth in SEQ ID NO: 17; or (xvi) the heavy chain nucleic acid sequence of antibody FS28-024 as set forth in SEQ ID NO: 7 or 6 and / or the light chain nucleic acid sequence of antibody FS28-024 as set forth in SEQ ID NO: 17 The nucleic acid molecule according to

[40] or

[41] ,

[0063]

[43] One or more vectors comprising one or more nucleic acid molecules according to any one of

[40] to

[42] .

[0064]

[44] A recombinant host cell comprising a nucleic acid molecule according to any one of

[40] to

[42] or a vector according to

[43] .

[0065]

[45] A method for producing an antibody molecule described in any one of [1] to

[37] , comprising culturing a recombinant host cell described in

[44] under conditions for the production of the antibody molecule.

[0066]

[46] The method according to

[45] , further comprising isolating and / or purifying the antibody molecule.

[0067]

[47] A pharmaceutical composition comprising the antibody molecule or conjugate according to any one of [1] to

[39] and a pharma- ceutical acceptable excipient.

[0068]

[48] ​​An antibody molecule or conjugate according to any one of [1] to

[39] for use in a method for treating cancer in an individual.

[0069]

[49] A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody molecule or conjugate described in any one of [1] to

[39] .

[0070]

[50] Use of an antibody molecule or conjugate according to any one of [1] to

[39] in the preparation of a medicament for the treatment of cancer.

[0071]

[51] The antibody molecule or conjugate, method or use according to any one of

[48] to

[50] , wherein cancer cells express MSLN on their cell surface.

[0072]

[52] The antibody molecule or conjugate, method or use according to any one of

[48] to

[50] , wherein the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer and lung cancer.

[0073]

[53] The antibody molecule or conjugate for use according to

[48] , wherein the method of treatment comprises administering the antibody molecule or conjugate to an individual in combination with a second therapeutic agent.

[0074]

[54] The method of

[49] , further comprising administering to the individual a therapeutically effective amount of a second therapeutic agent.

[0075]

[55] An antibody molecule or conjugate according to any one of [1] to

[37] or

[39] for use in a method for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual.

[0076]

[56] A method for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual, the method comprising the use of an antibody molecule or conjugate described in any one of [1] to

[37] or

[39] .

[0077]

[57] Use of an antibody molecule or conjugate according to any one of [1] to

[37] or

[39] in the manufacture of a diagnostic agent for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual.

[0078]

[58] A kit for use in a method for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual, comprising an antibody molecule or conjugate according to any one of [1] to

[37] or

[39] .

[0079]

[59] The antibody molecule or conjugate, method, use or kit for use according to any one of

[55] to

[58] , wherein the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer and lung cancer. [Brief description of the drawings]

[0080] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Binding of anti-MSLN mAbs to NCI-H226 cells is shown. Anti-human MSLN mAbs FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256 (all in IgG1 LALA format) showed dose-dependent binding to NCI-H226 cells. FS28-024, FS28-026 and FS28-091 showed high affinity binding to cell surface MSLN with low nanomolar EC50 values, similar to the positive control anti-MSLN mAb SS1. mAbs FS28-185 and FS28-256 showed weaker binding to cell surface MSLN with EC50 values ​​above 30 nM and lower Emax values ​​compared to FS28-024, FS28-026 and FS28-091. [Diagram 2]Figure 1 shows the ADCC activity of anti-MSLN mAb2. Anti-human MSLN mAb2 FS28-024-052 and FS28-256-271 as well as the positive control antibody SS1 were tested in hIgG1 LALA format (no effector function) and in the effector-competent backbone hIgG1. FS28-024-052 has the highest ADCC activity of the antibodies tested, and FS28-256-271 has ADCC activity comparable to the control antibody. In all cases, introduction of the LALA mutation into the IgG1 backbone completely abrogated ADCC activity as expected. [Diagram 3] Individual tumor volume measurements are shown in CT26.G10 syngeneic tumor models treated with (A) G1-AA / HelD1.3 (human IgG1 control), (B) FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in mAb2 format), (C) FS22m-063-AA / HelD1.3 in combination with G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN Fab), (D) FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2) (E) FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in mAb2 format), and (F) G1-AA / FS28m-228-010 (anti-mouse MSLN Fab). FS22m-063-AA / FS28m-228-010 showed reduced tumor growth compared to the isotype control as well as the other treatment groups. [Figure 4]Kaplan-Meier survival curves for CT26.G10 syngeneic tumor models treated with G1-AA / HelD1.3 (IgG control), FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in mAb2 format), FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in mAb2 format), G1-AA / FS28m-228-010 (anti-mouse MSLN Fab), a combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN Fab) and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2). The results show that FS22m-063-AA / FS28m-228-010 treatment resulted in significantly improved survival compared to mice treated with isotype control or other treatment groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Detailed Description The present invention relates to an antibody molecule that binds to MSLN. The antibody molecule preferably binds to human MSLN, more preferably to human and cynomolgus MSLN. The antibody molecule of the present invention is preferably capable of binding to MSLN expressed on the surface of a cell. The cell is preferably a tumor cell.

[0082] The antibody molecule preferably specifically binds to MSLN. The term "specific" may refer to the situation where the antibody molecule does not show significant binding to molecules other than its specific binding partner (here MSLN). The term "specific" also applies when the antibody molecule is specific for a particular epitope, such as an epitope on MSLN carried by several antigens, in which case the antibody molecule will be able to bind to various antigens carrying the epitope. The antibody molecule preferably does not bind or does not show significant binding to other molecules involved in cell adhesion, such as CEACAM-5, E-cadherin, thrombomodulin and / or EpCAM.

[0083] As explained in the background section above, mature MSLN is shed from tumor cells and cleared from the tumor site. This shed MSLN may act as a sink for anti-MSLN binding molecules, which then bind to the shed MSLN and are cleared from the tumor site. To select molecules that preferentially bind to MSLN present on the surface of tumor cells, we selected antibody molecules with high avidity for MSLN. Specifically, we selected antibody molecules that bound to immobilized MSLN with higher affinity than MSLN in solution. It is believed that antibody molecules that bind to MSLN with high avidity preferentially bind to MSLN present on tumor cells, where multiple copies of MSLN are expected to be present and available for bivalent binding by antibody molecules, in contrast to mesothelin shed from tumor cells, which is expected to be in a monomeric form. Thus, without wishing to be bound by theory, it is expected that the antibody molecules of the present invention will have a longer period of time to exert their therapeutic effect because they are slowly cleared from the tumor site.

[0084] The antibody molecule preferably binds to the immobilized MSLN with higher affinity than the MSLN in solution.The immobilized MSLN can be the MSLN immobilized on a surface, such as a chip for use in surface plasmon resonance.The MSLN in solution is also referred to herein as soluble MSLN and is not immobilized.The soluble MSLN is preferably in monomeric form, i.e., monomeric mesothelin.

[0085] The affinity of an antibody for its cognate antigen is determined by the equilibrium dissociation constant (K D ) can be expressed as K D The higher the value, the lower the affinity of the antibody molecule for the antigen.

[0086] The antibody molecule preferably has an affinity (K D ) or higher affinity to immobilized MSLN. Preferably, the antibody molecule binds to immobilized MSLN with an affinity (KD ) or binds with higher affinity to immobilized MSLN.

[0087] The antibody molecule preferably has an affinity (K D ) or lower affinity to immobilized MSLN. More preferably, the antibody molecule binds to immobilized MSLN with an affinity (K D ) or binds with lower affinity to immobilized MSLN.

[0088] In a preferred embodiment, the antibody molecule has an affinity (K D ) or higher affinity to immobilized MSLN, with an affinity of 18 nM (K D ) or binds with lower affinity to MSLN in solution.

[0089] The binding affinity of an antibody molecule to cells containing surface-bound MSLN is expressed as the half-maximal binding of the antibody molecule to the cells (EC 50 The half-maximal binding of an antibody molecule to a cell can be measured by determining the concentration of the antibody molecule required to achieve half-maximal binding (EC) of the antibody molecule to a cell. Suitable methods for determining the concentration of the antibody molecule required to achieve half-maximal binding of the antibody molecule to a cell are known in the art and are disclosed in the Examples of the present invention (see, for example, Example 4). As explained above, antibody molecules whose binding to tumor cells containing surface-bound MSLN is not affected or is less affected by the presence of soluble mesothelin are preferred in view of the presence of shed MSLN in the tumor environment. Thus, in a preferred embodiment, the half-maximal binding (EC) of an antibody to cells (e.g., tumor cells) containing surface-bound MSLN in the presence of soluble MSLN is measured. 50 ) is the concentration of antibody molecules required to achieve half-maximal binding of the antibody to the cells in the absence of soluble MSLN (EC 50 ) is less than 20-fold, less than 15-fold, less than 10-fold, less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, or less than 3-fold higher than the concentration of antibody molecules required to achieve the desired antibody titer.

[0090] It has been shown that the binding of antibody molecules that do not block MUC16 binding to MSLN to cells containing cell-bound MSLN is not significantly affected by the presence of soluble MSLN. Thus, antibody molecules that are not capable of or do not block MUC16 binding to MSLN may be preferred.

[0091] Alternatively, the antibody molecule may block binding of MUC16 to MSLN.

[0092] The immobilized MSLN may have the sequence set forth in SEQ ID NO: 169. The MSLN in solution may have the sequence set forth in SEQ ID NO: 169.

[0093] The antibody molecules of the invention have also been shown to bind to cynomolgus monkey MSLN. Binding to cynomolgus monkey MSLN as well as human MSLN may be useful for performing efficacy and toxicity testing of the antibody molecules in cynomolgus monkeys, which may be predictive of efficacy and toxicity of the antibody molecules in humans.

[0094] The antibody molecules may bind to immobilized human MSLN and immobilized cynomolgus MSLN with similar affinity. Furthermore, the antibody molecules may bind to human MSLN in solution and cynomolgus MSLN in solution with similar affinity. This may be beneficial in ensuring that efficacy and toxicity studies performed with the antibody molecules in cynomolgus monkeys are predictive of efficacy and toxicity of the antibody molecules in humans.

[0095] Thus, in a preferred embodiment, the antibody molecule binds to immobilized cynomolgus MSLN with an affinity that is at least 10-fold, preferably at least 5-fold, and more preferably at least 2-fold lower or higher than the affinity with which the antibody molecule binds to immobilized human MSLN. Furthermore, the antibody molecule preferably binds to cynomolgus MSLN in solution with an affinity that is at least 10-fold, preferably at least 5-fold, and more preferably at least 2-fold lower or higher than the affinity with which the antibody molecule binds to human MSLN in solution.

[0096] The binding affinity of antibody molecules to a cognate antigen, such as human or cynomolgus MSLN, can be determined, for example, by surface plasmon resonance (SPR), such as Biacore.

[0097] The antibody molecule may be a chimeric, humanized or human antibody molecule. Preferably, the antibody molecule is a human antibody molecule.

[0098] The antibody molecules are preferably monoclonal.

[0099] The antibody molecule may be isolated in the sense of being free of contaminating substances, such as antibodies capable of binding to other polypeptides and / or serum components.

[0100] The antibody molecule may be natural or partly or wholly synthetically produced, for example, the antibody molecule may be a recombinant antibody molecule.

[0101] The antibody molecule comprises one or more CDR-based antigen-binding sites for MSLN.

[0102] The antibody molecule may be an immunoglobulin or an antigen-binding fragment thereof. For example, the antibody molecule may be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule, more preferably an IgG1 or IgG2 molecule, most preferably an IgG1 molecule or a fragment thereof. In a preferred embodiment, the antibody molecule is a complete immunoglobulin molecule.

[0103] In other embodiments, the antibody molecule can be an antigen-binding fragment that comprises a CDR-based antigen-binding site for MSLN. For example, the antigen-binding fragment can be a fragment antigen-binding (Fab), IgGΔCH2, F(ab')2, single chain Fab (scFab), disulfide stabilized variable fragment (dsFv), single chain variable fragment (scFv), (scFv)2, scFv-CH3 (minibody), scFv-Fc, scFv-zipper, diabody, triabody, tetrabody, or single domain antibody (sdAb), such as V HThe antigen-binding fragment may be an H domain or a nanobody. Preferred antigen-binding fragments include two or more CDR-based antigen-binding sites for MSLN, i.e., they may be multivalent. Thus, the antigen-binding fragment may preferably be an IgGΔCH2, F(ab')2, diabody, triabody or tetrabody (Brinkmann and Kontermann, (2017) and Powers et al, (2012)).

[0104] Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger and Hudson, 2005. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing the CDRs or variable regions of one antibody molecule into a different antibody molecule (EP-A-184187, GB-A-2188638A and EP-A-239400).

[0105] A CDR-based antigen-binding site is an antigen-binding site in an antibody variable region. A CDR-based antigen-binding site can be formed by three CDRs, for example three light chain variable domain (VL) CDRs or three heavy chain variable domain (VH) CDRs. Preferably, a CDR-based antigen-binding site is formed by six CDRs, three VL CDRs and three VH CDRs. The contribution of different CDRs to the binding of antigen can be different in different antigen-binding sites.

[0106] The three VH domain CDRs of an antigen-binding site can be located within an immunoglobulin VH domain, and the three VL domain CDRs can be located within an immunoglobulin VL domain. For example, a CDR-based antigen-binding site can be located in an antibody variable region.

[0107] An antibody molecule has one or preferably two or more, e.g., two, CDR-based antigen-binding sites for MSLN. Thus, an antibody molecule may contain one VH and one VL domain, but preferably contains two VH and two VL domains, i.e., two VH / VL domain pairs, e.g., as in a natural IgG molecule.

[0108] The CDR-based antigen binding site comprises the three VH CDRs or the three VL CDRs of antibody FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably antibody FS28-256-271 or FS28-024-052, most preferably antibody FS28-256-271. It may comprise CDRs, preferably three VH CDRs and three VL CDRs.

[0109] The sequences of the CDRs can be readily determined from the VH and VL domain sequences of the antibody molecule using routine techniques. The VH and VL domain sequences of antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 are described herein and therefore the three VH and three VL domain CDRs of said antibodies may be determined from said sequences. CDR sequences can be determined, for example, according to Kabat et al., 1991 or the International Immunogenetics Information System (IMGT) (Lefranc et al., 2015).

[0110] The VH domain CDR1, CDR2 and CDR3 sequences of an antibody molecule according to the IMGT numbering may be sequences located at positions 27 to 38, 56 to 65 and 105 to 117, respectively, of the VH domain of the antibody molecule.

[0111] The VH domain CDR1, CDR2 and CDR3 sequences of an antibody molecule according to the Kabat numbering may be the sequences located at positions 31 to 35, 50 to 65 and 95 to 102 of the VH domain, respectively.

[0112] The VL domain CDR1, CDR2 and CDR3 sequences of an antibody molecule according to the IMGT numbering may be sequences located at positions 27 to 38, 56 to 65 and 105 to 117 of the VL domain, respectively.

[0113] The VL domain CDR1, CDR2 and CDR3 sequences of an antibody molecule according to the Kabat numbering may be the sequences located at positions 24 to 34, 50 to 56 and 89 to 97 of the VL domain, respectively.

[0114] for example, (i) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-271 may be set forth in SEQ ID NOs: 98, 73 and 99, respectively; (ii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-052 may be set forth in SEQ ID NOs: 10, 11 and 41, respectively; (iii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-021 may be set forth in SEQ ID NOs: 98, 73 and 99, respectively; (iv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-012 may be set forth in SEQ ID NOs: 98, 73 and 99, respectively; (v) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-023 may be set forth in SEQ ID NOs: 101, 73 and 103, respectively; (vi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-024 may be set forth in SEQ ID NOs: 98, 73 and 99, respectively; (vii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-026 may be set forth in SEQ ID NOs: 101, 73 and 103, respectively; (viii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-027 may be set forth in SEQ ID NOs: 98, 73 and 99, respectively; (ix) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-001 may be set forth in SEQ ID NOs: 85, 73 and 75, respectively; (x) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-005 may be set forth in SEQ ID NOs: 85, 73 and 75, respectively; (xi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-014 may be set forth in SEQ ID NOs: 111, 73 and 113, respectively; (xii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-018 may be set forth in SEQ ID NOs: 101, 73 and 103, respectively; (xiii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256 may be set forth in SEQ ID NOs: 71, 73 and 75, respectively; (xiv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-051 may be set forth in SEQ ID NOs: 10, 11 and 32, respectively; (xv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-053 may be set forth in SEQ ID NOs: 10, 11 and 51, respectively; (xvi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024 may be set forth in SEQ ID NOs: 10, 11 and 12, respectively; Herein, the CDR sequences are defined according to the IMGT numbering scheme.

[0115] (i) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-271 may be set out in SEQ ID NOs: 20, 21 and 44, respectively; (ii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-052 may be set out in SEQ ID NOs: 20, 21 and 22, respectively; (iii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-021 may be set forth in SEQ ID NOs: 20, 21 and 34, respectively; (iv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-012 may be set forth in SEQ ID NOs: 20, 21 and 25, respectively; (v) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-023 may be set forth in SEQ ID NOs: 20, 21 and 34, respectively; (vi) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-024 may be set forth in SEQ ID NOs: 20, 21 and 43, respectively; (vii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-026 may be set forth in SEQ ID NOs: 20, 21 and 43, respectively; (viii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-027 may be set forth in SEQ ID NOs: 20, 21 and 44, respectively; (ix) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-001 may be set forth in SEQ ID NOs: 20, 21 and 34, respectively; (x) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-005 may be set forth in SEQ ID NOs: 20, 21 and 43, respectively; (xi) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-014 may be set forth in SEQ ID NOs: 20, 21 and 25, respectively; (xii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-018 may be set forth in SEQ ID NOs: 20, 21 and 25, respectively; (xiii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256 may be set forth in SEQ ID NOs: 20, 21 and 25, respectively; (xiv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-051 may be set forth in SEQ ID NOs: 20, 21 and 22, respectively; (xv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-053 may be set forth in SEQ ID NOs: 20, 21 and 22, respectively; (xvi) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024 may be set forth in SEQ ID NOs: 20, 21 and 22, respectively; Herein, the CDR sequences are defined according to the IMGT numbering scheme.

[0116] for example, (i) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-271 may be set forth in SEQ ID NOs: 97, 182 and 100, respectively; (ii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-052 may be set forth in SEQ ID NOs: 13, 14 and 42, respectively; (iii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-021 may be set forth in SEQ ID NOs: 97, 74 and 100, respectively; (iv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-012 may be set forth in SEQ ID NOs: 97, 74 and 100, respectively; (v) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-023 may be set forth in SEQ ID NOs: 102, 74 and 104, respectively; (vi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-024 may be set forth in SEQ ID NOs: 97, 74 and 100, respectively; (vii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-026 may be set forth in SEQ ID NOs: 102, 74 and 104, respectively; (viii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-027 may be set forth in SEQ ID NOs: 97, 74 and 100, respectively; (ix) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-001 may be set forth in SEQ ID NOs: 86, 74 and 76, respectively; (x) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-005 may be set forth in SEQ ID NOs: 86, 74 and 76, respectively; (xi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-014 may be set forth in SEQ ID NOs: 112, 74 and 114, respectively; (xii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256-018 may be set forth in SEQ ID NOs: 102, 74 and 104, respectively; (xiii) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-256 may be set forth in SEQ ID NOs: 72, 74 and 76, respectively; (xiv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-051 may be set forth in SEQ ID NOs: 13, 14 and 33, respectively; (xv) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024-053 may be set forth in SEQ ID NOs: 13, 14 and 52, respectively; (xvi) the sequences of the VH domain CDR1, CDR2 and CDR3 of FS28-024 may be set forth in SEQ ID NOs: 13, 14 and 15, respectively; Herein, the CDR sequences are defined according to the Kabat numbering scheme.

[0117] (i) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-271 may be set out in SEQ ID NOs: 23, 24 and 44, respectively; (ii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-052 may be set out in SEQ ID NOs: 23, 24 and 22, respectively; (iii) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-021 may be set forth in SEQ ID NOs: 23, 24 and 34, respectively; (iv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-012 may be set forth in SEQ ID NOs: 23, 24 and 25, respectively; (v) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-256-023 may be set forth in SEQ ID NOs: 23, 24 and 34, respectively; (vi) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-024 may be set forth in SEQ ID NOs: 23, 24 and 43, respectively; (vii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-026 may be set forth in SEQ ID NOs: 23, 24 and 43, respectively; (viii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-027 may be set forth in SEQ ID NOs: 23, 24 and 44, respectively; (ix) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-001 may be set forth in SEQ ID NOs: 23, 24 and 34, respectively; (x) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-005 may be set forth in SEQ ID NOs: 23, 24 and 43, respectively; (xi) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-014 may be set forth in SEQ ID NOs: 23, 24 and 25, respectively; (xii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256-018 may be set forth in SEQ ID NOs: 23, 24 and 25, respectively; (xiii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-256 may be set forth in SEQ ID NOs: 23, 24 and 25, respectively; (xiv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-051 may be set forth in SEQ ID NOs: 23, 24 and 22, respectively; (xv) the sequences of the VL domains CDR1, CDR2 and CDR3 of FS28-024-053 may be set forth in SEQ ID NOs: 23, 24 and 22, respectively; (xvi) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS28-024 may be set forth in SEQ ID NOs: 23, 24 and 22, respectively; Herein, the CDR sequences are defined according to the Kabat numbering scheme.

[0118] The CDR-based antigen-binding sites are expressed in the antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-2 Preferably the VH or VL domain, preferably the VH and VL domains, of antibody FS28-256-271 or FS28-024-052, most preferably antibody FS28-256-271.

[0119] Antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28- 256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014 , FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 may have the sequences set forth in SEQ ID NOs: 180, 39, 109, 109, 121, 109, 121, 109, 63, 63, 115, 121, 69, 30, 49 and 8, respectively.

[0120] Antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS2 8-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256 The VL domains of FS28-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 may have the sequences set out in SEQ ID NOs: 56, 18, 93, 79, 93, 53, 53, 56, 93, 53, 79, 79, 79, 18, 18 and 18, respectively.

[0121] The antibody molecule may comprise the heavy or light chain, preferably the heavy and light chains, of antibody FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably antibody FS28-256-271 or FS28-024-052, most preferably antibody FS28-256-271.

[0122] Antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-25 6-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28 The heavy chains of FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 (with the LALA mutation) may have the sequences set forth in SEQ ID NOs: 178, 37, 107, 107, 125, 107, 125, 107, 89, 89, 119, 125, 67, 28, 47, 5, respectively.

[0123] Antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256 -024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28- The heavy chains of 256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 (without the LALA mutation) may have the sequences set forth in SEQ ID NOs: 176, 35, 105, 105, 123, 105, 123, 105, 81, 87, 117, 123, 65, 26, 45 and 4, respectively.

[0124] The light chain of antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 may have the sequence set forth in SEQ ID NOs: 95, 16, 83, 77, 83, 90, 90, 95, 83, 90, 77, 77, 77, 16, 16 and 16, respectively.

[0125] The antibody molecule may also comprise variants of the CDR, VH domain, VL domain, heavy or light chain sequences disclosed herein. Suitable variants can be obtained by methods of sequence variation or mutation and screening. In a preferred embodiment, the antibody molecule comprising one or more such variant sequences retains one or more functional properties of the parent antibody molecule, such as binding specificity and / or binding affinity to MSLN, preferably human and / or cynomolgus MSLN. For example, the antibody molecule comprising one or more variant sequences preferably binds to MSLN with the same affinity or higher affinity than the (parent) antibody molecule. The parent antibody molecule is an antibody molecule that does not contain the amino acid substitutions, deletions and / or insertions incorporated in the variant antibody molecule.

[0126] Antibody molecules comprising CDRs 1-6, a VH domain and / or a heavy chain of antibody FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018 or FS28-256 may comprise an amino acid substitution at position 55 or 57 of the VH domain, wherein numbering of the amino acid residues is according to the IMGT numbering scheme.

[0127] For example, the antibody molecule may comprise CDRs 1-6, a VH domain and / or a heavy chain of antibody FS28-256-027, wherein the antibody molecule comprises an amino acid substitution at position 55 of the VH domain, wherein numbering of the amino acid residues is according to the IMGT numbering scheme.

[0128] The antibody molecule of the present invention may be the antibody FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably the antibody FS28-256-271 or FS28-024-052, most preferably the antibody FS2 The VH domain, VL domain, heavy chain or light chain may comprise a VH domain, VL domain, heavy chain or light chain having at least 70%, at least 75%, 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 VH domain, VL domain, heavy chain or light chain of 8-256-271.

[0129] Sequence identity is generally defined in relation to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximize the number of matches and minimize the number of gaps. Generally, default parameters are used, with a gap creation penalty equal to 12 and a gap extension penalty equal to 4. Although the use of GAP may be preferred, other algorithms may be used, such as BLAST (using the method of Altschul et al., 1990), FASTA (using the method of Pearson and Lipman, 1988), generally using default parameters, or the Smith-Waterman algorithm (Smith and Waterman, 1981), or the TBLASTN program of Altschul et al., 1990, supra. In particular, the psi-Blast algorithm (Altschul et al., 1997) may be used.

[0130] The antibody molecule may comprise the VH CDR1, VH CDR2, VH CDR3, VH CDR4, VH CDR5, VH CDR6, VH CDR7, VH CDR8, VH CDR9, VH CDR10, VH CDR11, VH CDR12, VH CDR13, VH CDR14, VH CDR15, VH CDR16, VH CDR17, VH CDR18, VH CDR19 ...9, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH CDR19, VH C The VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 may comprise one or more amino acid sequence mutations (addition, deletion, substitution and / or insertion of amino acid residues) compared to VH CDR1, VL CDR2 and / or VL CDR3, preferably no more than three mutations, no more than two mutations or one mutation.

[0131] The antibody molecule may be the antibody FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably may comprise a VH domain, VL domain, heavy chain or light chain which has one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues), preferably no more than 20 mutations, no more than 15 mutations, no more than 10 mutations, no more than 5 mutations, no more than 4 mutations, no more than 3 mutations, no more than 2 mutations or 1 mutation, compared to the VH domain, VL domain, heavy chain or light chain of antibody FS28-256-271 or FS28-024-052, most preferably antibody FS28-256-271. In particular, the amino acid sequence alterations may be located in one or more framework regions of the antibody molecule, such as one or more framework regions of the heavy and / or light chain of the antibody molecule.

[0132] In preferred embodiments where one or more amino acids are replaced with another amino acid, the replacement may be a conservative replacement, for example as represented in the table below: In some embodiments, amino acids of the same category in the middle column are replaced with each other, i.e., a non-polar amino acid is replaced with, for example, another non-polar amino acid. In some embodiments, amino acids in the same row in the right-most column are replaced with each other.

[0133] [Table 1]

[0134] In certain embodiments, substitutions can be functionally conservative, i.e., in certain embodiments, the substitution does not affect (or does not substantially affect) one or more functional properties (e.g., binding affinity) of an antibody molecule that contains the substitution, compared to a comparable unsubstituted antibody molecule.

[0135] The heavy chain of the antibody molecule may optionally include an additional lysine residue (K) immediately adjacent to the C-terminus of the heavy chain CH3 domain sequence.

[0136] The CH2 domain is known to bind to Fcγ receptors and complement. Binding of the CH2 domain to Fcγ receptors is required for antibody-dependent cell-mediated cytotoxicity (ADCC), while binding to complement is required for complement-dependent cytotoxicity (CDC). When the antibody molecule is used to treat cancer and is not in the form of an ADC, ADCC activity is expected to result in the killing of cancer cells, and therefore should preferably be retained. However, when the antibody molecule is in the form of an ADC and conjugated to a biologically active molecule, mutations that reduce or suppress ADCC activity are expected to be beneficial in avoiding the killing of target cancer cells via ADCC before the biologically active molecule is delivered to the cell. Furthermore, mutations that reduce or suppress ADCC and / or CDC activity are expected to be beneficial when the antibody molecule contains a second antigen-binding site for an immune cell antigen as described herein, in which case ADCC and / or CDC-mediated killing of immune cells bound by the antibody molecule should be avoided.

[0137] Thus, the CH2 domain of the antibody molecule may comprise one or more mutations that reduce or inhibit the binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγRlla, FcγRllb, FcγRIII, and / or complement. The inventors hypothesize that reducing or inhibiting the binding to Fcγ receptors reduces or eliminates ADCC mediated by the antibody molecule. Similarly, reducing or inhibiting the binding to complement is expected to reduce or eliminate CDC mediated by the antibody molecule. Mutations that reduce or inhibit the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). These mutations include the "LALA mutation" described in Bruhns et al., 2009 and Hezareh et al., 2001, which comprises the replacement of leucine residues at positions 1.3 and 1.2 of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, the generation of a-glycosyl antibodies through mutation of the conserved N-linked glycosylation site by mutating the asparagine (N) at position 84.4 of the CH2 domain to alanine, glycine or glutamine (N84.4A, N84.4G or N84.4Q) is also known to reduce IgG1 effector function (Wang et al., 2018). As a further alternative, complement activation (C1q binding) and ADCC are known to be reduced by mutation of the proline at position 114 of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can also be combined to generate antibody molecules with further reduced or no ADCC or CDC activity.

[0138] Thus, the antibody molecule may comprise a CH2 domain, wherein the CH2 domain preferably comprises (i) alanine residues at positions 1.3 and 1.2; and / or (ii) alanine or glycine at position 114; and / or (iii) alanine, glutamine or glycine at position 84.4 Includes; Here, the numbering of amino acid residues follows the IMGT numbering scheme.

[0139] In a preferred embodiment, the antibody molecule comprises a CH2 domain, wherein the CH2 domain comprises: (i) an alanine residue at position 1.3; and (ii) an alanine residue at position 1.2 Includes; Here, the numbering of amino acid residues follows the IMGT numbering scheme.

[0140] For example, the CH2 domain may contain a LALA mutation and have the sequence set forth in SEQ ID NO:173.

[0141] In another preferred embodiment, the antibody molecule comprises a CH2 domain, wherein the CH2 domain comprises: (i) an alanine residue at position 1.3; (ii) an alanine residue at position 1.2; and (iii) alanine at position 114 Includes; Here, the numbering of amino acid residues follows the IMGT numbering scheme.

[0142] For example, the CH2 domain can contain a LALA-PA mutation and have the sequence set forth in SEQ ID NO:174.

[0143] Also contemplated is an antibody molecule that comprises a CDR-based antigen binding site for MSLN and competes with the antibody molecule described herein or binds to the same epitope in MSLN as the antibody molecule described herein.Methods for determining the competition for an antigen by two antibodies are known in the art.For example, the competition of binding to an antigen by two antibodies can be determined by surface plasmon resonance, for example, using a Biacore instrument.Methods for mapping the epitope bound by an antibody are also known.

[0144] The N-terminal region of MSLN has been reported to interact with MUC16, and this interaction may play a role in cancer cell adhesion.

[0145] Antibody molecules have been shown to have a range of activity for ligand binding. For example, the antibody molecule may be capable of blocking the binding of MUC16 to MSLN, or may not be capable of blocking the binding of MUC16 to MSLN. Alternatively, the antibody molecule may be capable of promoting the binding of MUC16 to MSLN. For example, the antibody molecule may comprise CDR1-6, VH domain, VL domain, heavy chain and / or light chain of antibody FS28-024-051, FS28-024-052, FS28-024-053 or FS28-024 or variants thereof, where the antibody molecule blocks the binding of MUC16 to MSLN.

[0146] In a preferred embodiment, the antibody molecule does not block binding of MUC16 to MSLN. For example, the antibody molecule may comprise CDRs 1-6, a VH domain, a VL domain, a heavy chain and / or a light chain of antibody FS28-256-271, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256 or a variant thereof, wherein the antibody molecule does not block binding of MUC16 to MSLN.

[0147] Suitable methods for determining the ability of an antibody molecule to block binding of MUC16 to MSLN are known in the art and include ELISAs and cell-based assays, such as assays in which the antibody competes with MUC16 for binding to cells expressing MSLN, such as NCI-H226 cells.

[0148] In a preferred embodiment, the antibody molecule may contain, in addition to the CDR-based antigen-binding site for MSLN, one or more additional antigen-binding sites that bind to one or more additional antigens, preferably specifically binding to their cognate antigens.

[0149] The one or more additional antigen binding sites preferably do not bind to MSLN. Thus, the antibody molecule may be a multispecific, such as a bispecific, trispecific or tetraspecific molecule, preferably a bispecific molecule. In a preferred embodiment, the antibody molecule is capable of simultaneously binding to MSLN and one or more additional antigens.

[0150] Antibody molecules are known to have a modular structure, with individual domains that can be combined in a number of different ways to form multispecific, e.g., bispecific, trispecific, or tetraspecific antibody formats. Exemplary multispecific antibody formats are described, for example, in Spiess et al. (2015) and Kontermann (2012). The antibody molecules of the present invention can be used in such multispecific formats.

[0151] For example, the antibody molecule of the present invention may be a heterodimeric antibody molecule, such as a heterodimeric complete immunoglobulin molecule or fragment thereof. In this case, a portion of the antibody molecule will have one or more sequences described herein. For example, if the antibody molecule of the present invention is a bispecific heterodimeric antibody molecule, the antibody molecule may comprise a heavy chain and a light chain as described herein paired with a heavy chain and a light chain comprising a VH domain and a VL domain, respectively, that binds to an antigen other than MSLN or further binds to another epitope on MSLN. Techniques for preparing heterodimeric antibodies are known in the art and include the knob-in-hole (KIH) technique, which involves engineering the CH3 domain of an antibody molecule to form either a "knob" or a "hole" to promote heterodimerization of the chains. Alternatively, heterodimeric antibodies may be prepared by the introduction of charge pairs into the antibody molecule to avoid homodimerization of the CH3 domains by electrostatic repulsion and direct heterodimerization by electrostatic attraction. Examples of heterodimeric antibody formats include CrossMab, mAb-Fv, SEED-body and kih IgG.

[0152] Alternatively, a multispecific antibody molecule may comprise a complete immunoglobulin molecule or a fragment thereof and one or more additional antigen-binding moieties. The antigen-binding moieties may be, for example, Fv, scFv or single domain antibodies and may be fused to a complete immunoglobulin molecule or a fragment thereof. Examples of multispecific antibody molecules comprising additional antigen-binding moieties fused to a complete immunoglobulin molecule include DVD-IgG, DVI-IgG, scFv4-IgG, IgG-scFv and scFv-IgG molecules (Spiess et al., 2015; Figure 1). Examples of multispecific antibody molecules comprising additional antigen-binding moieties fused to an immunoglobulin fragment include, for example, BiTE molecules, bispecific antibodies and DART molecules (Spiess et al., 2015; Figure 1). Other suitable formats will be readily apparent to the skilled artisan.

[0153] In a preferred embodiment, the antibody molecule comprises a second antigen-binding site that binds to a second antigen, and the second antigen-binding site is located in the constant domain of the antibody molecule. For example, the antibody molecule can be a mAb2(TM) bispecific antibody. The mAb2 bispecific antibody referred to herein is an IgG immunoglobulin that comprises a CDR-based antigen-binding site in each of its variable regions and at least one antigen-binding site in the constant domain of the antibody molecule.

[0154] In a preferred embodiment, the antibody is an antibody molecule that binds to MSLN and a second antigen, the antibody molecule comprising: (i) two CDR-based antigen-binding sites for MSLN, formed by an immunoglobulin VH domain and an immunoglobulin VL domain, respectively; and (ii) two antigen-binding sites that bind to a second antigen located in the two CH3 domains of the antibody molecule; Includes.

[0155] In a more preferred embodiment, the antibody is a complete immunoglobulin molecule, e.g., a complete IgG1 molecule that binds MSLN and a second antigen, and the antibody molecule comprises: (i) two CDR-based antigen-binding sites for MSLN, formed by an immunoglobulin VH domain and an immunoglobulin VL domain, respectively; and (ii) two antigen-binding sites that bind to a second antigen located in the two CH3 domains of the antibody molecule; Includes; Here, the immunoglobulin molecule further comprises a CH1, a CH2 and a CL domain.

[0156] The antigen-binding site for the second antigen may be located in any constant domain of the antibody molecule. For example, the antigen-binding site for the second antigen may be located in one or more of the CH4, CH3, CH2, CH1 or CL domains, preferably in the CH3 or CH2 domain, most preferably in the CH3 domain.

[0157] An antigen-binding site may be composed of one or more, such as one, two, three or more, structural loops of the constant domain of an antibody molecule.

[0158] The structural loops of the antibody constant domain include the AB, BC, CD, DE, EF and FG structural loops. The antigen-binding site may comprise two or more of the AB, BC, CD, DE, EF and FG structural loops of the constant domain, preferably the AB and EF structural loops or the AB, CD and EF structural loops.

[0159] The location of structural loops in antibody constant domains is well known in the art. For example, the structural loops of the CH3 domain are located between positions 10 and 19 (AB loop), 28 and 39 (BC loop), 42 and 79 (CD loop), 82 and 85 (DE loop), 91 and 102 (EF loop) and 106 and 117 (FG loop) of the CH3 domain, where the residues are numbered according to the IMGT numbering scheme. The location of the structural loops in other constant domains can be easily determined.

[0160] The structural loop of the constant domain may comprise one or more amino acid modifications to form an antigen-binding site for a second antigen. The one or more amino acid modifications may comprise amino acid substitutions, additions or deletions. The introduction of amino acid modifications into the structural loop region of an antibody constant domain to form an antigen-binding site for a target antigen is well known in the art and is described, for example, in Wozniak-Knopp G et al., 2010, WO 2006 / 072620 and WO 2009 / 132876. Examples of constant domain binding sites are shown below.

[0161] In a preferred embodiment, the antibody molecule comprises one or more amino acid modifications (substitutions, additions and / or deletions) in the AB, CD and / or EF structural loops, preferably in the AB and EF structural loops or in the AB, CD and EF structural loops. For example, the antibody molecule may comprise one or more amino acid modifications (substitutions, additions and / or deletions) at positions 11-18, 43-78 and / or 92-101 of the CH3 domain, preferably at positions 11-18 and 92-101 or 11-18, 43-78 and 92-101 of the CH3 domain, to provide an antigen binding site for a second antigen as described herein. More preferably, the antibody molecule comprises one or more amino acid modifications (substitutions, additions and / or deletions) at positions 12-18, 45.1-78, 92-94 and / or 97-98 of the CH3 domain, more preferably at positions 12-18, 92-94 and 97-98 or at positions 12-18, 45.1-78, 92-94 and 97-98 of the CH3 domain to provide an antigen binding site for a second antigen as described herein. The unmodified CH3 domain preferably comprises or consists of the sequence set forth in SEQ ID NO: 172. Residue numbering is according to the IMGT numbering scheme.

[0162] The second antigen bound by the second antigen-binding site of the antibody molecule can be an immune cell antigen, preferably a member of the tumor necrosis factor receptor superfamily (TNFRSF). TNFRSF receptors are membrane-bound cytokine receptors that contain an extracellular cysteine-rich domain that binds to one or more ligands of the tumor necrosis factor superfamily (TNFSF).

[0163] TNFRSF receptors are preferably located on the surface of immune cells, such as T cells, antigen-presenting cells (APCs), NK cells and / or B cells, preferably T cells. Upon binding of TNFRSF ligands, TNFRSF receptors form clusters on the immune cell surface, which activate the immune cells. For example, ligand-bound TNFRSF receptors can form multimers, such as trimers, or clusters of multimers. The presence of clusters of ligand-bound TNFRSF receptors stimulates intracellular signaling pathways that activate the immune cells.

[0164] Without wishing to be bound by theory, it is believed that by engaging both MSLN on the tumor cell surface and TNFRSF receptors on the immune cell surface, the antibody molecule is bridged by binding to MSLN, thereby causing clustering and activation of the TNFRSF receptor and thus activation of the immune cell. In other words, the antibody molecule acts as a TNFRSF receptor agonist when both targets are bound. The activated immune cell can then initiate, promote or participate in an immune response against cancers expressing MSLN bound to their cell surface. A review of the role the immune system plays in recognizing and eradicating cancer cells is provided by Chen and Mellman (2013).

[0165] Although antibody molecules can be cross-linked by binding to Fcγ receptors, this is inefficient and cannot be targeted to specific locations, such as disease sites, as Fcγ receptor expressing cells are present throughout the human body. Thus, in a preferred embodiment, the antibody molecule comprising a second antigen binding site for a TNFRSF receptor comprises one or more mutations that reduce or abolish binding to one or more of the Fcγ receptors described herein.

[0166] The present inventors have developed a bispecific molecule that contains binding sites for both the MSLN and TNFRSF receptors, in particular a mAb that contains two constant domain binding sites for the TNFRSF receptor and two CDR-based antigen binding sites for MSLN. 2 Using this molecule, we have shown that binding of antibody molecules to both the MSLN and TNFRSF receptors induces or promotes T cell activation.

[0167] An antibody molecule that contains a second antigen binding site for the TNFRSF receptor, which activates immune cells such as T cells only upon binding to the MSLN and TNFRSF receptor, or whose immune cell activation activity is enhanced upon binding to the MSLN and TNFRSF receptor, is also called a conditional agonist. This immune cell activation activity is independent of the antibody molecule's binding to an Fcγ receptor and / or an external cross-linking agent, such as protein A or G or a secondary antibody, thus allowing the conditional agonist activity of the antibody molecule to be targeted to sites where both MSLN and TNFRSF are present. When MSLN is a tumor antigen, the antibody molecule can selectively activate immune cells such as T cells at the site of the tumor and not elsewhere in the individual.

[0168] Antibody molecules that activate immune cells such as T cells only upon binding to MSLN and TNFRSF receptors may have increased immune cell activation activity compared to antibody molecules that rely on cross-linking by other mechanisms such as external cross-linking agents or cross-linking by Fcγ receptor interactions. Because activation of the TNFRSF receptor is more efficient, immune cell activation may be achieved at lower concentrations of the antibody molecules described herein compared to other anti-TNFRSF antibody molecules.

[0169] When the antibody molecule of the present invention comprises a second antigen binding site for the TNFRSF receptor present on T cells, the antibody molecule preferably induces increased activation of immune cells, such as T cells, when the antibody molecule is cross-linked, e.g., by binding to MSLN, than when the antibody molecule is not cross-linked.

[0170] The ability of antibody molecules to activate T cells can be measured using T cell activation assays.T cells release IL-2 upon activation.Therefore, T cell activation assays can measure IL-2 release to determine the level of T cell activation induced by antibody molecules.

[0171] For example, the ability of an antibody molecule to activate T cells can be determined by measuring the concentration of the antibody molecule required to achieve half-maximal release of IL-2 by T cells in a T cell activation assay when the antibody molecule is cross-linked. This is also referred to as the EC50 of the antibody molecule. A lower EC50 indicates that a lower concentration of the antibody molecule is required to achieve half-maximal release of IL-2 by T cells in a T cell activation assay, and thus the antibody molecule has a higher T cell activation activity. The antibody molecule can be cross-linked, for example, with an anti-CH2 antibody.

[0172] Additionally or alternatively, the ability of the antibody molecule to activate T cells may be determined by measuring the maximal concentration of IL-2 released by T cells in a T cell activation assay in the presence of the antibody molecule, where the antibody molecule is cross-linked.

[0173] In a preferred embodiment, an antibody molecule (e.g., a mAb comprising Fcab FS22-172-003) is 2 format), for example, the EC of FS22-172-003-AA / FS28-256-271 in the same assay when crosslinked by binding to NCI-H226 cells. 50 EC in a T cell activation assay that is within 50-fold, 40-fold, 30-fold, 20-fold, 10-fold, or 5-fold of 50 wherein FS22-172-003-AA / FS28-256-271 consists of a heavy chain of SEQ ID NO:187 and a light chain of SEQ ID NO:188.

[0174] For example, the antibody molecules when crosslinked have an EC in a primary T cell activation assay of 30 nM or less, 25 nM or less, 20 nM or less, 14 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1.5 nM, 1 nM or 0.5 nM or less, preferably 1.5 nM or less, more preferably 1 nM and most preferably 0.5 nM or less, when crosslinked. 50 may have:

[0175] Additionally or alternatively, the ability of the antibody molecule to activate T cells may be determined by measuring the maximum concentration of IL-2 released by T cells in a T cell activation assay in the presence of the crosslinked antibody molecule.

[0176] In a preferred embodiment, antibody molecules (e.g., mAb including Fcab FS22-172-003) are crosslinked, e.g., by binding to NCI-H226 cells. 2 In one embodiment, the maximal concentration of IL-2 released by T cells in the presence of FS22-172-003-AA / FS28-256-271 (in a format) is within 20% or 10% of the maximal concentration of IL-2 released by T cells in the presence of FS22-172-003-AA / FS28-256-271 in the same assay, where FS22-172-003-AA / FS28-256-271 consists of a heavy chain of SEQ ID NO: 187 and a light chain of SEQ ID NO: 188.

[0177] For example, the T cell assay can be a pan-T cell activation assay or a CD8+ T cell activation assay depending on the TNFRSF receptor bound by the second antigen-binding site. For example, if the TNFRSF receptor is OX40, a pan-T cell assay is preferred, whereas if the TNFRSF receptor is CD137, a CD8+ T cell assay is preferred.

[0178] For example, pan-T cell activation assay can include isolating human PBMC from leukodepleted pyramidal cells.Methods for isolating PBMC are known in the art.T cells can then be isolated from PBMC.Methods for isolating T cells from PBMC are also known in the art.

[0179] The T cell activation assay may include preparing the required number of T cells in a suitable medium, such as, for example, T cell medium. The required number of T cells may be 1.0×10 6The isolated T cells may be prepared at a concentration of 2.0×10 cells / ml. The T cells may then be stimulated with a suitable T cell activation reagent that provides the signal required for T cell activation. For example, the T cell activation reagent may be a reagent that contains CD3 and CD28, such as beads that contain CD3 and CD28. The isolated T cells may be incubated overnight with the T cell activation reagent to activate the T cells. After this, the activated T cells are washed to separate the T cells from the T cell activation reagent and then diluted with 2.0×10 6 The activated T cells can then be added to a plate coated with an anti-human CD3 antibody.

[0180] Suitable dilutions of each test antibody molecule can be prepared and added to the wells. T cells can then be incubated with the test antibody for 24 hours at 37°C, 5% CO2. Supernatants can be collected and assayed to determine the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in a solution are known in the art and are described in the examples of the present invention. The concentration of human IL-2 can be plotted against the log concentration of the antibody molecule. The resulting curve can be fitted using the equation log(agonist) vs. response.

[0181] For example, CD8+ T cell activation assay can include isolating human PBMC from leukodepleted pyramidal cells. Methods for isolating PBMC are known in the art. Then, CD8+ T cells can be isolated from PBMC. Methods for isolating CD8+ T cells from PBMC are also known in the art.

[0182] CD8+ T cells can then be added to the multi-layer plate coated with anti-human CD3 antibody. Suitable dilutions of each test antibody molecule can be prepared and added to the wells. T cells can then be incubated with the test antibody for 24 hours at 37°C, 5% CO2. Supernatants can be collected and assayed to determine the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in a solution are known in the art and are described in the examples of the present invention. The concentration of human IL-2 can be plotted against the log concentration of the antibody molecule. The resulting curve can be fitted using the equation of log(agonist) vs. response.

[0183] TNFRSF receptors include CD27, CD40, EDA2R, EDAR, FAS, LTBR, ​​RELT, TNFRSF1A, TNFRSF1B, TNFRSF4 (OX40), TNFRSF6B, TNFRSF8, TNFRSF9 (CD137), TNFRSF10A-10D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21 and TNFRSF25.

[0184] In a preferred embodiment, the TNFRSF receptor is TNFRSF4 (OX40).

[0185] In another preferred embodiment, the TNFRSF receptor is TNFRSF9 (CD137).

[0186] CD27 (TNFRSF7: Gene ID 939) has a reference amino acid sequence of NP_001233.1 and may be encoded by a reference nucleotide sequence of NM_001242.4. CD40 (TNFRSF5: Gene ID 958) has a reference amino acid sequence of NP_001241.1 and may be encoded by a reference nucleotide sequence of NM_001250.5. EDA2R (TNFRSF27: Gene ID 60401) has a reference amino acid sequence of NP_001186616.1 and may be encoded by a reference nucleotide sequence of NM_001199687.2. EDAR (Gene ID 10913) has a reference amino acid sequence of NP_071731.1 and may be encoded by a reference nucleotide sequence of NM_022336,3. FAS (TNFRSF6: Gene ID 355) has a reference amino acid sequence of NP_000034.1 and may be encoded by a reference nucleotide sequence of NM_000043.5. LTBR (TNFRSF3: Gene ID 4055) has a reference amino acid sequence of NP_001257916.1 and may be encoded by a reference nucleotide sequence of NM_001270987.1. RELT (TNFRSF19L: Gene ID 84957) has a reference amino acid sequence of NP_116260.2 and may be encoded by a reference nucleotide sequence of NM_032871.3. TNFRSF1A (Gene ID 7132) has a reference amino acid sequence of NP_001056.1 and may be encoded by a reference nucleotide sequence of NM_001065.3. TNFRSF1B (gene ID 7133) has a reference amino acid sequence of NP_001057.1 and may be encoded by a reference nucleotide sequence of NM_001066.2. TNFRSF4 (gene ID 7293) has a reference amino acid sequence of NP_003318 and may be encoded by a reference nucleotide sequence of NM_003327. TNFRSF6B (gene ID 8771) has a reference amino acid sequence of NP_003814.1 and may be encoded by a reference nucleotide sequence of NM_003823.3. TNFRSF8 (gene ID 943) has a reference amino acid sequence of NP_001234.3 and may be encoded by a reference nucleotide sequence of NM_001243.4.TNFRSF9 (Gene ID 3604) has the reference amino acid sequence of NP_001552 and may be encoded by the reference nucleotide sequence of NM001561). TNFRSF10A (Gene ID 8797) has the reference amino acid sequence of NP_003835.3 and may be encoded by the reference nucleotide sequence of NM_003844.3. TNFRSF10B (Gene ID 8795) has the reference amino acid sequence of NP_003833.4 and may be encoded by the reference nucleotide sequence of NM_003842.4. TNFRSF10C (Gene ID 8794) has the reference amino acid sequence of NP_003832.2 and may be encoded by the reference nucleotide sequence of NM_003841.4. TNFRSF10D (gene ID 8793) has the reference amino acid sequence of NP_003831.2 and may be encoded by the reference nucleotide sequence of NM_003840.4. TNFRSF11A (gene ID 8792) has the reference amino acid sequence of XP_011524547.1 and may be encoded by the reference nucleotide sequence of XM_11526245.2. TNFRSF11B (gene ID 4982) has the reference amino acid sequence of NP_002537.3 and may be encoded by the reference nucleotide sequence of NM_002546.3. TNFRSF12A (gene ID 51330) has the reference amino acid sequence of NP_057723.1 and may be encoded by the reference nucleotide sequence of NM_016639.2. TNFRSF13B (gene ID 23495) has the reference amino acid sequence of NP_0036584.1 and may be encoded by the reference nucleotide sequence of NM_012452.2. TNFRSF13C (gene ID 115650) has the reference amino acid sequence of NP_443177.1 and may be encoded by the reference nucleotide sequence of NM_052945.3. TNFRSF14 (gene ID 8764) has the reference amino acid sequence of NP_001284534.1 and may be encoded by the reference nucleotide sequence of NM_001297605.1. TNFRSF17 (gene ID 608) has the reference amino acid sequence of NP_001183.2 and may be encoded by the reference nucleotide sequence of NM_001192.2.TNFRSF18 (Gene ID 8784) has a reference amino acid sequence of NP_004195.2 and may be encoded by a reference nucleotide sequence of NM_004186.1. TNFRSF19 (Gene ID 55504) has a reference amino acid sequence of NP_001191387.1 and may be encoded by a reference nucleotide sequence of NM_001204458.1. NFRSF21 (Gene ID 27242) has a reference amino acid sequence of NP_055267.1 and may be encoded by a reference nucleotide sequence of NM_014452.4. TNFRSF25 (DR3: Gene ID 8718) binds to the ligand TNFSF15 (TL1A) and has a reference amino acid sequence of NP_001034753.1 and may be encoded by a reference nucleotide sequence of NM_001039664.1.

[0187] In certain embodiments, the antibody molecule does not comprise an antigen binding site in a constant domain, such as the CH3 domain, of the antibody molecule. For example, the antibody molecule does not comprise an antigen binding site that binds to CD137 in a constant domain, such as the CH3 domain, of the antibody molecule. In particular, the antibody molecule does not comprise a CD137 antigen binding site in a constant domain of the antibody molecule, where the antigen binding site comprises a modification in one or more structural loops of the constant domain, such as one or more modifications in the AB, CD and / or EF structural loops of the constant domain. In certain embodiments, the antibody molecule does not comprise a CD137 antigen binding site located in the CH3 domain of the antibody molecule comprising a first sequence and a second sequence located in the AB and EF structural loops, respectively, of the CH3 domain, where the first and second sequences have the sequences set forth in SEQ ID NOs: 198 and 199 [FS22-172-003], respectively. For example, the antibody molecule does not comprise the light and heavy chain sequences set forth in SEQ ID NOs: 200 and 201 [FS22-172-003-AA / FS28-256-271].

[0188] The antibody molecule may be conjugated to a biologically active molecule or a detectable label. In this case, the antibody molecule may be referred to as a conjugate. Such conjugates are utilized in the treatment and / or diagnosis of diseases as described herein.

[0189] For example, the bioactive molecule may be an immune system regulator, such as a cytokine, preferably a human cytokine. For example, the cytokine may be a cytokine that stimulates T cell activation and / or proliferation. Examples of cytokines for conjugation to antibody molecules include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-γ.

[0190] Alternatively, the bioactive molecule may be a ligand trap, such as a ligand trap for a cytokine, for example a ligand trap for TGF-β or IL-6.

[0191] As a further alternative, the bioactive molecule may be a ligand such as CD137L, OX40L, TRAIL, CD40L, CD27L or GITRL.

[0192] As a further alternative, the biologically active molecule may be a drug, such as an inhibitor of tubulin polymerization (e.g., auristatins), a tubulin depolymerizing agent (e.g., maytansine), a DNA strand break inducer (e.g., a calicheamicin), a DNA alkylating agent (e.g., a duocarmycin) or an RNA polymerase inhibitor (such as α-amanitin).

[0193] As yet another alternative, the biologically active molecule may be a therapeutic radioisotope.

[0194] Radioimmunotherapy is used, for example, in cancer treatment. Suitable therapeutic radioisotopes for radioimmunotherapy are known in the art and include yttrium-90, iodine-131, bismuth-213, astatine-211, lutetium-177, rhenium-188, copper-67, actinium-225, and iodine-125.

[0195] Suitable detectable labels that can be conjugated to the antibody molecule are known in the art and include radioisotopes, such as iodine-125, iodine-131, yttrium-90, indium-111 and technetium-99; fluorescent dyes, such as fluorescein, rhodamine, phycoerythrin, Texas Red and cyanine dye derivatives, such as Cy7 and Alexa750; chromogenic dyes, such as diaminobenzidine; latex beads; enzyme labels, such as horseradish peroxidase; fluorescent or laser dyes with spectrally resolved absorption or emission characteristics; and chemical moieties such as biotin, which can be detected via binding to specific cognate detectable moieties, such as labeled avidin.

[0196] The antibody molecule can be conjugated to the biologically active molecule or detectable label by any suitable covalent or non-covalent bond, such as a disulfide or peptide bond. If the biologically active molecule is a cytokine, the cytokine can be linked to the antibody molecule by a peptide linker. Suitable peptide linkers are known in the art and can be 5 to 25, 5 to 20, 5 to 15, 10 to 25, 10 to 20, or 10 to 15 amino acids in length.

[0197] In certain embodiments, the biologically active molecule may be conjugated to the antibody molecule by a cleavable linker. The linker may allow for release of the biologically active molecule from the antibody molecule at the site of treatment. The linker may comprise an amide bond (e.g., a peptide linker), a disulfide bond, or a hydrazone. The peptide linker may be cleaved, for example, by a site-specific protease, the disulfide bond may be cleaved by the reducing environment of the cytosol, and the hydrazone may be cleaved by acid-mediated hydrolysis.

[0198] The conjugate may be a fusion protein comprising an antibody molecule and a biologically active molecule. In this case, the biologically active molecule may be conjugated to the antibody molecule by a peptide linker or a peptide bond. If the antibody molecule is a multi-chain molecule, for example, if the antibody molecule is or comprises an Fcab or is an mAb2, the biologically active molecule may be conjugated to one or more chains of the antibody molecule. For example, the biologically active molecule may be conjugated to one or both heavy chains of the mAb2 molecule. Fusion proteins have the advantage of being easy to produce and purify, facilitating the production of clinical grade materials.

[0199] The present invention also provides one or more isolated nucleic acid molecules encoding the antibody molecules of the present invention. A person skilled in the art will have no difficulty in preparing such nucleic acid molecules using methods well known in the art.

[0200] The one or more nucleic acid molecules may be selected from the group consisting of antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, It may encode the VH domain and / or the VL domain, preferably the VH domain and VL domain, of FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably the antibody FS28-256-271 or FS28-024-052, most preferably the antibody FS28-256-271.

[0201] For example, antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014 Nucleic acid molecules encoding the VH domain of FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 are set forth in SEQ ID NOs: 181, 40, 110, 110, 122, 110, 122, 110, 64, 64, 116, 122, 70, 31, 50 and 9, respectively.

[0202] Antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28 -256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-0 Nucleic acid molecules encoding the VL domains of FS28-14, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 are set forth in SEQ ID NOs: 57, 19, 94, 80, 94, 54, 54, 57, 94, 54, 80, 80, 80, 19, 19 and 19, respectively.

[0203] In a preferred embodiment, the nucleic acid molecule is selected from the group consisting of antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS Preferably the heavy and / or light chains, preferably the heavy and light chains, of antibody FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 or FS28-024, preferably antibody FS28-256-271 or FS28-024-052, most preferably antibody FS28-256-271.

[0204] For example, antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28 Nucleic acid molecules encoding the heavy chains of FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 (with the LALA mutation) are set forth in SEQ ID NOs: 179, 38, 108, 108, 126, 108, 126, 108, 84, 84, 120, 126, 68, 29, 48 and 7, respectively.

[0205] For example, antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28 Nucleic acid molecules encoding the heavy chains of FS28-256-018, FS28-256, FS28-024-051, FS28-024-053 and FS28-024 (without the LALA mutation) are set forth in SEQ ID NOs: 177, 36, 106, 106, 124, 106, 124, 106, 82, 88, 118, 124, 66, 27, 46 and 6, respectively.

[0206] Nucleic acid molecules encoding the light chains of antibodies FS28-256-271, FS28-024-052, FS28-256-021, FS28-256-012, FS28-256-023, FS28-256-024, FS28-256-026, FS28-256-027, FS28-256-001, FS28-256-005, FS28-256-014, FS28-256-018, FS28-256, FS28-024-051, FS28-024-053, and FS28-024 are set forth in SEQ ID NOs: 96, 17, 92, 78, 92, 91, 91, 96, 92, 91, 78, 78, 78, 17, 17, and 17, respectively.

[0207] When a nucleic acid encodes the VH and VL domains or the heavy and light chains of an antibody molecule of the invention, the two domains or chains may be encoded on two separate nucleic acid molecules.

[0208] The isolated nucleic acid molecule can be used to express the antibody molecule of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Thus, another aspect of the present invention provides a vector comprising the nucleic acid described above. A suitable vector can be selected or constructed, containing appropriate control sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences, as needed. Preferably, the vector contains appropriate control sequences to cause expression of the nucleic acid in a host cell. The vector can be a plasmid, virus, such as a phage or phagemid, as needed.

[0209] The nucleic acid molecules or vectors described herein may be introduced into a host cell. Techniques for the introduction of nucleic acids or vectors into a host cell are well established in the art and any suitable technique may be used. A variety of host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NS0 or HEK cells, such as HEK293 cells.

[0210] Another aspect of the invention provides a method of producing an antibody molecule of the invention, comprising expressing a nucleic acid encoding the antibody molecule in a host cell and optionally isolating and / or purifying the antibody molecule so produced. Methods for culturing host cells are well known in the art. The method may further comprise isolating and / or purifying the antibody molecule. Techniques for purification of recombinant antibody molecules are well known in the art and include, for example, HPLC, FPLC or affinity chromatography, for example using Protein A or Protein L. In certain embodiments, purification may be performed using an affinity tag on the antibody molecule. The method may also optionally comprise formulating the antibody molecule into a pharmaceutical composition using a pharma- ceutically acceptable excipient or other substances as described below.

[0211] As described above, MSLN is expressed on the surface of tumor cells, and high expression levels of soluble MSLN have been correlated with poor prognosis in some cancers. Anti-MSLN antibodies are being investigated as anti-cancer therapeutic agents. These anti-MSLN antibodies induce direct cell killing by their ADCC activity or are used in the form of ADC.

[0212] It is therefore anticipated that the antibody molecules described herein will find use in the treatment of cancer. Accordingly, related aspects of the invention provide: (i) an antibody molecule as described herein for use in a method of treating cancer in an individual; (ii) use of an antibody molecule as described herein in the manufacture of a medicament for use in treating cancer in an individual; and (iv) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody molecule described herein.

[0213] The individual may be a patient, preferably a human patient.

[0214] The antibody molecule of the present invention has been shown to preferentially bind to MSLN present on the surface of cancer cells compared to soluble MSLN.Therefore, the cancer to be treated with the antibody molecule of the present invention preferably expresses or is known to express MSLN.More preferably, the cells of the cancer to be treated contain or are known to contain MSLN on their cell surface, i.e. are known to contain MSLN bound to the cell surface.

[0215] Where the antibody molecule comprises a second antigen binding site for an immune cell antigen, such as a TNFRSF member, for example in the constant domain of the antibody molecule, the cancer preferably comprises or is known to comprise tumor infiltrating lymphocytes (TILs) that express the TNFRSF member. In particular, the TILs preferably comprise or are known to comprise a TNFRSF member on their cell surface.

[0216] Methods for determining the presence of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

[0217] The cancer may be a primary cancer or a secondary cancer. Thus, the antibody molecules described herein may be for use in a method of treating cancer in an individual, wherein the cancer is a primary tumor and / or a tumor metastasis.

[0218] The cancer to be treated with the antibody molecule of the present invention may be a solid tumor.

[0219] Cancers include mesothelioma, pancreatic cancer, ovarian cancer, lung cancer (such as small cell lung cancer and non-small cell lung cancer), esophageal cancer, breast cancer, gastric cancer, bile duct cancer, colon cancer, thymic cancer, endometrial cancer, head and neck cancer, sarcoma (biphasic synovial sarcoma, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, or soft tissue sarcoma), desmoplastic small cell tumor, leukemia (acute lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, hairy cell leukemia, or myeloid leukemia), adrenocortical carcinoma, bladder cancer, brain tumor, cervical cancer, cervical hyperplasia, testicular choriocarcinoma, The cancer may be selected from the group consisting of essential thrombocythemia, genitourinary cancer, glioma, glioblastoma, lymphoma (such as Hodgkin's disease or non-Hodgkin's lymphoma), malignant carcinoid carcinoma, malignant hypercalcemia, melanoma (also called malignant melanoma), malignant pancreatic insulinoma, medullary thyroid carcinoma, multiple myeloma, mycosis fungoides, neuroblastoma, polycythemia vera, primary brain tumor, primary macroglobulinemia, prostate cancer, renal cell carcinoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, and Wilms' tumor.

[0220] Preferably, the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, bile duct cancer, colon cancer, thymic cancer, endometrial cancer, head and neck cancer, biphasic synovial sarcoma and desmoplastic small cell tumor.

[0221] More preferably, the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer and lung cancer.

[0222] Cancer is characterized by the abnormal proliferation of malignant cancer cells. When a specific type of cancer is mentioned, such as breast cancer, this refers to the abnormal proliferation of malignant cells in related tissues, such as breast tissue. A secondary cancer that is located in the breast but is the result of the abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, is ovarian cancer, not breast cancer, as referred to herein.

[0223] With respect to cancer, treatment may include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis, as well as inhibiting cancer recurrence. Cancer growth generally refers to any one of several indicators that indicate changes within a cancer to a more developed form. Thus, indicators for measuring inhibition of cancer growth include reduced cancer cell survival, reduced tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasonography, or other imaging methods), delayed tumor growth, destruction of tumor vasculature, improved performance of delayed-type hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and reduced levels of tumor-specific antigens. Activating or promoting an immune response against cancerous tumors in an individual may improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency of cancer growth in the individual.

[0224] Although the antibody molecule may be administered alone, the antibody molecule will usually be administered in the form of a pharmaceutical composition that may contain at least one component in addition to the antibody molecule. Accordingly, another aspect of the present invention provides a pharmaceutical composition comprising the antibody molecule described herein. Also provided is a method comprising formulating the antibody molecule into a pharmaceutical composition.

[0225] A pharmaceutical composition may include, in addition to the antibody molecule, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those of skill in the art. The term "pharmacologically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk-benefit ratio, within the scope of sound medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection, or any other suitable route, as discussed below.

[0226] For parenteral, e.g., subcutaneous or intravenous, administration, e.g., by injection, pharmaceutical compositions containing antibody molecules may be in the form of parenterally acceptable aqueous solutions that are pyrogen-free and have suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Buffers such as phosphate, citric acid and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids. preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as needed, including monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0227] In certain embodiments, the antibody molecule may be provided in lyophilized form for reconstitution prior to administration, for example, lyophilized antibody molecules may be reconstituted with sterile water and mixed with saline prior to administration to an individual.

[0228] The administration may be a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the method of administration, the scheduling of administration, and other factors known to physicians. Prescription of treatment, such as decisions regarding dosage, falls within the responsibility of general practitioners and other physicians, and may depend on the severity of the condition and / or the progression of the disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann et al., 1991; Bagshawe et al., 1991). Specific doses as set forth herein or in the Physician's Desk Reference (2003), as appropriate for the antibody molecule being administered, may be used. Therapeutically effective or suitable doses of antibody molecules may be determined by comparing in vitro and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dose will depend on several factors, including the size and location of the area to be treated, as well as the precise nature of the antibody molecule.

[0229] Typical antibody doses range from 100 μg to 1 g for systemic applications and 1 μg to 1 mg for local applications. An initial larger loading dose may be administered, followed by one or more smaller doses. This is the dose for a single treatment of an adult individual, which may be adjusted proportionally for children and infants, and for other antibody formats proportional to molecular weight.

[0230] Treatments may be repeated daily, twice weekly, weekly or monthly at the physician's discretion. The treatment schedule for an individual will depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

[0231] Treatment can be periodic, with the period between administrations being about 2 weeks or more, e.g., about 3 weeks or more, about 4 weeks or more, about once a month or more, about 5 weeks or more, or about 6 weeks or more. For example, treatment can be every 2 to 4 weeks or every 4 to 8 weeks. Suitable formulations and routes of administration are described above.

[0232] With respect to cancer treatment, the antibody molecules described herein may be administered to an individual in combination with another anti-cancer therapy or treatment, such as an anti-cancer therapy or treatment shown to be suitable or expected to be suitable for the treatment of cancer. For example, the antibody molecules may be administered to an individual in combination with a chemotherapeutic agent, a radiotherapy, an immunotherapeutic agent, an anti-tumor vaccine, an oncolytic virus, an adoptive cell transfer (ACT) therapy (such as adoptive NK cell therapy, or a therapy using chimeric antigen receptor (CAR) T cells, autologous tumor infiltrating lymphocytes (TIL) or gamma / delta T cells, or an agent for hormonal therapy.

[0233] Without wishing to be bound by theory, it is believed that the antibody molecules described herein that contain a second antigen binding site for an immune cell antigen, such as a TNFRSF receptor, may serve as an adjunct in anti-cancer therapeutics. In particular, it is believed that administration of the antibody molecule to an individual in combination with chemotherapy and / or radiation therapy or in combination with an anti-tumor vaccine will elicit an immune response against cancer that is greater than that achieved, for example, with chemotherapy and / or radiation therapy or with an anti-tumor vaccine alone.

[0234] The one or more chemotherapeutic agents for administration in combination with the antibody molecules described herein may be selected from the group consisting of taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B-Raf enzyme inhibitors, MEK inhibitors, c-MET inhibitors, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogs, nucleoside analogs, antifolates, thalidomide derivatives, antitumor chemotherapeutic agents, and others. Taxanes include docetaxel, paclitaxel, and nab-paclitaxel; cytotoxic antibiotics include actinomycin, bleomycin, and anthracyclines, such as doxorubicin, mitoxantrone, and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobimetinib, and trametinib; PARP inhibitors include niraparib. B-Raf enzyme inhibitors include vemurafenib and dabrafenib; alkylating agents include dacarbazine, cyclophosphamide and temozolomide; platinum analogs include carboplatin, cisplatin and oxaliplatin; nucleoside analogs include azacitidine, capecitabine, fludarabine, fluorouracil and gemcitabine; antifolates include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in the present invention include defactinib, entinostat, eribulin, irinotecan and vinblastine.

[0235] Preferred therapeutic agents for administration with the antibody molecules described herein are pentostatin, cyclophosphamide, cisplatin, pemetrexed, paclitaxel, carboplatin, gemcitabine, doxorubicin, vinorelbine, docetaxel or etoposide.

[0236] Radiation therapy for administration with the antibody molecules described herein can be external beam radiation therapy (such as intensity modulated radiation therapy (IMRT), stereotactic body radiation therapy (SBRT), image-guided radiation therapy (IGRT), intraoperative radiation therapy (IORT), electron or electron beam therapy (EBT), superficial radiation therapy (SRT)) or internal radiation therapy (such as brachytherapy, radioisotope or radionuclide therapy, SIRT, etc. Preferably, the radiation therapy is conventional external beam radiation therapy, external beam radiation therapy (EBRT), stereotactic radiation therapy or brachytherapy.

[0237] The immunotherapeutic agent for administration in combination with the antibody molecules described herein may be a therapeutic antibody molecule, a nucleic acid, a cytokine, or a cytokine-based therapy. For example, the therapeutic antibody molecule may bind to an immune-modulating molecule, such as an inhibitory checkpoint molecule or an immune co-stimulatory molecule, a receptor of the innate immune system, or a tumor antigen, such as a cell surface tumor antigen or a soluble tumor antigen. Examples of immune-modulating molecules to which the therapeutic antibody molecule may bind include inhibitory checkpoint molecules, such as CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, PD-L1, PD-1, or KIR, immune co-stimulatory molecules, such as OX40, CD40, CD137, GITR, CD27, or ICOS, and other immune-modulating molecules, such as CD47, CD73, CSF-1R, HVEM, TGFB, or CSF-1. Examples of receptors of the innate immune system to which therapeutic antibody molecules may bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5) and STING.

[0238] The nucleic acid for administration in combination with the antibody molecules described herein may be an siRNA.

[0239] The cytokine or cytokine-based therapy may be selected from the group consisting of IL-2, a conjugated prodrug of IL-2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferons.

[0240] Antitumor vaccines for the treatment of cancer have been realized in the clinic and are well described in the scientific literature (e.g. Rosenberg, S. 2000). This mainly involves the use of autologous or allogeneic cancer cells as a vaccination strategy to induce the immune system to respond to various cell markers expressed by these cells, with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF works particularly well when used in this strategy, eliciting a strong response in antigen presentation.

[0241] The agent for chemotherapy, radiation therapy, immunotherapy, antitumor vaccine, oncolytic virus, ACT therapy or hormone therapy is preferably an agent for chemotherapy, radiation therapy, immunotherapy, antitumor vaccine, oncolytic virus, ACT therapy or hormone therapy for cancer, i.e., an agent for chemotherapy, radiation therapy, immunotherapy, antitumor vaccine, oncolytic virus, ACT therapy or hormone therapy that has been shown to be effective in the treatment of cancer. The selection of a suitable agent for chemotherapy, radiation therapy, immunotherapy, antitumor vaccine, oncolytic virus, ACT therapy or hormone therapy that has been shown to be effective in cancer is well within the capabilities of a person skilled in the art.

[0242] The antibody molecules of the present invention may be useful for detecting MSLN, particularly for detecting immobilized MSLN, such as cell surface-bound MSLN. The antibody molecules may be conjugated to a detectable label as described elsewhere herein.

[0243] Thus, the present invention relates to the use of antibody molecules to detect the presence of immobilized MSLN, preferably cells containing MSLN on their cell surface, in a sample.

[0244] Also provided is an in vitro method of detecting MSLN, which comprises incubating an antibody molecule with a sample of interest and detecting binding of the antibody molecule to the sample, where binding of the antibody to the sample indicates the presence of immobilized MSLN. Binding of the antibody molecule to the sample can be detected, for example, using ELISA.

[0245] In a preferred embodiment, the present invention relates to an in vitro method for detecting cells containing MSLN on their cell surface, comprising incubating an antibody molecule with a cell sample of interest and determining binding of the antibody molecule to cells present in the sample, where binding of the antibody to cells present in the sample indicates the presence of cells containing MSLN on their cell surface. Methods for detecting binding of antibody molecules to cells are known in the art and include ELISA and flow cytometry.

[0246] The antibody molecule of the present invention may be used for the detection, diagnosis and / or prognosis of cancer. The cancer may be any cancer that may be treated with the antibody molecule of the present invention as described herein.

[0247] Accordingly, related aspects of the present invention provide: (i) an antibody molecule as described herein for use in a method for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual; (ii) use of an antibody molecule as described herein in the manufacture of a diagnostic agent for use in detecting, diagnosing, prognosing or monitoring the prognosis of cancer; (iii) a method of detecting, diagnosing, prognosing, or monitoring the prognosis of cancer in an individual using the antibody molecules described herein; and (iv) A kit for use in a method for detecting, diagnosing, prognosing or monitoring the prognosis of cancer in an individual, the kit comprising an antibody molecule as described herein.

[0248] The method may comprise administering an antibody molecule of the invention to an individual and determining the presence of the antibody molecule at a site in the individual's body, where the presence of the antibody molecule at the site in the body indicates the presence of a tumor, particularly a tumor comprising cells expressing MSLN on their cell surface.

[0249] In a preferred embodiment, the method includes determining the presence of cells expressing MSLN on their cell surface in a sample obtained from the individual, where the presence of cells expressing MSLN on their cell surface indicates that the individual has cancer.

[0250] In another preferred embodiment, the method includes determining the presence of tumor cells that express MSLN on their cell surface in a tumor sample obtained from an individual, where the presence of tumor cells that express MSLN on their cell surface indicates that the individual has a worse prognosis (e.g., has a higher risk of cancer metastasis) than an individual having the same cancer that does not contain cells that express MSLN on their cell surface.

[0251] The cancer may be any cancer mentioned herein. Preferably, the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, bile duct cancer, colon cancer, thymic cancer, endometrial cancer, head and neck cancer, biphasic synovial sarcoma and desmoplastic small cell tumor. More preferably, the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer and lung cancer.

[0252] Further aspects and embodiments of the present invention will be apparent to those of skill in the art in light of this disclosure including the following experimental exemplification.

[0253] All documents mentioned herein are hereby incorporated by reference in their entirety.

[0254] As used herein, "and / or" should be construed as a specific disclosure of each of the two stated features or components with or without the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0255] Unless the context requires otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0256] Other aspects and embodiments of the present invention provide those aspects and embodiments described above with the term "comprising" being replaced by the terms "consisting of" or "consisting essentially of" unless the context requires otherwise.

[0257] Certain aspects and embodiments of the present invention are illustrated by way of example and with reference to the figures described above. EXAMPLES

[0258] Working Example Example 1: Isolation of anti-human MSLN antibodies: antigen, selection and screening Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein that is synthesized as a 69 kDa precursor and proteolytically processed to a 30 kDa NH2-terminal secreted form (called megakaryocyte potentiating factor or MPF) and a 40 kDa membrane-bound mesothelin (MSLN). Soluble forms of MSLN, shed from the tumor cell surface and generated by alternative splicing of membrane-bound MSLN or by tumor necrosis factor-α converting enzyme (TACE), are found in patient serum. This tumor-shedding antigen is known to form a "sink" that can serve as a decoy for therapeutic antibodies (Lee et al., 2018), and therefore this must be overcome to allow antibodies to bind to MSLN on tumors. To circumvent this sink effect, we set out to isolate novel anti-mesothelin antibodies that bound preferentially to immobilized MSLN compared to soluble MSLN, with the intention that this would lead to preferential binding to membrane-bound MSLN over soluble MSLN. To this end, different forms of the MSLN antigen were used in the phage selection and subsequent screening work.

[0259] 1.1 Production of human, cynomolgus monkey, and mouse mesothelin antigens Recombinant biotinylated human MSLN-His antigen, designated "hMSLN-His Acro", lacking the C-terminal 18 amino acids, was obtained from Acrobiosystems (catalog number MSN-H8223). To maximize the diversity of binders selected across the antigen, full-length monomeric human MSLN antigen was generated and biotinylated in-house for phage selection. Cynomolgus and mouse MSLN were produced to allow for the isolation of binders that were able to bind to human and cynomolgus MSLN, respectively, and further for the isolation of mouse MSLN binders.

[0260] Briefly, MSLN antigen was produced by cloning DNA encoding human (SEQ ID NO: 169) (hMSLN-His-Avi), cynomolgus (SEQ ID NO: 170) (cMSLN-His-Avi) or mouse (SEQ ID NO: 171) (mMSLN-His-Avi) MSLN, along with six C-terminal histidine residues and the Avi sequence, into a modified pFUSE vector (Invivogen catalog number pfuse-mg2afc2) using EcoRI-HF and BamHI-HF restriction enzymes. The vector was transfected into HEK293-6E cells (National Research Council of Canada) and the expressed MSLN was purified using a HisTrap™ Excel Nickel column (GE LifeSciences, 29048586). Each of the antigens was biotinylated using a BirA Biotin-Biotin Protein Ligase Reaction Kit (Avidity LLC, BirA500) to produce monomeric MSLN antigens labeled with a single biotin molecule. Specifically, 5 to 10 mg of antigen was mixed with BirA enzyme mix to a molar ratio of enzyme to substrate of 1:50. Additives were then added according to the manufacturer's recommendations and incubated overnight at room temperature, after which recombinant human, cynomolgus monkey or mouse MSLN was purified using a HisTrap™ Excel Nickel column (GE LifeSciences, 29048586) to remove excess free biotin.

[0261] The biophysical properties of each antigen were characterized by SEC-HPLC analysis to determine if aggregates were present and PAGE to confirm the size of the molecules. SEC-HPLC of these antigens showed less than 10% aggregation and PAGE confirmed that the antigens were monomeric. ELISA and surface plasmon resonance (SPR) were used to confirm that the biotinylated MSLN antigens could be bound by an MSLN-specific positive control antibody (SS1, section 1.3; see Hassan et al 2002, and MOR6626, section 7.1, patent publication number WO 2009 / 068204 A1). Based on this data, all antigens were considered suitable for naive selection.

[0262] 1.2 Phagemid library selection A synthetic naive phagemid library (MSM Technologies) displaying germline human Fab domains with randomized amino acids in CDR1, CDR2 and CDR3 was used for selections with the MSLN antigen as described in section 1.1.

[0263] Fab libraries were initially selected in multiple runs of 3 or 4 with streptavidin Dynabeads (Thermo Fisher, 11205D), neutravidin binding protein coupled to Dynabeads (Thermo Fisher, 31000) or anti-His Dynabeads (Thermo Fisher, 10103D) to isolate phages that bound to biotinylated human, cynomolgus or mouse MSLN-His-Avi or hMSLN-His Acro. Selection runs were also performed with full-length MSLN antigen produced in-house, where human MSLN selection rounds were alternated with cynomolgus MSLN antigen in an effort to isolate human and cyno cross-reactive clones. Selections for binders to mouse MSLN (R&D mMSLN-His 8604-MS) were also performed. Standard phage selection and phage recovery procedures were used.

[0264] To obtain clones that bind to different regions of the MSLN antigen, an epitope masking approach was employed using anti-MSLN antibodies from the earlier selection run described above. Briefly, the first round of selection of the naive Fab library was performed with biotinylated human MSLN-His-Avi at 500 nM. In the second and third rounds, phage binding of biotinylated cynomolgus MSLN-His-Avi at 500 nM (round 2) or 100 nM (round 3) was tested in the presence of a mixture of naive anti-mesothelin mAb proteins isolated from the earlier selection run (FS28-004, FS28-024, FS28-026, FS28-091 and FS28-97, each mAb at 500 nM). These epitope masking selections reduced the output titers, indicating that the selection strategy was working as fewer binders were identified. This led to the identification of clones (FS28-243, FS28-255 and FS28-256, see section 2.1.3) that target additional regions of the MSLN compared to clones from the previous selection run.

[0265] 1.3 Screening to identify anti-MSLN antibodies Approximately 2000 clones from the third and fourth output of all selections were screened by phage ELISA for binding to 25 nM immobilized biotinylated hMSLN-His Acro, full-length biotinylated human or cynomolgus MSLN-His-Avi, matching the antigens used in the selection rounds. Streptavidin plates or plates immobilized with unrelated biotinylated His-tagged antigen were included as negative controls. Clones with MSLN binding signals at least 4-fold higher than the signal to the negative controls were selected and their variable regions were sequenced to identify 156 unique VH / VL sequence combinations, which were subsequently selected for soluble expression. Clones were selected from all selection runs, including epitope masking selections. For each clone, the VH and VL were individually cloned into pTT5 expression vectors (National Research Council of Canada) containing the CH1, CH2 (with a LALA mutation in the CH2 domain (Bruhns et al., 2009; Hezareh et al., 2001) and either the CH3 or CL domains, respectively. The resulting pTT5-FS28 VH and pTT5-FS28 VL vectors with the LALA mutation (AA) were transiently co-transfected into HEK293-6E cells and the clones were produced as intact IgG1 molecules. The antibodies were either retained in the supernatant or purified by a mAb Select SuRe Protein A column and subjected to further testing as described below. Using the same method, the VH and VL regions of SS1 and the anti-hen egg white lysozyme antibody HelD1.3 were cloned and expressed as IgG1 Expression in the LALA format gave G1-AA / SS1 (SEQ ID NOs: 167 and 168) and G1-AA / HelD1.3 (SEQ ID NOs: 165 and 166), which were used as positive and negative controls, respectively.

[0266] Example 2: Identification of a panel of naive anti-MSLN mAbs Anti-mesothelin mAbs have a wide range of potential applications, including their use as therapeutics capable of inducing ADCC, as tumor targeting arms for the delivery of immunotoxins, ADCs, and for the generation of bispecific antibodies, among others. The desired properties of anti-mesothelin mAbs depend on the application, and therefore the inventors set out to identify a series of mAbs that bound membrane-bound human and cynomolgus MSLN with different affinities and were able to target different regions of MSLN. To this end, a series of screening assays were performed, including ELISA, Biacore blocking assays, and cell binding, as well as assays comparing the binding regions to MSLN.

[0267] 2.1 Screening for binding to recombinant MSLN 2.1.1 Binding ELISA HEK293-6E supernatants containing soluble anti-MSLN binding clones or purified clones were screened for binding to human MSLN-His Acro, hMSLN-His-Avi, and for some work, cMSLN-His-Avi by ELISA. Briefly, hMSLN-His Acro, hMSLN-His-Avi, cMSLN-His-Avi, or unrelated His-tagged antigens were coated on maxisorp plates at 25 nM overnight at 4°C. The next day, plates were blocked with 300 μl of PBS containing 0.05% Tween 20 and 2% Marvel milk (Marvel milk powder). Supernatants or purified proteins containing anti-MSLN mAb were incubated for 1 h at room temperature, and their binding was detected using a mouse anti-human Fc-IgG antibody conjugated to horseradish peroxidase (HRP). Clones that showed binding to unrelated antigens or were not cross-reactive with both human and cynomolgus antigens were discarded. Additionally, clones that bound to the truncated MSLN antigen, hMSLN-His Acro, but not the full-length MSLN antigen, MSLN-His-Avi, were not pursued further, as it is anticipated that the full-length antigen may be more representative of the antigen conformation on the cell surface of MSLN-expressing cells.

[0268] Mesothelin is a glycoprotein, whose glycosylation pattern can vary depending on species and tissue. To ensure that the antibodies have specificity for binding to MSLN, anti-MSLN binding clones were also tested for differential binding to glycosylated and deglycosylated MSLN. Biotinylated hMSLN-His-Avi was deglycosylated with PNGase F enzyme (NEB, P0704L) for 24 hours at 37°C, purified with Amicon ultra centrifugal filters (Millipore, UFC901024), and coated on maxisorp plates at 25 nM. ELISA binding of unique anti-mesothelin mAbs to MSLN was detected with mouse anti-human Fc-IgG-HRP (Sigma, A0170). Clones that showed more than a two-fold reduction in binding signal to deglycosylated MSLN antigen compared to glycosylated MSLN antigen were excluded from the set of naive anti-MSLN binding mAbs.

[0269] 2.1.2 BIAcore Screening Human MSLN-His-Avi was immobilized on flow cell 2 in a CM5 Series S BIAcore sensor chip (GE Healthcare, BR100530) using an amine coupling kit (GE Healthcare, BR10050) to approximately 200 response units (RU). Flow cell 1 was left blank for subtraction. HEK293-6E supernatants or purified proteins were adjusted to approximately 50 nM anti-MSLN mAb per sample using HBS-EP+ (GE Healthcare). Samples were injected over flow cells 1 and 2 at 30 μl / min for 2.5 min and then dissociated in HBS-EP buffer for 2.5 min. Regeneration was performed by injecting 10 mM glycine pH 1.5 (GE Healthcare, BR100354) for 30 s at a rate of 30 μl / min. The subtracted data (flow cell 2 minus flow cell 1) was analyzed using BIAevaluation 3.2 Software (GE Healthcare). Thirty-nine of the 86 clones tested in this assay from the binding ELISA showed binding responses of greater than 10 RU at 50 nM and were therefore selected for re-expression, purification and further screening.

[0270] 2.1.3 Binning of antibodies based on MSLN-bound regions Based on the ELISA and Biacore screening data, clones were then tested in a binning assay, where the binding of the mAb to human MSLN was tested in the presence of another mAb by biolayer interferometry (BLI) in an Octet (ForteBio).

[0271] Briefly, biotinylated hMSLN-His-Avi (5 μg / ml) was bound to a streptavidin chip (ForteBio, 18-5020) for 5 min. G1-AA / SS1 was diluted to 200 nM in 1× kinetic buffer (ForteBIO, 18-1092) and bound to hMSLN-His-Avi for 5 min. Binding of a mixture containing 200 nM of test mAb and 200 nM of G1-AA / SS1 was then assessed for 5 min. This was compared to the binding of the test mAb to hMSLN-His-Avi in ​​the absence of bound G1-AA / SS1 to determine the maximum degree of binding possible in the absence of SS1 (i.e., when there was no competition for binding). If both antibodies compete for binding to the same region of MSLN, the test antibody will not be able to bind.

[0272] These binning experiments with G1-AA / SS1 showed that the majority of FS28-lineage antibodies tested (19 out of 23) were unable to bind MSLN in the presence of G1-AA / SS1 and could therefore be attributed to binding to a similar region as G1-AA / SS1. The MSLN binding site for the SS1 antibody in the Fab format has been reported (Ma et al., 2012) and is defined as the N-terminal region including amino acids 7 to 64, which are also involved in MUC16 binding. Most of the anti-MSLN binders isolated bound to this or a similar region, suggesting that these amino acids are well exposed in the recombinant antigen. Four other clones, FS28-185, FS28-243, FS28-255 and FS28-256, were identified that showed partial or no competition with G1-AA / SS1 for binding to MSLN. Binning of these clones against each other showed that they represented two additional independent bins, i.e., they were able to bind to two additional distinct regions of MSLN. FS28-185, FS28-243 and FS28-255 were assigned to one bin ("Bin 2"), while FS28-256 was assigned to another bin ("Bin 3"). These results indicated that the epitope masking selection in section 1.2 was successful, as antibodies that bound to multiple regions of MSLN were identified.

[0273] 2.1.4 Affinity For each bin described in 2.1.3, binding kinetics were determined using the same method described in section 2.1.2, except that human or cynomolgus MSLN-His-Avi was immobilized at 50 or 100 RU. Clones were tested in a concentration range of 81 nM to 0.33 nM in 3-fold dilutions. Binders were ranked and the best from each bin were selected: FS28-024, FS28-026 and FS28-091, all from bin 1, FS28-185 from bin 2 and FS28-256 from bin 3. All of these clones were shown to be cynomolgus cross-reactive, but affinities were not calculated under these test conditions. The affinities of the selected antibodies are shown in Table 1, which shows that the affinities obtained at 50 RU of immobilized MSLN were lower than those at 100 RU of immobilized MSLN antigen, indicating increased binding at higher levels of MSLN.

[0274] [Table 2]

[0275] 2.2 MUC16-MSLN blocking assay The N-terminal region of MSLN (amino acids 296-359) has been reported to interact with the glycoprotein MUC16, and this interaction may play a role in cancer cell adhesion (Kaneko et al., 2009). FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256 were tested for their ability to block MUC16 binding to mesothelin in a blocking assay. SS1 is known from the literature to block MUC16 binding to MSLN (Ma et al., 2012). As mentioned above, we converted it to the G1-AA / SS1 format, expecting that this would not affect its ability to block MUC16 binding. G1-AA / SS1 and a control IgG1 antibody (G1-AA / HelD1.3) were included as positive and negative controls, respectively.

[0276] Briefly, recombinant human MUC16 (R&D Systems, 5809-MU-050) was coated onto maxisorp plates at 0.65 μg / ml in 1× PBS overnight at 4° C. Plates were washed 3 times with 1× PBS and blocked with 300 μl of PBS containing 2% Tween 20 and 2% Marvel Milk. A concentration of anti-MSLN mAb (0.23 nM to 500 nM, 3-fold dilutions) was premixed with biotinylated hMSLN-His-Avi antigen (final concentration 2 μg / ml) in a volume of 100 μl for 1 h at room temperature. After removal of the blocking solution, the mAb / MSLN mixture was added to the plate and incubated at room temperature for 1 h. Plates were washed three times with PBST (1x PBS and 0.05% Tween 20) and incubated with streptavidin-HRP (Thermo Scientific, 21126, 1:1000 dilution in 1x PBS) for 1 h at room temperature. Finally, plates were washed three times with PBST and three times with PBS. MSLN bound to MUC16 was visualized by adding 100 μl TMB followed by 100 μl 1 M sulfuric acid solution for 15 min. Absorbance was read at 450-630 nm (Gen5 software, BioTek).

[0277] [Table 3]

[0278] Bin 1 clones FS28-024, FS28-026 and FS28-091 had IC of 2.9 nM, 5.2 nM and 4.4 nM, respectively. 50FS28-256, G1-AA / SS1, and G1-AA / HelD1.3 showed dose-dependent blocking of MUC16-MSLN interaction (Table 2). The observed blocking activity was similar to that of G1-AA / SS1. FS28-256, as well as the negative control G1-AA / HelD1.3, showed no blocking activity. On the other hand, FS28-185 promoted the binding of MUC16 to MSLN. This phenomenon has been reported (U.S. Patent No. 8,911,732 B2). These results were consistent with the binning data in section 2.1.3, where clones bound to three different regions of MSLN showed three different behaviors in the ligand blocking assay.

[0279] In conclusion, the results show that a series of clones were selected that bind to three different regions (bins) of MSLN; antibodies that bind to one region of MSLN block MUC16 binding to MSLN, whereas antibodies that bind to the other two regions do not.

[0280] 2.3 Specificity Considering the different regions of MSLN bound by the series of antibodies, their specificity for binding to MSLN was tested. The specificity of FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256 was tested by ELISA by comparing their binding to MSLN with that to other molecules involved in cell adhesion, such as CEACAM-5, E-cadherin, thrombomodulin and EpCAM.

[0281] A similar protocol was used as described in section 1.2.1, where maxisorp plates were coated with 1 μg / ml of recombinant human MSLN-His-Avi, human CEACAM-5-His-Fc (Sino Biological, 1077-H03H), human E-cadherin (R&D systems, 8505-EC), human thrombomodulin (Peprotech, 100-58) or human EpCAM-hFc (produced in-house). Binding of anti-MSLN mAbs tested at concentrations ranging from 0.02 to 1000 nM (3-fold dilutions) was detected using anti-human Fab-HRP (Sigma, A0293). Positive controls for each molecule included human EpCAM antibody (clone 2G8 from US Patent No. 8236308 B2), CEACAM-5 antibody (clone hMN15 from US Patent No. 8771690 B2), human E-cadherin antibody, mouse IgG2b clone 180215 (R&D systems, MAB1838), human thrombomodulin antibody, mouse IgG1, clone 501733 (R&D systems, MAB3947). The latter two were detected using goat anti-mouse Fc-HRP (Sigma, A9309) as secondary mAb.

[0282] FS28-024, FS28-026, FS28-091, and FS28-185 bound to human MSLN-His-Avi (EC 50 and 3 maximum binding signals), no binding was observed to any of the cell adhesion molecules tested up to 1000 nM. The positive control antibodies bound to their respective targets as expected. Thus, the anti-MSLN antibodies demonstrated a high level of specificity.

[0283] 2.4 Cell binding A panel of five selected anti-mesothelin mAbs was analyzed for binding to endogenous cell surface MSLN in the human lung cancer cell line NCI-H226.

[0284] Briefly, NCI-H226 cells (ATCC CRL-5826) were harvested from T175 cell culture flasks using StemPro Accutase (Gibco, A11105-01). Cells were centrifuged at 1200 rpm for 3 min and 2 × 10 6 Cells were resuspended in ice-cold FACS buffer consisting of DPBS (Life Technologies, 14190169) and 1% BSA (Sigma-Aldrich, A7906) at 100 cells / ml and 50 μl per well and plated into 96-well V-bottom plates (Costar, 3894). All mAbs tested were diluted in 120 μl of FACS buffer at concentrations ranging from 0.01 to 200 nM (4-fold dilutions). NCI-H226 cells were then centrifuged, the supernatant removed, and the cells were resuspended in 100 μl of each mAb dilution and incubated at 4° C. for 45 min. Cells were washed twice by centrifugation with 150 μl of FACS buffer, resuspended in 100 μl containing goat anti-human IgG (γ-chain specific) F(ab')2 fragment-R-phycoerythrin antibody (Sigma, P8047) diluted 1:1000 in FACS buffer, and incubated for 45 min at 4° C. Cells were washed once with 150 μl of FACS buffer and then 150 μl of DPBS, resuspended in 150 μl of DPBS containing DAPI (Biotium, 40043) at 1:10,000, and read on a BDCantoII or iQue (Intellicyt). Data was analyzed using FlowJo v10 to determine the PE signal geometric mean for live cells in each well.

[0285] Cell binding data (Table 3) showed that FS28-024, FS28-026 and FS28-091 all had ECs ranging from 0.62 to 1.22 nM, as did the positive control G1-AA / SS1. 50 In comparison, FS28-185 and FS28-256 bound to cell surface MSLN in NCI-H226 with an EC 50 and low maximum binding signal (E max ) showed weak binding. A representative binding assay is shown in FIG.

[0286] [Table 4]

[0287] 2.5 Overview of naive screening procedures From 156 mAbs identified by the initial screen of the naive phage library, five anti-human MSLN mAb clones (FS28-024, FS28-026, FS28-091, FS28-185, and FS28-256) were selected based on a series of screening assays that first confirmed their binding to full-length, deglycosylated recombinant MSLN and their ability to bind to cynomolgus MSLN. Second, the clones were classified based on the diversity of the regions (bins) of MSLN that they bound and on MUC16 blocking activity, and the highest affinity binders were selected from these groups. The resulting set of mAb clones, FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256, bind to three distinct regions of MSLN, one of which (bin 1 containing FS28-024, FS28-026 and FS28-091) blocks MUC16 binding to MSLN in vitro. A set of five anti-MSLN mAbs showed specific binding to MSLN, different affinities for recombinant and cell-surface MSLN and were selected for further characterization and / or optimization, as described in Examples 3 and 4 below.

[0288] Example 3: Affinity maturation and sequence optimization of naive anti-MSLN mAb 3.1 Affinity maturation of FS28-185 and FS28-256 Compared to FS28-024, FS28-026 and FS28-091, FS28-185 and FS28-256 had weaker affinity for both recombinant and cell surface MSLN and were therefore subjected to affinity maturation.

[0289] 3.1.1 Affinity maturation and screening of FS28-185 and FS28-256 The VH and VL CDR3 regions were affinity matured in parallel in scFv format by randomizing overlapping cassettes of 5 to 6 amino acids using NNK primers. For FS28-185 the randomized regions were VH G95-M100F and VL S91-A95, and for FS28-256 they were VH Y95-L100B and VL S91-I96 (Kabat numbering). Prior to library generation, parsimonious mutagenesis was performed at potential methionine oxidation and deamidation sites in the CDR1 and CDR3 regions (except for the FS28-256 VL CDR3 library). Phagemid libraries were generated independently and pooled to generate one VH CDR3 and one VL CDR3 library for each clone. Two rounds of selection were performed as described for the naive run, the first with 20 nM biotinylated human MSLN-His-Avi and the second with 20 or 2 nM cynomolgus MSLN-His-Avi. Soluble scFv (single point concentration) were then tested for binding to the ovarian cancer cell line OVCAR-3 (ATCC® HTB-161™). OVCAR-3 cells were harvested with StemPro Accutase (Gibco, A11105-01), centrifuged at 1200 rpm for 3 min, and 2×10 6Cells were resuspended in FACS buffer (DPBS containing 2% BSA) at 100 μl / ml. 100 μl of OVCAR-3 cells were added to a 96-well V-bottom plate. The plate was centrifuged at 1200 rpm for 3 minutes and the buffer was discarded. 150 μl of scFv was added to the cells and incubated at 4° C. for 1 hour. Parental clone FS28-185 and 256 ScFv were included as controls. After washing, cells were resuspended in 100 μl Penta His Alexa-Fluor 647 (Qiagen, 109-546-098), washed, and then resuspended in 100 μl DPBS containing Sytox Green Nucleic Acid Stain (Invitrogen S7020, 1:10000 dilution). Samples were run on an iQue (Intellicyt Corporation, IQue Plus) and the geometric mean of APC was recorded.

[0290] For both FS28-185 and FS28-256, affinity matured clones with improved binding to OVCAR-3 cells were identified. Based on cell binding (MFI >850) and sequence diversity, 10 clones were selected from the FS28-256 VH CDR3 and 9 were selected from the VL CDR3 selection. Of the 38 FS28-185 affinity matured clones tested in this assay, 14 were selected from the VH CDR3 selection and 1 was selected from the VL CDR3 selection. The selected clones were used as mAbs. 2 It was further characterized in a bispecific antibody format.

[0291] 3.1.2 FS28-185 and FS28-256 based mAbs 2 Generation of For further characterization of anti-MSLN binders, affinity matured VH or VL regions of FS28-185 or FS28-256 as well as the parental clones were used as mAbs. 2 The resulting mAb was produced in the 2are IgG1 antibodies that contain the CDRs of either the FS28-185 or FS28-256 clones or affinity matured variants derived therefrom, a LALA mutation in the CH2 domain, and a human CD137 receptor-binding site in the CH3 domain. 2 The molecules were designated FS22-053-008-AA / FS28-185 (SEQ ID NOs: 154 and 195) and FS22-053-008-AA / FS28-256 (SEQ ID NOs: 156 and 77) and for the affinity matured progeny FS22-053-008-AA / FS28-256-x. 2 were produced by transient expression in HEK293-6E cells and, where indicated, purified using a mAb Select SuRe Protein A column.

[0292] 3.1.3 Affinity Screening of FS28-185 and FS28-256 Affinity-Matured Clones Against Fixed MSLN Next, the mAb containing the affinity matured CDR regions 2 The binding of mAb was screened for binding to immobilized human and cynomolgus MSLN-His-Avi by Biacore. HEK293-6E supernatants containing FS22-053-008-AA / FS28-185 and FS22-053-008-AA / FS28-256 affinity matured clones were analyzed for binding to immobilized MSLN at 200 RU as described in section 2.1.2. Two concentrations of mAb, namely 50 and 100 nM, were assayed for binding to immobilized MSLN. 2 The binding was similarly evaluated using mAb 2 The binding of the parental antibodies in the same format was compared to that of the parental antibodies in the same format. The binders were ranked and the top six cynomolgus cross-reactive binders from each line were re-expressed, purified, and tested in cell binding assays as described in Section 3.1.4 below. Next, for the FS28-256 lineage, clones with improved VH CDR3 were shuffled with clones with improved VL CDR3 to form nine additional VH / VL pairings, which were then used as mAbs. 2 and was also tested.

[0293] 3.1.4 Cell binding in the presence of soluble MSLN As described above in section 1, soluble MSLN can serve as a decoy for the binding of any anti-MSLN antibody. Therefore, affinity matured clones were screened for binding to recombinant and cell surface MSLN in the presence and absence of soluble human MSLN. Because soluble shed MSLN lacks either 7 or 13 C-terminal residues (Zhang et al., 2011), a commercially available human MSLN antigen, MSLN-His Acro, lacking 18 C-terminal amino acids, was used as a mimic. This antigen was used at a concentration of 20 nM, which is approximately 10-20 times the level of soluble MSLN that was found to have diagnostic value in defining malignant mesothelioma and lung cancer patients as MSLN positive (Cui et al., 2014).

[0294] Cell binding assays using OVCAR-3 cells were performed similarly to the assay described in section 2.4. The effect of the presence of soluble MSLN was tested by preincubating the antibodies in the presence or absence of soluble MSLN before mixing with the cells to determine whether preincubation affected binding to MSLN at the cell surface. Affinity matured clones and parental molecules (all mAbs as described above) were incubated with 100 mM NaCl and 100 mM MSLN for 1 h. 2 format) was diluted in FACS buffer to give a 2x final concentration in 96-well V-bottom plates. 60μl from each well was then added to either FACS buffer alone or FACS buffer containing 60μl of 40nM recombinant hMSLN (R&D systems, 3265-MS-050) (to give a final concentration of 20nM hMSLN) and pre-incubated for 1 hour at room temperature before adding 100μl to the cells. Bound antibody was detected using goat anti-human anti-Fcγ Alexa-Fluor 488 (Jackson Immunoresearch, 109-546-098).

[0295] For the FS28-185 lineage, all affinity matured clones showed improved cell binding (EC50 However, this binding was reduced in the presence of 20 nM soluble MSLN, with a 3- to 7-fold higher EC 50 For the FS28-256 lineage, five clones were selected as representative of the lineage, all with EC values ​​ranging from 0.9 to 8.6 nM. 50 Most importantly, binding affinity was maintained, changing less than 2-fold when binding in the presence of 20 nM soluble MSLN compared to its absence. From this data, clones selected for further testing included FS28-256-012, FS28-256-021, FS28-256-023 and FS28-256-024.

[0296] 3.2 Affinity maturation of clone FS28-024 using the NNK walk method While FS28-024 bound to human MSLN with sub-nanomolar affinity, its affinity to cynomolgus MSLN was approximately 5-fold lower (see Example 4, Table 4). To improve binding to cynomolgus MSLN, a NNK walk was used on five residues in the VH CDR3 region. The sequences of FS28-024 VH and VL were compared with the same mAb 10A1111 described in Section 3.1.2. 2The format was optimized. A parsimonious mutagenesis library was generated by diversifying one amino acid residue at the RATLF residues (Kabat numbering 95-99) in the VH CDR3, resulting in a total of five individual libraries. The libraries were generated using low-redundancy NNK codons to represent all possible amino acids at the targeted positions. Forward and reverse primers were designed according to the guidelines of the Quickchange Lightning Site-Directed Mutagenesis Kit (Agilent, 200518) and used to generate the libraries. Each mutant was expressed at small scale in HEK293-6E cells and the supernatants were screened by BIAcore for retained or improved binding to human and cynomolgus MSLN-His-Avi. Of the 84 clones screened, only a few retained binding, most of which had a substitution of the T98 residue. Four clones, FS28-024-51, FS28-024-52, FS28-024-53 and FS28-024-060, were expressed again, purified and their affinity to human and cynomolgus MSLN was determined. Only one clone, FS28-024-53, showed improved cynomolgus cross-reactivity achieved by a single T98V mutation (Kabat numbering, see Table 4, Example 4). All four clones were taken further as they may offer alternative sequences and properties depending on the application.

[0297] 3.3 Overview of affinity maturation Overall, the affinity maturation and sequence optimization approach for the FS28-024, FS28-185 and FS28-256 lines was successful by further expanding the panel and diversity of anti-MSLN mAbs.

[0298] Example 4: Characterization of anti-MSLN clones 4.1 Affinity mAbs 2Binding of selected anti-MSLN clones in the format to recombinant human and cynomolgus MSLN-His-Avi antigens was measured by SPR using a Biacore T200 processing unit (GE Healthcare). As described above, it was desirable to bind more strongly to immobilized antigen than to soluble antigen. To evaluate the binding properties of the clones, the binding kinetics to immobilized MSLN antigen was determined as described in Section 2.1.2 and compared to the kinetics obtained when MSLN antigen in solution was bound to the captured clones.

[0299] For capture experiments, mAb 2 Clones in the format were captured using a BIAcore Sensor Series S chip Protein G (GE Healthcare, 29179315). mAbs diluted in HBS-EP buffer (GE Healthcare, BR100188) containing 900 mM NaCl2 at 1 μg / ml. 2 were injected individually at 30 μl / min on flow cells 2, 3 and 4 to achieve a response of approximately 100RU. G1-AA / HelD1.3 was captured on flow cell 1. Recombinant human and cynomolgus MSLN-His-Avi (section 1.1) diluted in HBS-EP buffer containing 900 mM NaCl2 were injected over flow cells 1, 2, 3 or 4 at 70 μl / min for 5 min in a concentration range of 1000 nM to 0.051 nM as required in 3-fold dilutions, followed by dissociation in buffer for 5 min. Regeneration was performed by injecting 10 mM glycine pH 1.5 (GE Healthcare, BR100354) and surfactant P20 (GE Healthcare, BR-1000-54) for 20 s at a rate of 30 μl / min. Subtracted data (flow cell 2-flow cell 1, flow cell 3-flow cell 1 or flow cell 4-flow cell 1) were analyzed using BIAevaluation 3.2 Software (GE Healthcare) to identify binding using a model 1:1 binding, local Rmax and a refractive index (RI) constant of 0.

[0300] [Table 5]

[0301] Kinetic data showed that, except for FS28-024-060, all clones tested were cross-reactive with cynomolgus MSLN-His-Avi, with affinities for the cynomolgus antigen within 5-fold of the affinity for human MSLN-His-Avi.

[0302] Furthermore, for each group of clones bound to a different MSLN binding region, clones were identified that bound to immobilized human MSLN-His-Avi with low nanomolar affinity. The progeny clones of FS28-256 had higher affinities than their respective parent antibodies, confirming that affinity maturation of these clones had been successful. Interestingly, all clones had higher affinity for immobilized MSLN than for human MSLN-His-Avi in ​​solution, which is consistent with the immobilized K D K in solution compared to D This feature was quantified by calculating the fold difference in binding affinity (see Table 4). Therefore, it can be hypothesized that the anti-MSLN antibodies do not bind to the target with high affinity, as observed by the low affinity for binding to soluble MSLN, but rather appear to bind more strongly to immobilized MSLN due to enhanced avidity interactions with the immobilized antigen. Avidity appears to be antibody specific.

[0303] 4.2 Cell binding to NCI-H226 in the presence of soluble MSLN All selected clones supplemented with other unshuffled FS28-256 affinity matured clones (see Table 5) were then tested for binding to cell surface MSLN in NCI-H226 cells in the absence and presence of 20 nM MSLN. The method used was exactly as described in section 3.1.4. EC 50 and E max Both values ​​were determined (Table 5).

[0304] [Table 6]

[0305] Data are for the mAbs tested. 2 The anti-MSLN binding Fab arms of FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256 bound with variable affinity to NCI-H226 cells ranging from 0.78 to >16 nM, with the majority of clones displaying low nanomolar cell binding affinity, consistent with the reported affinity for immobilized recombinant MSLN. The ranking of cell binding affinity of naive FS28-024, FS28-026, FS28-091, FS28-185 and FS28-256 was also consistent with the ranking data obtained in the mAb format (Table 3, section 2.4). Interestingly, the effect of 20 nM recombinant MSLN on cell binding affinity was significantly lower than the EC values ​​observed for most clones. 50 The cell binding affinity of FS28-256-derived clones, such as FS28-256-001, FS28-256-005, FS28-256-012, FS28-256-014, FS28-256-018, FS28-256-023, FS28-256-024 and FS28-256-026, was not affected by the presence of soluble MSLN. This indicated that most clones preferentially bound to membrane-bound MSLN even in the presence of excess soluble MSLN.

[0306] For clones derived from FS28-024, the more variable effect of soluble MSLN ranged from 3.4 to 17.6-fold with EC 50 This was observed accompanied by an increase in

[0307] The two clones with the highest affinity for recombinant soluble MSLN (see Table 4), namely FS28-024-060 and FS28-256-027 (both with single-digit nM K for MSLN-His-Avi in ​​solution) D(having a β-amyloid β-amyloid complex) was most affected when bound to cells in the presence of soluble MSLN, indicating that higher affinity binders are more likely to be affected by shed MSLN. As a result, EC 50 The increase in EC was less favorable, because the final EC in the presence of soluble MSLN is most likely a reflection of the affinity of the mAb for these tumor cells in patients. 50 The actual affinity required of the anti-MSLN antibody depends on the application.

[0308] Example 5: Sequence optimization of FS28-256 affinity matured clones 5.1 Sequence optimization of FS28-256 affinity matured clones All FS28-256 lineage clones contained potential N-linked glycosylation sites in VH CDR2 (IMGT numbering N55-X-S57, where X is any residue). In addition, FS28-256-001, FS28-256-021 and FS28-256-023 had potential deamidation sites in the VL CDR3 region at positions N116-T117 (IMGT numbering). Similar to the procedure described in section 3.2, the NNK walk method was used to identify amino acid substitutions in clone FS28-256-021 that could remove these potential glycosylation and deamidation sites. First, the VH CDR2 residue N55 and the VL CDR3 N116 residue were mutated in each clone and mutants were screened for retention of optimized binding to human and cynomolgus MSLN. For FS28-256-021, changes to the VL CDR3 N116 residue resulted in loss of antigen binding. Of the VH CDR2 changes at N55, only four mutants (N55A, N55H, N55S, and N55T) retained binding to human MSLN, but their binding was weaker compared to the parental clone.

[0309] Since we were unable to remove potential deamidation sites in VL CDR3 by mutating the sequence, we took a different approach. Some of the other affinity matured clones derived from FS28-256 shared the same heavy chain sequence as FS28-256-021, but had different light chain sequences, so we further investigated these other clones. Specifically, FS28-256-027 was selected for testing. As described above, FS28-256-027 had a higher affinity for soluble MSLN than FS28-256-021 (6.0 nM, Table 4), and in the presence of soluble MSLN (Table 5), it resulted in reduced binding to MSLN expressed on the cell surface, and therefore was not selected as a preferred clone at that time. To investigate whether this clone could be optimized for binding to MSLN expressed on the cell surface, the N55A, N55H, N55S or N55T mutations identified for the FS28-256-021 clone were introduced into the heavy chain of FS28-256-027 and binding of the four resulting clones was measured by SPR similar to the protocol used in Example 4.1, except that about 40 RU was used instead of 100 for binding to immobilized and in solution MSLN. The results are shown in Table 6. Introduction of the mutation N55T into FS28-256-027 resulted in a clone, FS28-256-274, which had a much weaker affinity for immobilized MSLN than the other clones and was not taken further. Introduction of the mutations N55H and N55S into FS28-256-027 yielded clones FS28-256-272 and FS28-256-273, respectively, which bound to soluble MSLN with higher or equal affinity to immobilized MSLN. Therefore, it was likely that the binding of both of these clones to cell surface-expressed MSLN was adversely affected by the presence of soluble MSLN. In contrast, introduction of the mutation N55A into FS28-256-027 yielded clone FS28-256-271, which showed the highest affinity to immobilized MSLN and weaker binding to soluble MSLN among the four clones tested.These results unexpectedly showed that the N55A mutation in VH CDR2 of the parent clone FS28-256-027 reduced the affinity of binding to both immobilized and soluble MSLN such that FS28-258-271 preferentially targets immobilized MSLN over soluble MSLN (K<10 nM). D and a K value of 10 nM or higher. D Consistent with other clones such as FS28-256-021, which bound to soluble MSLN in situ, it is expected that binding of FS28-258-271 to MSLN at the cell surface will not be significantly affected by the presence of soluble MSLN.

[0310] [Table 7]

[0311] Cynomolgus cross-reactivity was determined for mAb2 FS22-172-003-AA / FS28-256-271 by SPR using steady-state kinetic analysis. CM5 chips (GE Healthcare BR-1005-30) were coated with hMSLN-His-Avi or cMSLN-His-Avi at approximately 50 RU according to the manufacturer's instructions. mAb2 was injected at a range of concentrations, a 3-fold dilution series starting at 243 nM, at a flow rate of 10 μl / min. Association time was 1000 seconds to steady state and dissociation time was 30 seconds. Running buffer was HBS-EP (GE Healthcare BR100188). The flow cell was regenerated by injecting glycine-HCl pH 1.5 at a flow rate of 30 μl / min for 30 seconds. Data was analyzed by double referencing against a flow cell that was intentionally left blank (no antigen coating). Kinetic data were analyzed using BiaEvaluation software version 3.2 using a steady-state affinity model. Binding to cynomolgus monkey MSLN was within 3-fold of binding to human MSLN.

[0312] 5.2 Antibody-dependent cytotoxicity (ADCC) activity To evaluate the effector function of the MLSN mAbs, the molecules were tested in an ADCC in vitro assay. To this end, the FS28-256-271 and FS28-024-052 antibodies were produced in human IgG1 format with or without the LALA mutation (LALA or G1, respectively).

[0313] Raji cells expressing human MSLN (Raji.hMSLN cells) were generated by lentiviral transduction using the Lenti-X HTX Packaging system (Takara, Catalog No. 631253). Lenti-X expression vector (pLVX) (Takara, Catalog No. 631253) containing cDNA encoding human MLSN was co-transfected with Lenti-X HTX Packaging Mix into Lenti-X 293T cell line (Takara, Catalog No. 632180) to generate virus. Raji cell line (ATCC® CCL-86™) was then transduced with these lentiviral vectors. Expression of human MLSN in these cells was confirmed by binding of G1 / SS1 positive control antibody for 1 hour, and then cell binding was detected using a fluorescently labeled anti-human Fc detection antibody (Stratech Scientific Ltd, Catalog No. 109-546-098-JIR).

[0314] The ADCC Reporter Bioassay Kit (Promega, Cat. No. G7010) was used according to the manufacturer's protocol. Effector cells from the ADCC kit were mixed with Raji.hMSLN cells at a ratio of 20:1. mAb2 or control antibody SS1 were titrated on a 96-well plate and incubated for 6 hours at 37°C 5% CO2. ADCC activity was measured by adding luciferase substrate from Bio-Glo Assay System Kit (Promega, Cat. No. G7941) according to the manufacturer's instructions. Luminescence signal was plotted against log concentration of antibody and the resulting curve was fitted using the equation of log(agonist) vs. response in GraphPad Prism. The results are shown in Figure 2 and show that both FS28-256-271 and FS28-024-052 were able to trigger ADCC activity when in human IgG1 format. As expected, ADCC activity was lost when the LALA mutation was introduced into the hIgG1 backbone of these antibodies. This data demonstrates that the MSLN antibodies described herein can be used to trigger effector functions and modulate immune responses.

[0315] 5.3 Potency of FS28-256-271 in Primary T Cell Assays 5.3.1 Potency of FS28-256-271 in OX40 mAb2 Format In Vitro in Primary T Cell Assays Pan T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from leukodepleted pyramidal cells, a by-product of platelet apheresis donations. Briefly, the contents of the leukodepleted pyramidal cells were flushed with PBS and layered on a Ficoll gradient (GE Lifesciences catalogue no. 17144002). PBMCs were isolated by centrifugation and collection of cells that did not pass through the Ficoll gradient. PBMCs were further washed with PBS and remaining red blood cells were lysed by the addition of 10 ml of red blood cell lysis buffer (eBioscience) according to the manufacturer's instructions. PBMCs were counted and cultured at 2.0 × 10 in T cell medium (RPMI medium (Life Technologies) containing 10% FBS (Life Technologies), 1 × penicillin-streptomycin (Life Technologies), sodium pyruvate (Gibco), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco) and 50 μM 2-mercaptoethanol (Gibco). 6 The cells were resuspended to 10 cells / ml.

[0316] T cells were then isolated from PBMCs using the Pan T cell isolation kit II (Miltenyi Biotec Ltd) according to the manufacturer's instructions. Human T-Activator CD3 / CD28 Dynabeads (Invitrogen 111.32D) were resuspended by vortexing. The beads were washed twice with T cell medium (RPMI medium (Life Technologies) containing 10% FBS (Life Technologies), 1x penicillin-streptomycin (Life Technologies), sodium pyruvate (Gibco), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco) and 50 μM 2-mercaptoethanol (Gibco). 1.0 × 10 cells in T cell medium were cultured at 10 °C for 1 h. 6T cells at a concentration of 1.0 × 10 cells / ml were stimulated with washed human T-Activator CD3 / CD28 Dynabeads in a T-25 flask (Sigma) at a cell-to-bead ratio of 2:1 and incubated overnight at 37°C, 5% CO2 to activate the T cells. Activated T cells were washed with Dynabeads and diluted to 2.0 × 10 6 Activated T cells were resuspended in T cell medium at a concentration of 6×10 cells / ml. 96-well flat-bottom plates were coated with anti-human CD3 antibody by incubation with 2.5 μg / ml anti-human CD3 antibody (R&D systems clone UHCT1) diluted in PBS for 2 hours at 37° C., 5% CO2, and then washed twice with PBS. Activated T cells were resuspended in T cell medium at a concentration of 6×10 cells / ml ...96-well flat-bottom plates were coated with anti-human CD3 antibody by incubation with 2.5 μg / ml anti-human CD3 antibody (R&D systems clone UHCT1) diluted in PBS for 2 hours at 37° C., 5% CO2, and then washed twice with PBS. 5 Cells were added to the plate at 10,000 cells / well. Mesothelin expressing cells, NCI-H226, were added at 20,000 cells / well. Anti-OX40 Fcab as well as mAb2 including anti-MSLN Fab FS28-256-271 were tested in this assay along with control antibodies. Assay plates were incubated for 72 hours at 37°C, 5% CO2. Supernatants were collected and IL-2 release was measured by human IL-2 ELISA (Life Technologies, 88-7025-88). Concentration of human IL-2 (hIL-2) was plotted against log concentration of antibody and the resulting curve was fitted using the equation for log(agonist) vs response in GraphPad Prism. The results are shown in Table 7 and indicate that mAb 1 was activated so that the OX40 binding site could bind and activate OX40. 2 This demonstrates that FS28-256-271 in this format is capable of binding to MSLN expressed on the cell surface and cross-linking antibodies.

[0317] 5.3.2 Potency of FS28-256-271 in CD137 mAb2 Format in In Vitro Primary T Cell Assays Peripheral blood mononuclear cells (PBMCs) were obtained as described in 5.2. CD8 + T cells were cultured according to the manufacturer's instructions using CD8 +T cells were isolated using the T cell isolation kit II (Miltenyi Biotec Ltd, 130-096-495). 96-well flat-bottom tissue culture plates were coated with 8 μg / ml of anti-CD3 antibody (Clone UCHT1, R&D systems, MAB100-SP) in PBS overnight at 4° C. The plates were then washed three times with 200 μl of PBS. NCI-H226 cells were cultured at 2 × 10 cells per well on 96-well flat-bottom plates coated with anti-CD3 antibody (8 μg / ml) in 100 μl of T cell culture medium (RPMI medium (Life Technologies, 61870-044)) containing 10% FBS (Life Technologies), 1 × penicillin-streptomycin (Life Technologies, 15140122), 1 mM sodium pyruvate (Gibco, 11360-070), 10 mM Hepes (Sigma-Aldrich, H0887), 2 mM L-glutamine (Sigma-Aldrich, G7513), and 50 μM 2-mercaptoethanol (Gibco, M6250). 4 After the cells had attached after 4 hours of incubation, all T cell culture medium was removed and 4.0 × 10 cells were plated. 5 Replace with 50 µl T cell culture medium containing T cells at a concentration of 2.0 x 10 cells / ml. 4Cells / well were obtained. Control antibodies or mAb2 including CD137 Fcab and MSLN Fab FS28-256-271 were diluted in T cell media at 4x final concentration starting at 60nM with 1:3 or 1:7 serial dilutions. 50μl of antibody titration was added to cells for a total assay volume of 200μl and 1x concentration of antibody. Assays were incubated for 72 hours at 37°C, 5% CO2. Supernatants were collected and assayed using the V-PLEX IL-2 kit from Meso Scale Discovery (K151QQD-4) according to the manufacturer's instructions. The concentration of human IL-2 (hIL-2) was plotted against the log concentration of antibody and the resulting curve was fitted using the log(agonist) vs response equation in GraphPad Prism. The results are shown in Table 7 and indicate that mAb 2 was activated so that the CD137 binding site can bind and activate CD137. 2 This demonstrates that FS28-256-271 in this format is capable of binding to MSLN on the cell surface and cross-linking antibodies.

[0318] 5.3.3 Overview of the Sequence-Optimized MSLN Clone FS28-256-271 and Its Use to Drive Agonism of TNFRSF Members Table 7 shows the results of the T cell assays described in 5.3.1 and 5.3.2, which show that mAbs crosslinking and activating different TNFSFRs, such as OX40- or CD137, induce agonism in T cells. 2 This demonstrates the ability of anti-MSLN Fabs such as FS28-256-271 in this format.

[0319] [Table 8]

[0320] Summary of Isolation of Anti-Human Mesothelin mAb (Examples 1 to 5) Phage selection and antibody screening approaches aimed at isolating anti-human MSLN antibodies that bind to cell surface MSLN led to the identification of a series of specific anti-mesothelin binding antibodies with a range of affinities, MUC16-MSLN blocking activities, MSLN domain binding bins and cell binding properties. Due to the screening cascade performed, most anti-MSLN binding Fabs were identified as mAbs or mAbs. 2 Regardless of which format was tested, it bound preferentially to cell surface MSLN.

[0321] Example 6: Selection and characterization of anti-mouse MSLN antibodies 6.1 Naive selection of anti-mouse MSLN mAbs The amino acid identity between mouse and human MSLN is low (60%). To enable in vivo proof-of-concept (PoC) studies in mice, we set out to isolate an anti-mouse MSLN mAb with similar properties to the anti-human MSLN mAbs described in Examples 1 to 5.

[0322] Phage selections using a synthetic naive phagemid library displaying human IgG1 germline Fab domains with randomizations in CDR1, CDR2 and CDR3 (MSM Technologies) were used for selections with biotinylated mouse MSLN-His-Avi (SEQ ID NO: 171, see section 1.1) as described in section 1.2. Four rounds of selections were performed with decreasing concentrations of biotinylated mMSLN-His-Avi and similar to the anti-human MSLN selections, and epitope masking procedures were performed in subsequent runs. Additionally, after the first round with recombinant antigen, HEK293-mMSLN cells were generated and used for rounds 2, 3 and 4.

[0323] Briefly, mouse MSLN sequences were subcloned into the pcDNA5 / FRT / TO vector (Life Technologies, V652020) and then co-transfected with the Flp recombinase expression plasmid, pOG44 (Life Technologies, V600520), into the Flp-In TREx 293 cell line (Life Technologies, R78007). Cells were grown in DMEM containing 10% FBS, 100 μg / ml hygromycin B (Melford Laboratories Ltd, Z2475) and 15 μg / ml blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until colonies of stably transformed cells formed. These colonies were expanded in the presence of 1 μg / ml doxycycline (Sigma Aldrich, D9891) and tested for expression of MSLN using anti-mouse MSLN (LS Bio, LS-C179484).

[0324] A total of 47 individual mAbs from the enriched population were screened for antigen binding, and 45 unique positive binders were subcloned and expressed as soluble mAbs in IgG1 LALA format as described above in Example 1.3. The mAbs were characterized for specific binding to immobilized mMSLN-His-Avi by ELISA and ranked based on their affinity to immobilized mMSLN-His-Avi at approximately 50 or 200 RU in kinetic experiments using Biacore analysis. This identified a series of mAbs including FS28m-194, FS28m-201, FS28m-209, FS28m-216, FS28m-228, FS28m-261 and FS28m-265 with affinities ranging from 1 to 25 nM. Furthermore, binding to different regions of MSLN was tested as described in Section 2.1.3. Mouse cross-reactive mAb G1-AA / MOR6626, generated by cloning the VH and VL of the MOR6626 clone (Patent Publication No. WO 2009 / 068204 A1), was used as a positive control. Most clones, especially FS28m-228, were unable to bind to MSLN already bound to MOR6626, while other clones such as FS28-194 or FS28-026 showed partial or complete binding, respectively. Thus, clones binding to different regions (bins) were isolated. The anti-MSLN binding region of MOR6626 has been used in in vivo PoC studies (Patent Publication No. US 2017 / 0342169 A1). Based on the data obtained, FS28m-228 may bind to a similar region of MSLN as MOR6626.

[0325] 6.2 Affinity maturation of anti-mouse MSLN mAb The VH and VL CDR3 regions of FS28m-228 were optimized in parallel in scFv format by randomizing overlapping cassettes of 5 amino acids using NNK primers as described in Example 3.1.1. Two rounds of selection were performed, the first with 50 nM biotinylated mMSLN-His-Avi and the second with 0.2 nM mMSLN-His-Avi. For the second round, off-rate selection pressure was also applied by adding a 1000-fold excess (200 nM) of mMSLN-His-Avi (non-biotinylated) and incubating the antigen / phage mixture for 2.5 hours at room temperature. Soluble scFv (single point concentration) was then tested for binding to mMSLN-His-Avi using Octet. Briefly, streptavidin sensors (ForteBio, 18-5019) were incubated with mMSLN-His-Avi (10 μg / ml) for 5 min. The association of soluble scFvs diluted to a final concentration of 1× buffer (ForteBio, 18-1092) to mMSLN was analyzed using 10× kinetic buffer for 5 min, followed by a 5 min dissociation step. Compared to the parental FS28m-228 scFv, about 66 of the 85 clones tested showed improved binding. Nine clones were identified as mAbs. 2 The Fab arm was then combined with an Fcab that binds mouse CD137 (called FS22-063-AA) using the same cloning and expression methods as described (section 3.1.2). The resulting mAb is shown. 2 were tested for their MSLN binding affinity.

[0326] 6.3 Anti-mouse MSLN mAb against fixed and soluble mouse MSLN 2 Binding affinity of Similar to the anti-human MSLN binders, affinity matured variants derived from FS28m-228 were tested for binding to immobilized and soluble MSLN using Biacore. The procedure for binding to immobilized MSLN was similar to that described in Section 2.1.4 for mMSLN-His-Avi immobilized at 50 RU. To determine affinity for soluble MSLN, mAb 2 was captured via anti-human Fc. Briefly, 25 μg / ml of anti-human IgG (Fc) antibody (GE Healthcare, Human Antibody Capture Kit, BR100839) was coated onto flow cells 1, 2, 3 and 4 of a Biacore sensor chip CM5 (GE Healthcare, BR100530) to achieve a final response of about 750 RU. mAb2 clone diluted in HBS-EP buffer (GE Healthcare, BR100188) at 50 nM was injected individually at 30 μl / min on flow cells 2, 3 and 4 to achieve a response of about 100 RU. Recombinant mMSLN-His-Avi antigen diluted in HBS-EP buffer was injected over flow cells 1, 2, 3 or 4 at concentrations ranging from 243 nM to 0.11 nM as required in 3-fold dilutions for 5 min at 70 μl / min, followed by dissociation in buffer for 5 min. Regeneration was performed by injecting 3M magnesium chloride (GE Healthcare, Human Antibody Capture Kit, BR100839) for 30 seconds at a rate of 30 μl / min.

[0327] Kinetic data for both immobilized and solution affinity (Table 8) showed that the affinity matured clones showed improved binding to mouse MSLN-His-Avi antigen compared to the parental FS28m-228 Fab arm. Furthermore, all clones showed binding kinetics with higher affinity to immobilized MSLN than to soluble MSLN in variable ratios. Similar to the human clones described in sections 4.1 and 5.1, it was believed that these clones may have stronger binding to membrane-bound MSLN than to soluble shed MSLN due to the enhanced strong binding interaction. Thus, affinity maturation of FS28m-228 resulted in a series of affinity matured clones with increased affinity to mouse MSLN, all of which bound more strongly to immobilized MSLN than to soluble MSLN. Clone FS28m-228-010 was selected as the preferred clone since it had the highest affinity to immobilized mouse MSLN and the lowest affinity to mouse MSLN in solution.

[0328] [Table 9]

[0329] 6.4 Overview of isolation of anti-mouse mesothelin mAb Phage selection and antibody screening procedures led to the identification of a series of anti-mouse mesothelin-binding clones with a range of affinities that bound to different regions of mMSLN. Similar to the anti-human MSLN binders, the clones exhibited binding properties that favored binding to immobilized mMSLN over soluble mMSLN, making them suitable molecules to test in a mouse in vivo PoC assay.

[0330] 6.5 In vivo efficacy of mAb2 containing anti-mouse MSLN mAb FS28m-228-010 Having shown that anti-MLSN mAbs of the hIgG1 isotype were capable of eliciting ADCC activity, it was desirable to demonstrate that anti-MLSN mAbs in the CD137 mAb2 format were capable of eliciting MLSN-dependent cross-linking in vivo.

[0331] A syngeneic mouse tumor model expressing mouse MSLN was constructed. CT26 colon cancer cells (ATCC, CRL-2638) expressing full-length mouse mesothelin (SEQ ID NO: 171) were produced by lipofection (Lipofectamine 3000, Thermo Fisher Scientific, Catalog No. L3000008) using pcDNA3.1 vector (+) (Thermo Fisher Scientific, Catalog No. V79020). CT26 cells were transfected with pcDNA3.1 vector containing mouse MSLN cDNA according to the manufacturer's protocol. Stable transfection was performed in complete medium (RPMI, 10% FBS) using Geneticin as selection antibiotic (at 600 μg / ml). Expression of mouse MSLN in CT26 cells was confirmed by flow cytometry by using positive control antibody MOR6626 (WO 2009 / 068204 A1). Specifically, cells were incubated with a positive control antibody for 1 hour, and then cell binding was detected using a fluorescently labeled anti-human IgG detection antibody (Stratech Scientific Ltd, Cat. No. 109-546-098-JIR). Clonal populations were expanded and then analyzed to determine relative expression levels using the same flow cytometry procedure, after which one clone was selected and named CT26.G10.

[0332] CT26.G10 tumor growth was confirmed in vivo. 8-10 week old Balb / c female mice (Charles River) were microchipped to provide a unique identifier. Each cohort had 17 mice, and each animal received 1 × 10 IgG subcutaneously injected in 100 μl of serum-free medium into the dorsal left flank. 5Tumor volume measurements were performed three times a week using calipers to determine the longest and shortest axes of the tumor. Tumor volume was calculated using the following formula: Volume = L x S22 (where L=longest axis; S=shortest axis).

[0333] In accordance with the United Kingdom Animal (Scientific Procedures) Act and EU Directive EU86 / 609, the study was stopped when tumor volume reached a humane endpoint. Tumor tissue was harvested at the end of the study and expression of membrane-bound mesothelin was confirmed by immunohistochemical staining in formalin-fixed paraffin-embedded (FFPE) tumor tissue as follows: 4 μm FFPE tissue sections were deparaffinized and antigen retrieved using low pH 6.1 at 97°C (Dako PT Link), followed by peroxidase and protein blocks before incubation with primary anti-mesothelin antibody at a concentration of 1 μg / ml (LifeSpan Biosciences, Cat. No. LS-C407883). Anti-mesothelin antibody was detected using a labeled polymer-HRP anti-rabbit secondary reagent and a DAB (3,3'-diaminobenzidine) chromogenic endpoint (Dako EnVision+ System).

[0334] To evaluate the antibody FS28m-228-010, the following molecules or combinations were tested in vivo: the FS28m-228-010 antibody of human IgG1 isotype with the LALA mutation (G1-AA / FS28m-228-010), two "mock" CD137 mAbs 2 (FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420), FS28m-228-010 antibody, and mock CD137 mAb with the LALA mutation 2(G1-AA / FS28m-228-010+FS22m-063-AA / HelD1.3), a human isotype control antibody (G1-AA / HelD1.3) and finally a CD137 / MSLN mAb2 with the LALA mutation (FS22m-063-AA / FS28m-228-010) (SEQ ID NOs: 196 and 197).

[0335] Balb / c female mice (Charles River), 8-10 weeks old and weighing 20-25 g each, were acclimated for 1 week before the start of the study. All animals were microchipped to provide a unique identifier. Each cohort contained 20 mice, except for FS22m-063-AA / 4420 (n=10 mice). The CT26.G10 colon cancer cell line was propagated and a cell bank was generated. Each animal received 1x10 6 cells injected subcutaneously in the left flank in 100 μl of serum-free medium. 5 Mice that did not have tumors 12 days after tumor cell inoculation were removed from the study.

[0336] A dose of 200 μg of each antibody (approximately 10 mg / kg) was prepared and injected intraperitoneally (IP) into mice. In addition, both G1-AA / FS28m-228-010+FS22m-063-AA / HelD1.3 were prepared at 200 μg per dose (approximately 10 mg / kg) for the combination group. A dose of 200 μl was administered to mice 12, 14, and 16 days after tumor inoculation (alternate days (q2d)×3). Tumor volume measurements were performed three times a week using calipers, and mice were closely monitored. Study endpoints were determined by humane endpoints based on tumor volume and condition of mice.

[0337] As shown in Figure 3, FS22m-063-AA / FS28m-228-010 mAb 2 significantly inhibited tumor growth compared to the G1-AA / HelD1.3 isotype control. Table 9 shows pairwise comparisons of tumor growth rates for all treatment groups over the course of the study using mixed model analysis, comparing all groups to the G1-AA / HelD1.3 isotype control.

[0338] 62.5mm at the end of the test 3 All animals bearing the following tumors were counted as full responders (see Table 10): Anti-CD137 / MSLN mAb 2 Thirty-five percent of animals treated with FS22m-063-AA / HelD1.3 demonstrated a complete response to treatment at the end of the study, compared with 0% in the G1-AA / HelD1.3 isotype control, FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420, and FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 combination groups.

[0339] Survival analysis (Figure 4 and Table 11) showed that FS22m-063-AA / FS28m-228-010 mAb 2 showed that FS22m-063-AA / FS28m-228-010 mAb induced a significant survival benefit compared to the G1-AA / HelD1.3 antibody, whereas G1-AA / FS28m-228-010, FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420 showed no survival benefit. 2 resulted in an improved median survival of 42.5 days compared with G1-AA / HelD1.3 (29 days), FS22m-063-AA / HelD1.3 (30 days), FS22m-063-AA / 4420 (29 days), G1-AA / FS28m-228-010 (30 days) and the combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010 (29 days).

[0340] These data suggest that mAbs mediate the MSLN 2This suggests that cross-linking of the mAb with the LALA mutation (G1-AA / FS28m-228-010) may induce CD137 agonism in tumors; a better effect of bispecific antibodies targeting CD137 and / or MSLN alone (and in combination), resulting in significantly improved survival of tumor-bearing mice. The mAb with the LALA mutation (G1-AA / FS28m-228-010) did not show any inherent activity in this study by itself. Without wishing to be bound by theory, it is expected that the effector-competent form of the antibody FS28m-228-010 (minus the LALA mutation) will show ADCC activity and antitumor efficacy, consistent with the in vitro results observed in section 5.2.

[0341] [Table 10]

[0342] [Table 11]

[0343] [Table 12]

[0344] 6.6 Anti-mouse CD137 / MSLN mAb containing anti-mouse MLSN mAb FS28m-228-010 2 Mechanism of action To further understand the pharmacology of the antitumor responses observed with FS22m-063-AA / FS28m-228-010, which contains the anti-mouse MSLN mAb FS28m-228-010, we performed a multicenter study comparing CD137 / MSLN mAbs in MSLN-positive syngeneic tumor models. 2 The mechanism of action was investigated.

[0345] Mice were prepared as described in Example 6.5 and inoculated with the CT26.G10 colon cancer cell line. Each cohort consisted of 20 mice. FS22m-063-AA / FS28-228-010 (CD137 / MSLN) mAb 2, a human IgG1 isotype control (G1-AA / 4420) and an anti-CD137 agonist antibody (clone 3H3; G1 / 3H3; Rickert et al., 2016) were also included for comparison. All three antibodies were prepared in DPBS + 1 mM arginine + 0.05% Tween 80 at 134 μg per dose (approximately 6.7 mg / kg for a 20 g mouse) and injected intraperitoneally (IP) into mice. Each mouse was administered antibody at a fixed dose of 134 μg by a single 200 μl IP injection on day 20 after tumor inoculation. Tumor volume measurements were performed three times a week using calipers as described in Example 6.5, and mice were closely monitored.

[0346] Six mice per group were sacrificed at 20 days after tumor inoculation, 24, 72, 144 and 192 hours after dosing. Spleen, blood and tumor tissues were collected for analysis from CT26.G10 tumor-bearing mice treated with either FS22m-063-AA / FS28-228-010, G1-AA / 4420 or G1 / 3H3. All samples were examined for T cell abundance and proliferation by flow cytometry, as T cell activation and proliferation markers are known to be downstream effects of CD137 agonism (Fisher et al., 2012). In addition, serum from blood was also collected for detection and quantification of soluble MSLN expression. Spleen and tumor tissues were disaggregated into single cell suspensions by standard mechanical and enzymatic methods, and red blood cells were lysed once in red blood cell lysis buffer (Miltenyi Biotec Ltd., 130-094-183). Blood was collected by terminal cardiac bleed, half was collected in tubes containing EDTA for single cell analysis by flow cytometry, and half of the blood was collected in clot activator / serum tubes for analysis of soluble MSLN. Whole blood collected in tubes containing EDTA was lysed three times in red blood cell lysis buffer (Miltenyi Biotec Ltd., 130-094-183) according to the manufacturer's instructions. Blood collected in serum tubes was fractionated by centrifugation, and serum was removed for analysis of soluble MSLN.

[0347] Single cells from spleen, tumor and blood were then treated similarly, cells were washed once with PBS and samples were stained with fixable viability dye (eBioscience, 65-0865-14). Cells were then stained for cell surface markers using the antibody staining panel shown in Table 12 (except intracellular markers, Ki67 and FoxP3) in the presence of Fc block (eBioscience, 16-0161-85 at 1:25) for 45 minutes at 4°C. Cells were then fixed and permeabilized with eBioscience FoxP3 staining kit (eBioscience, 00-5523-00) according to the manufacturer's instructions. Cells were resuspended in 100 μl of permeabilization buffer with intracellular markers Ki67 and FoxP3 antibodies and incubated overnight at 4°C in the dark. Cells were washed once with permeabilization buffer and resuspended in 120 μl of PBS containing 0.5% BSA before acquisition on a BD Fortessa flow cytometer. Data were acquired using BD FACS Diva software and analyzed using FlowJo (V10) and Microsoft Excel. Data are expressed as percentages of the parent population and are expressed as CD8 + T cell abundance and proliferation are shown.

[0348] [Table 13]

[0349] As shown in Table 13, CD8 + The increase in the percentage of T cells was greater in the G1 / 3H3 and FS22m-063-AA / FS28-228-010 mAbs compared to the control treatment group (G1-AA / 4420). 2 The CD8 + The mean percentage of T cells increased from 32.1% (G1-AA / 4420) to 56.1% in G1 / 3H3 and to 58.4% in FS22m-063-AA / FS28m-228-010 at 144 h post-dose.

[0350] In addition, CD8 + Increased abundance of T cells was also observed in the blood and spleen, but only with G1 / 3H3 compared to the IgG1 control. + The mean percentage of T cells increased from 22.6% (G1-AA / 4420) to 57.0% (G1 / 3H3), but this increase was not observed in FS22m-063-AA / FS28m-228-010 (25.8%). + The mean percentage of T cells increased from 28.8% (G1-AA / 4420) to 38.0% in G1 / 3H3, but this increase was not observed in FS22m-063-AA / FS28m-228-010 (29%).

[0351] This is FS22m-063-AA / FS28m-228-010 mAb 2 However, when MSLN is expressed, CD8 + While this suggests that CD137-targeting antibody, G1 / 3H3, also increases CD8 + Shown is an increase in T cells in the periphery (blood and spleen).

[0352] After administration, CD8 + To determine whether there were differences in T cell proliferation, we measured the proliferation marker, Ki67, in tumors, blood, and spleen compared with CD8+ cells. + As shown in Table 14, a high percentage of CD8 + T cells were significantly higher in the control group than in the control group. + (mean expression of 75.1%), which correlates with high levels of proliferating CD8 + This suggests that CD8 T cells in the tumors were significantly higher in the treated groups than in the control group. + This may lead to unclear differences in Ki67 expression in T cells.

[0353] In comparison, CD8 in blood and spleen + Ki67 in T cells +A clear increase in expression was observed 144 hours after administration of G1 / 3H3 compared to the IgG1 control. In the blood, isotype control-treated mice had 10.4% of CD8 + Mean Ki67 in T cells + While the expression of G1 / 3H3 was not observed in + The mean expression of Ki67 in T cells is shown to be 86.3% at 144 hours post-administration. In comparison, this increase was not observed in FS22m-063-AA / FS28m-228-010, where CD8 + Mean Ki67 in T cells + Expression of mAb in blood 2 Similarly, in the spleen, the mean Ki67 + Expression was significantly higher in CD8+ cells after treatment with G1 / 3H3 compared to 8.1% observed after treatment with the isotype control and 11.4% observed after treatment with FS22m-063-AA / FS28m-228-010. + It was observed in 36.1% of T cells.

[0354] [Table 14]

[0355] [Table 15]

[0356] Taken together, these data indicate that the anti-mouse MLSN mAb FS28m-228-010 and mAb2 containing FS22m-063-AA / FS28m-228-010 inhibited cytotoxic CD8 + We show that this CD137-targeted agonist mediates tumor-specific expansion of T cells, as also observed with G1 / 3H3, but not with CD8 T cells in the blood and spleen. + It also promotes peripheral expansion of T cells. + T cells also show increased proliferation following administration of G1 / 3H3.

[0357] Sequence Listing Heavy chain amino acid sequence of parental clone FS28-256 showing the "master" CDR2 sequence applicable to the parental clone and affinity matured clones FS28-256-001, FS28-256-005, FS28-256-012, FS28-256-014, FS28-256-018, FS28-256-021, FS28-256-023, FS28-256-024, FS28-256-026 and FS28-256-027. The double underlined "X" represents all potential substitutions to eliminate possible N-linked glycosylation. The resulting clones may contain either one or the other substitutions or both. SEQ ID NO:1 Heavy chain AA (no LALA) [ka] SEQ ID NO:2 Heavy chain AA (with LALA) [ka]

[0358] Master light chain amino acid sequence applicable to clones FS28-256-001, FS28-256-021 and FS28-256-023. The double underlined "X" represents all potential substitutions to remove possible deamidation sites. The resulting clones may contain either one or the other substitution or both. SEQ ID NO:3 Light chain [ka]

[0359] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-024 mAb and amino acid sequences of the CDRs SEQ ID NO: 4 Heavy chain AA (no LALA) [ka] SEQ ID NO:6 Heavy chain DNA (without LALA) [ka] SEQ ID NO:5 Heavy chain AA (with LALA) [ka] SEQ ID NO: 7 Heavy chain DNA (having LALA) [ka] SEQ ID NO:8 Variable domain AA [ka] SEQ ID NO:9 Variable domain DNA [ka] [ka]

[0360] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-024 mAb and amino acid sequences of the CDRs SEQ ID NO: 16 Light chain AA [ka] SEQ ID NO:17 Light chain DNA [ka] SEQ ID NO: 18 Variable domain AA [ka] SEQ ID NO: 19 Variable domain DNA [ka] [ka]

[0361] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-024-051 mAb and amino acid sequences of the CDRs SEQ ID NO:26 Heavy chain AA (without LALA) [ka] SEQ ID NO:27 Heavy chain DNA (without LALA) [ka] SEQ ID NO:28 Heavy chain AA (with LALA) [ka] SEQ ID NO:29 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 30 Variable domain AA [ka] SEQ ID NO:31 Variable domain DNA [ka] [ka]

[0362] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-024-051 mAb and amino acid sequences of the CDRs SEQ ID NO: 16 Light chain AA [ka] SEQ ID NO:17 Light chain DNA [ka] SEQ ID NO: 18 Variable domain AA [ka] SEQ ID NO: 19 Variable domain DNA [ka] [ka]

[0363] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-024-052 mAb and amino acid sequences of the CDRs SEQ ID NO:35 Heavy chain AA (without LALA) [ka] SEQ ID NO:36 Heavy chain DNA (without LALA) [ka] SEQ ID NO:37 Heavy chain AA (with LALA) [ka] SEQ ID NO:38 Heavy chain DNA (having LALA) [ka] SEQ ID NO: 39 Variable domain AA [ka] SEQ ID NO: 40 Variable domain DNA [ka] [ka]

[0364] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-024-052 mAb and amino acid sequences of the CDRs SEQ ID NO:16 Light chain AA [ka] SEQ ID NO:17 Light chain DNA [ka] SEQ ID NO: 18 Variable domain AA [ka] SEQ ID NO: 19 Variable domain DNA [ka] [ka]

[0365] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-024-053 mAb and amino acid sequences of the CDRs SEQ ID NO: 45 Heavy chain AA (without LALA) [ka] SEQ ID NO: 46 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 47 Heavy chain AA (with LALA) [ka] SEQ ID NO:48 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 49 Variable domain AA [ka] SEQ ID NO:50 Variable domain DNA [ka] [ka]

[0366] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-024-053 mAb and amino acid sequences of the CDRs SEQ ID NO: 16 Light chain AA [ka] SEQ ID NO:17 Light chain DNA [ka] SEQ ID NO: 18 Variable domain AA [ka] SEQ ID NO: 19 Variable domain DNA [ka] [ka]

[0367] Amino acid sequences of the heavy and light chains of FS28-024-060 mAb (with LALA) SEQ ID NO:55 Heavy chain AA (with LALA) [ka] SEQ ID NO: 16 Light chain AA [ka]

[0368] Amino acid sequences of the heavy and light chains of FS28-026 mAb (with LALA) SEQ ID NO:58 Heavy chain AA (with LALA) [ka] SEQ ID NO:59 Light chain AA [ka]

[0369] Amino acid sequences of the heavy and light chains of FS28-091 mAb (with LALA) SEQ ID NO:60 Heavy chain AA (with LALA) [ka] SEQ ID NO:61 Light chain AA [ka]

[0370] Amino acid sequences of the heavy and light chains of FS28-185 mAb (with LALA) SEQ ID NO:62 Heavy chain AA (with LALA) [ka] SEQ ID NO: 195 Light chain AA [ka]

[0371] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256 mAb and amino acid sequences of the CDRs SEQ ID NO:65 Heavy chain AA (without LALA) [ka] SEQ ID NO:66 Heavy chain DNA (without LALA) [ka] SEQ ID NO:67 Heavy chain AA (with LALA) [ka] SEQ ID NO:68 Heavy chain DNA (with LALA) [ka] SEQ ID NO:69 Variable domain AA [ka] SEQ ID NO: 70 Variable domain DNA [ka] [ka]

[0372] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256 mAb and amino acid sequences of the CDRs SEQ ID NO:77 Light chain AA [ka] SEQ ID NO:78 Light chain DNA [ka] SEQ ID NO: 79 Variable domain AA [ka] SEQ ID NO: 80 Variable domain DNA [ka] [ka]

[0373] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-001 mAb and amino acid sequences of the CDRs SEQ ID NO: 81 Heavy chain AA (without LALA) [ka] SEQ ID NO:82 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 89 Heavy chain AA (with LALA) [ka] SEQ ID NO:84 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 63 Variable domain AA [ka] SEQ ID NO:64 Variable domain DNA [ka] [ka]

[0374] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-001 mAb and amino acid sequences of the CDRs SEQ ID NO:83 Light chain AA [ka] SEQ ID NO:92 Light chain DNA [ka] SEQ ID NO: 93 Variable domain AA [ka] SEQ ID NO:94 Variable domain DNA [ka] [ka]

[0375] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-005 mAb and amino acid sequences of the CDRs SEQ ID NO: 87 Heavy chain AA (without LALA) [ka] SEQ ID NO:88 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 89 Heavy chain AA (with LALA) [ka] SEQ ID NO:84 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 63 Variable domain AA [ka] SEQ ID NO:64 Variable domain DNA [ka] [ka]

[0376] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-005 mAb, and amino acid sequences of the CDRs SEQ ID NO: 90 Light chain AA [ka] SEQ ID NO:91 Light chain DNA [ka] SEQ ID NO:53 Variable domain AA [ka] SEQ ID NO:54 Variable domain DNA [ka] [ka]

[0377] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-012 mAb and amino acid sequences of the CDRs SEQ ID NO: 105 Heavy chain AA (without LALA) [ka] SEQ ID NO: 106 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 107 Heavy chain AA (with LALA) [ka] SEQ ID NO: 108 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 109 Variable domain AA [ka] SEQ ID NO:110 Variable domain DNA [ka] [ka]

[0378] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-012 mAb and amino acid sequences of the CDRs SEQ ID NO:77 Light chain AA [ka] SEQ ID NO:78 Light chain DNA [ka] SEQ ID NO: 79 Variable domain AA [ka] SEQ ID NO: 80 Variable domain DNA [ka] [ka]

[0379] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-014 mAb and amino acid sequences of the CDRs SEQ ID NO: 117 Heavy chain AA (without LALA) [ka] SEQ ID NO:118 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 119 Heavy chain AA (with LALA) [ka] SEQ ID NO:120 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 115 Variable domain AA [ka] SEQ ID NO: 116 Variable domain DNA [ka] [ka]

[0380] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-014 mAb and amino acid sequences of the CDRs SEQ ID NO:77 Light chain AA [ka] SEQ ID NO:78 Light chain DNA [ka] SEQ ID NO: 79 Variable domain AA [ka] SEQ ID NO: 80 Variable domain DNA [ka] [ka]

[0381] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-018 mAb and amino acid sequences of the CDRs SEQ ID NO: 123 Heavy chain AA (without LALA) [ka] SEQ ID NO:124 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 125 Heavy chain AA (with LALA) [ka] SEQ ID NO: 126 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 121 Variable domain AA [ka] SEQ ID NO: 122 Variable domain DNA [ka] [ka]

[0382] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-018 mAb and amino acid sequences of the CDRs SEQ ID NO:77 Light chain AA [ka] SEQ ID NO:78 Light chain DNA [ka] SEQ ID NO: 79 Variable domain AA [ka] SEQ ID NO: 80 Variable domain DNA [ka] [ka]

[0383] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-021 mAb and amino acid sequences of the CDRs SEQ ID NO: 105 Heavy chain AA (without LALA) [ka] SEQ ID NO: 106 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 107 Heavy chain AA (with LALA) [ka] SEQ ID NO: 108 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 109 Variable domain AA [ka] SEQ ID NO:110 Variable domain DNA [ka] [ka]

[0384] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-021 mAb and amino acid sequences of the CDRs SEQ ID NO:83 Light chain AA [ka] SEQ ID NO:92 Light chain DNA [ka] SEQ ID NO: 93 Variable domain AA [ka] SEQ ID NO:94 Variable domain DNA [ka] [ka]

[0385] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-023 mAb and amino acid sequences of the CDRs SEQ ID NO: 123 Heavy chain AA (without LALA) [ka] SEQ ID NO:124 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 125 Heavy chain AA (with LALA) [ka] SEQ ID NO: 126 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 121 Variable domain AA [ka] SEQ ID NO: 122 Variable domain DNA [ka] [ka]

[0386] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-026 mAb and amino acid sequences of the CDRs SEQ ID NO: 123 Heavy chain AA (without LALA) [ka] SEQ ID NO:124 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 125 Heavy chain AA (with LALA) [ka] SEQ ID NO: 126 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 121 Variable domain AA [ka] SEQ ID NO: 122 Variable domain DNA [ka] [ka]

[0387] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-023 mAb and amino acid sequences of the CDRs SEQ ID NO:83 Light chain AA [ka] SEQ ID NO:92 Light chain DNA [ka] SEQ ID NO: 93 Variable domain AA [ka] SEQ ID NO:94 Variable domain DNA [ka] [ka]

[0388] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-024 mAb and amino acid sequences of the CDRs SEQ ID NO: 105 Heavy chain AA (without LALA) [ka] SEQ ID NO: 106 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 107 Heavy chain AA (with LALA) [ka] SEQ ID NO: 108 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 109 Variable domain AA [ka] SEQ ID NO:110 Variable domain DNA [ka] [ka]

[0389] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-024 mAb and amino acid sequences of the CDRs SEQ ID NO: 90 Light chain AA [ka] SEQ ID NO:91 Light chain DNA [ka] SEQ ID NO:53 Variable domain AA [ka] SEQ ID NO:54 Variable domain DNA [ka] [ka]

[0390] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-026 mAb and amino acid sequences of the CDRs SEQ ID NO: 90 Light chain AA [ka] SEQ ID NO:91 Light chain DNA [ka] SEQ ID NO:53 Variable domain AA [ka] SEQ ID NO:54 Variable domain DNA [ka] [ka]

[0391] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-027 mAb and amino acid sequences of the CDRs SEQ ID NO: 105 Heavy chain AA (without LALA) [ka] SEQ ID NO: 106 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 107 Heavy chain AA (with LALA) [ka] SEQ ID NO: 108 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 109 Variable domain AA [ka] SEQ ID NO:110 Variable domain DNA [ka] [ka]

[0392] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-027 mAb and amino acid sequences of the CDRs SEQ ID NO: 95 Light chain AA [ka] SEQ ID NO:96 Light chain DNA [ka] SEQ ID NO:56 Variable domain AA [ka] SEQ ID NO:57 Variable domain DNA [ka] [ka] SEQ ID NO: 175 Human MSLN preprotein [ka]

[0393] Amino acid and cDNA sequences of the heavy chain and its variable domain of FS28-256-271 mAb and amino acid sequences of the CDRs SEQ ID NO: 176 Heavy chain AA (without LALA) [ka] SEQ ID NO: 177 Heavy chain DNA (without LALA) [ka] SEQ ID NO: 178 Heavy chain AA (with LALA) [ka] SEQ ID NO: 179 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 180 Variable domain AA [ka] SEQ ID NO: 181 Variable domain DNA [ka] [ka]

[0394] Amino acid and cDNA sequences of the light chain and its variable domain of FS28-256-271 mAb and amino acid sequences of the CDRs SEQ ID NO: 95 Light chain AA [ka] SEQ ID NO:96 Light chain DNA [ka] SEQ ID NO:56 Variable domain AA [ka] SEQ ID NO:57 Variable domain DNA [ka] [ka]

[0395] FS22-053-008 / FS28-024 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 143 Heavy chain AA (without LALA) [ka] SEQ ID NO: 144 Heavy chain AA (with LALA) [ka] SEQ ID NO: 16 Light chain AA [ka]

[0396] FS22-053-008 / FS28-024-051 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 145 Heavy chain AA (without LALA) [ka] SEQ ID NO: 146 Heavy chain AA (with LALA) [ka] SEQ ID NO: 16 Light chain AA [ka]

[0397] FS22-053-008 / FS28-024-052 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 147 Heavy chain AA (without LALA) [ka] SEQ ID NO: 148 Heavy chain AA (with LALA) [ka] SEQ ID NO: 16 Light chain AA [ka]

[0398] FS22-053-008 / FS28-024-053 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 149 Heavy chain AA (without LALA) [ka] SEQ ID NO: BO:150 Heavy chain AA (with LALA) [ka] SEQ ID NO:16 Light chain AA [ka]

[0399] FS22-053-008-AA / FS28-024-060 mAb 2 Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 151 Heavy chain AA (with LALA) [ka] SEQ ID NO:16 Light chain AA [ka]

[0400] FS22-053-008-AA / FS28-026 mAb 2 Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 152 Heavy chain AA (with LALA) [ka] SEQ ID NO:59 Light chain AA [ka]

[0401] FS22-053-008-AA / FS28-091 mAb 2Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 153 Heavy chain AA (with LALA) [ka] SEQ ID NO:61 Light chain AA [ka]

[0402] FS22-053-008-AA / FS28-185 mAb 2 Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 154 Heavy chain AA (with LALA) [ka] SEQ ID NO: 195 Light chain AA [ka]

[0403] FS22-053-008 / FS28-256 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 155 Heavy chain AA (without LALA) [ka] SEQ ID NO: 156 Heavy chain AA (with LALA) [ka] SEQ ID NO:77 Light chain AA [ka]

[0404] FS22-053-008 / FS28-256-001 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 157 Heavy chain AA (without LALA) [ka] SEQ ID NO: 158 Heavy chain AA (with LALA) [ka] SEQ ID NO:83 Light chain AA [ka]

[0405] FS22-053-008 / FS28-256-005 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 159 Heavy chain AA (without LALA) [ka] SEQ ID NO: 160 Heavy chain AA (with LALA) [ka] SEQ ID NO: 90 Light chain AA [ka]

[0406] FS22-053-008 / FS28-256-012 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 127 Heavy chain AA (without LALA) [ka] SEQ ID NO: 128 Heavy chain AA (with LALA) [ka] SEQ ID NO:77 Light chain AA [ka]

[0407] FS22-053-008 / FS28-256-014 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 129 Heavy chain AA (without LALA) [ka] SEQ ID NO: 130 Heavy chain AA (with LALA) [ka] SEQ ID NO:77 Light chain AA [ka]

[0408] FS22-053-008 / FS28-256-018 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 131 Heavy chain AA (without LALA) [ka] SEQ ID NO: 132 Heavy chain AA (with LALA) [ka] SEQ ID NO:77 Light chain AA [ka]

[0409] FS22-053-008 / FS28-256-021 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 133 Heavy chain AA (without LALA) [ka] SEQ ID NO: 134 Heavy chain AA (with LALA) [ka] SEQ ID NO:83 Light chain AA [ka]

[0410] FS22-053-008 / FS28-256-023 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 135 Heavy chain AA (without LALA) [ka] SEQ ID NO: 136 Heavy chain AA (with LALA) [ka] SEQ ID NO:83 Light chain AA [ka]

[0411] FS22-053-008 / FS28-256-024 mAb 2 Amino acid and cDNA sequences of the heavy and light chains of SEQ ID NO: 137 Heavy chain AA (without LALA) [ka] SEQ ID NO: 138 Heavy chain AA (with LALA) [ka] SEQ ID NO: 90 Light chain AA [ka]

[0412] Amino acid sequences of the heavy and light chains of FS22-053-008 / FS28-256-026 SEQ ID NO: 139 Heavy chain AA (without LALA) [ka] SEQ ID NO: 140 Heavy chain AA (with LALA) [ka] SEQ ID NO: 90 Light chain AA [ka]

[0413] FS22-053-008 / FS28-256-027 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 141 Heavy chain AA (without LALA) [ka] SEQ ID NO: 142 Heavy chain AA (with LALA) [ka] SEQ ID NO: 95 Light chain AA [ka]

[0414] FS22-172-003-AA / FS28-256-271 mAb 2 The amino acid sequences of the heavy and light chains of SEQ ID NO: 187 Heavy chain AA (with LALA) [ka] SEQ ID NO: 188 Light chain AA [ka]

[0415] OX40(FS20-022-049) / FS28-256-271 mAb 2 The amino acid sequences of the heavy and light chains of SEQ ID NO: 189 Heavy chain AA (with LALA) [ka] SEQ ID NO: 190 Light chain AA [ka]

[0416] Amino acid sequences of the heavy and light chains of FS28m-228 mAb (with LALA) SEQ ID NO: 161 Heavy chain AA (with LALA) [ka] SEQ ID NO: 162 Light chain AA [ka]

[0417] FS22-063-AA / FS28m-228 mAb with LALA 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 163 Heavy chain AA (with LALA) [ka] SEQ ID NO: 164 Light chain AA [ka]

[0418] Amino acid sequences of the heavy and light chains of FS22m-063-AA / FS28m-228-010 mAb2 SEQ ID NO: 196 Heavy chain AA (with LALA) [ka] SEQ ID NO: 197 Light Chain AA [ka]

[0419] FS22m-063-AA / HelD1.3 mAb 2 Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 191 Heavy chain AA (with LALA) [ka] SEQ ID NO:192 Light chain AA [ka]

[0420] FS22m-063-AA / 4420 mAb 2 Amino acid sequences of the heavy and light chains of (with LALA) SEQ ID NO: 193 Heavy chain AA (with LALA) [ka] SEQ ID NO:194 Light chain AA [ka]

[0421] Amino acid sequences of the heavy and light chains of G1-AA / HelD1.3 mAb (with LALA) SEQ ID NO: 165 Heavy chain AA (with LALA) [ka] SEQ ID NO: 166 Light chain AA [ka]

[0422] Amino acid sequences of the heavy and light chains of G1-AA / SS1 mAb (with LALA) SEQ ID NO: 167 Heavy chain (with LALA) [ka] SEQ ID NO:168 Light chain [ka]

[0423] MSLN-His-Avi Mesothelin (without MPF and C-terminus) (shown) His and Avi tags (not shown) SEQ ID NO: 169 Human [ka] SEQ ID NO:170 Cynomolgus monkey [ka] SEQ ID NO:171 Mouse [ka]

[0424] Amino acid sequence of wild-type CH3 domain SEQ ID NO:172 [ka]

[0425] Amino acid sequence of the CH2 domain containing the LALA mutation (LALA mutations are shown in bold and underlined) SEQ ID NO:173 [ka]

[0426] Amino acid sequence of the CH2 domain containing the LALA-PA mutation (LALA-PA mutations are shown in bold and underlined) SEQ ID NO:174 [ka]

[0427] Amino acid sequence of the CH3 domain loop sequence of Fcab FS22-172-003 FS22-172-003 First sequence - PYIIPPY (SEQ ID NO: 198) FS22-172-003 Second sequence - GADRWLE (SEQ ID NO: 199)

[0428] Amino acid and cDNA sequences of the light chain of FS22-172-003-AA / FS28-256-271 SEQ ID NO:200 Light chain [ka] SEQ ID NO: 201 Heavy chain AA (with LALA) [ka]

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Claims

1. An antibody molecule that binds to mesothelin (MSLN), the antibody molecule being a human IgG1 molecule, and an antigen-binding site of the antibody molecule comprising: (i) FS28-024-052, as set forth in SEQ ID NOs: 10, 11, 41, 20, 21 and 22, respectively; (ii) FS28-024-051, as set forth in SEQ ID NOs: 10, 11, 32, 20, 21 and 22, respectively; (iii) FS28-024-053, as set forth in SEQ ID NOs: 10, 11, 51, 20, 21, and 22, respectively; or (iv) FS28-024, which is described in SEQ ID NOs: 10, 11, 12, 20, 21 and 22, respectively. and / or comprising complementarity determining regions (CDRs) 1 to 6 of:

4. The antigen binding site of the antibody molecule comprising an antibody: (i) FS28-024-052, as set forth in SEQ ID NOs: 13, 14, 42, 23, 24 and 22, respectively; (ii) FS28-024-051, as set forth in SEQ ID NOs: 13, 14, 33, 23, 24 and 22, respectively; (iii) FS28-024-053, as set forth in SEQ ID NOs: 13, 14, 52, 23, 24, and 22, respectively; or (iv) FS28-024, which is described in SEQ ID NOs: 13, 14, 15, 23, 24 and 22, respectively. wherein the CDR sequences are defined according to the Kabat numbering scheme, antibody molecule.

2. antibody: (i) FS28-024-052, as set forth in SEQ ID NOs: 39 and 18, respectively; (ii) FS28-024-051, as set forth in SEQ ID NOs: 30 and 18, respectively; (iii) FS28-024-053, as set forth in SEQ ID NOs: 49 and 18, respectively; or (iv) FS28-024, as described in SEQ ID NOs: 8 and 18, respectively. The antibody molecule of claim 1, comprising a VH domain and a VL domain of

3. The antibody molecule of claim 1, wherein the antigen-binding site of the antibody molecule comprises CDRs 1 to 6, as defined according to the IMGT numbering scheme, and / or CDRs 1 to 6, as defined according to the Kabat numbering scheme, of antibody FS28-024-052.

4. The antibody molecule of claim 2, wherein the antibody molecule comprises the VH and VL domains of antibody FS28-024-052.

5. The antibody molecule of claim 4, comprising the heavy and light chains of antibody FS28-024-052, as set forth in SEQ ID NOs: 37 and 16, respectively.

6. The antibody molecule of claim 5, wherein the heavy chain comprises a CH3 domain, the CH3 domain comprising a lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

7. The antibody molecule described in claim 1 or 2, wherein the antibody molecule comprises a heavy chain comprising a CH3 domain, and the CH3 domain comprises a lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

8. An antibody molecule described in any one of claims 1 to 7, wherein the antibody molecule is a multispecific antibody molecule and comprises a second antigen binding site that binds to a second antigen.

9. The antibody molecule of claim 8 , wherein the second antigen binding site is located in a constant domain of the antibody molecule.

10. The antibody molecule of claim 9, wherein the constant domain is a CH3 domain.

11. The antibody molecule of claim 8 , wherein the second antigen-binding site binds to an immune cell antigen.

12. The antibody molecule of claim 11, wherein the immune cell antigen is a member of the tumor necrosis factor receptor superfamily (TNFRSF).

13. The antibody molecule of claim 12, wherein the member of the TNFRSF is CD137.

14. the second antigen-binding site comprises a first sequence, a second sequence and / or a third sequence; The antibody molecule according to any one of claims 9 to 13, wherein the first sequence, the second sequence and the third sequence are located in the AB structural loop, the CD structural loop and the EF structural loop of the constant domain, respectively.

15. An antibody molecule described in any one of claims 11 to 14, wherein the antibody molecule is capable of activating immune cells in the presence of MSLN.

16. The antibody molecule of claim 15, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell or a dendritic cell (DC). (i) the antibody molecule has been modified to reduce or inhibit binding of the CH2 domain of the antibody molecule to one or more Fcγ receptors; and / or (ii) the antibody molecule does not bind to one or more Fcγ receptors; An antibody molecule according to any one of claims 1 to 16.

18. A conjugate comprising an antibody molecule according to any one of claims 1 to 17 and a biologically active molecule.

19. 18. One or more nucleic acid molecules encoding an antibody molecule according to any one of claims 1 to 17.

20. 20. One or more vectors comprising one or more nucleic acid molecules according to claim 19.

21. 21. A recombinant host cell comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. 28. A method for producing an antibody molecule according to any one of claims 1 to 17, comprising culturing a recombinant host cell according to claim 21 under conditions for the production of said antibody molecule.

23. The method of claim 22, further comprising isolating and / or purifying the antibody molecule.

24. 19. A pharmaceutical composition comprising an antibody molecule or conjugate according to any one of claims 1 to 18 and a pharma- ceutically acceptable excipient.

25. A pharmaceutical composition for treating cancer in an individual, comprising an antibody molecule or a conjugate according to any one of claims 1 to 18.

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