Mesothelin and CD137 binding molecules

Abstract

To solve the problem in which clinical development of CD137 agonist molecules has been held back because of treatment being associated with either dose-limiting high-grade liver inflammation (urelumab) or low clinical efficacy (utomilumab).SOLUTION: The present invention relates to antibody molecules that bind both mesothelin (MSLN) and CD137. The antibody molecules comprise a CDR-based binding site for MSLN, and a CD137 antigen-binding site located in a constant domain of the antibody molecule. The antibody molecules find application in the treatment of cancer, for example.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present invention relates to antibody molecules that bind to both mesothelin (MSLN) and CD137. The antibody molecules contain a CDR-based binding site for MSLN and a CD137 antigen-binding site located in the constant domain of the antibody molecule. The antibody molecules find use, for example, in the treatment of cancer. [Background technology]

[0002] Background of the Invention Cell signaling is an essential part of life in all organisms and typically involves cell surface receptors interacting with soluble or surface-expressed ligands. This interaction results in changes in the receptor, the ligand, or both. For example, ligand binding can induce conformational changes in the receptor, causing the receptor to cluster into dimers or oligomers. This clustering effect then leads to the activation of intracellular signaling pathways. Many receptors are activated in this manner, including members of the tumor necrosis factor receptor superfamily (TNFRSF), such as CD137.

[0003] CD137 (4-1BB; TNFRSF9) is a costimulatory molecule of the tumor necrosis factor receptor superfamily (TNFRSF). CD137 mediates CD8 activation after activation. + It is widely known to be upregulated in T cells and activated CD4 + It can also be expressed in helper T cells, B cells, regulatory T cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells (DCs) (Bartkowiak & Curran, 2015). The primary functional role of CD137 in enhancing tumor toxicity was first described in 1997 (Shuford et al., 1997), and soon thereafter, anti-CD137 mAbs were proposed as anti-cancer therapeutic agents.

[0004] CD137 is a transmembrane protein with four extracellular cysteine-rich domains, designated CRD1-4, and a cytoplasmic region involved in CD137 signaling. The ligand for CD137 is CD137L. Although no crystal structure of the CD137 / CD137L complex exists, CD137 is predicted to form a trimeric / trimeric complex with CD137L (Won et al., 2010). CD137L engagement leads to the formation of receptor trimers and subsequent clustering of multiple receptor trimers, activating the CD137 signaling cascade. This signaling cascade provides T cells with a survival signal against activation-induced cell death (Hurtado et al., 1997), thereby playing an important role in maintaining effective T cell immune responses and generating immunological memory (Bartkowiak & Curran, 2015).

[0005] The role of CD137 in leukocyte biology is generally well understood, with a clear biological rationale behind its role in tumor immunology. CD137 is expressed by activated T cells and binds antigen-specific CD4 + and CD8 + It is used as a marker to identify T cells. Usually, CD137 expression is associated with CD4 + CD8 rather than T cells + High in T cells (Wen et al., 2002). CD8 + In T cells, proliferation, survival, and cytotoxic effector function via production of interferon gamma and interleukin 2 have been attributed to CD137 cross-linking. CD137 cross-linking is also responsible for the activation of memory CD8 + It also contributes to the differentiation and maintenance of T cells. + In some subsets of T cells, CD137 cross-linking similarly triggers proliferation and activation, leading to the release of cytokines such as interleukin-2 ( Makkouk et al., 2016 ).

[0006] It has been demonstrated that natural killer (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) via tumor-targeting mAbs is enhanced as a result of CD137 stimulation via agonistic anti-CD137 monoclonal antibodies in vitro and in vivo (Bartkowiak & Curran, 2015). NK cells bind antibodies via Fc receptors and respond to antibodies with specific isotypes. In response, it can trigger the activation of NK cells, leading to the release of cytotoxic granules and lysis of target cells (Kohrt et al., 2012). We have demonstrated that anti-CD137 agonistic antibodies enhance the antitumor activity of the therapeutic antibodies rituximab, trastuzumab, and cetuximab by enhancing ADCC against tumor cells (Kohrt et al., 2014; Kohrt et al., 2011). Furthermore, human NK cells upregulate CD137 expression after encountering cell-bound antibodies via their FcγRs. Subsequent stimulation of these NK cells with anti-CD137 antibodies has been shown to enhance ADCC against tumor cells (Chester et al., 2015; Chester et al., 2016).

[0007] B lymphocytes also express CD137 upon activation. Binding of CD137 ligand to CD137 enhances B cell proliferation, survival, and cytokine production. CD137 expression is also induced on normal and malignant human B cells after CD40 binds to its ligand CD154 (CD40 ligand), resulting in enhanced B cell survival when CD137 is subsequently activated.

[0008] CD137 has also been demonstrated to be expressed on tumor-responsive subsets of tumor-infiltrating lymphocytes (TILs). CD137 monotherapy has been shown to be effective in several preclinical immunogenic tumor models, including MC38, CT26, and B-cell lymphoma. Engagement of CD137 with other anticancer agents, such as chemotherapy, cytokines, and other checkpoint regulators, has been demonstrated to reduce the growth of established tumors. Specifically, the combination of anti-CD137 antibodies with anti-CD20, anti-EGFR, and anti-HER-2 antibodies has been shown to synergistically reduce tumor growth in various preclinical xenograft models (Kohrt et al., 2014; Kohrt et al., 2012; Kohrt et al., 2011).

[0009] Combining tumor-targeted monoclonal antibody therapy with anti-CD137 agonist antibody treatment has shown promising results in preclinical models of lymphoma (Kohrt et al., 2011), head and neck cancer, colorectal cancer (Kohrt et al., 2014), and breast cancer (Kohrt et al., 2012). However, clinical development has been slowed by the dose-limiting high-grade liver inflammation associated with CD137 agonist antibody treatment. Urelumab (BMS-663513), a nonligand-blocking human IgG4 isotype antibody (Chester et al., 2018), was the first anti-CD137 antibody to enter clinical trials, but was discontinued after significant on-target, dose-dependent hepatotoxicity was observed (Chester et al., 2018). More recently, clinical trials of urelumab in the treatment of solid tumors have resumed, and urelumab treatment is being considered in combination with radiation therapy (NCT03431948). , or rituximab (NCT01775631), cetuximab (NCT02110082), the anti-PD-1 antibody nivolumab (NCT02253992, NCT02534506, NCT02845323), and nivolumab plus anti-LAG-3 It has been combined with other therapeutic antibodies, such as the combination of antibodies BMS986016 (NCT02658981). However, to reduce hepatotoxicity associated with urelumab treatment, the administration of urelumab in these trials had to be limited, and efficacy results were disappointing (Chester et al., 2018).

[0010] Pfizer's anti-CD137 antibody utomilumab (PF-05082566), a human IgG2 isotype antibody, was administered at doses ranging from 0.03 mg / kg to 10 mg / kg in a Phase I clinical trial for advanced cancer. No dose-limiting toxicities (DLTs) have been observed in a dose range up to 1 / kg (Chester et al. 2016; Segal et al., 2018). However, the overall objective response rate with this antibody was only 3.8% in patients with solid tumors, suggesting that utomilumab may have weaker potency and clinical efficacy than urelumab, but a more favorable safety profile. Utomilumab has been shown to be effective in combination with radiation therapy (NCT03217747) or chemotherapy, as well as with other antibody therapies, including the anti-PD-L1 antibody avelumab (NCT02554812) and the anti-PD-1 antibody pembrolizumab (NCT02179918) (Chester et al., 2018; Segal et al., 2018). Combinations have been studied to evaluate the safety, tolerability, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), and efficacy of different treatment combinations. These studies are ongoing, and early results show no DLTs at doses up to 5 mg / kg and a 26% patient response rate for the combination of utomilumab and pembrolizumab. Triple combinations of utomilumab with avelumab and other immuno-oncology therapies are also being tested (NCT02554812, NCT03217747).

[0011] MSLNs are the mesothelial cells that line the pleura, peritoneum, and pericardium in healthy individuals (Hassan et al., 2005). Although it is expressed at relatively low levels on the cytoplasm of the cytoplasm, it 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 unknown. In the context of cancer, high expression levels of MSLN correlate with poor prognosis in ovarian cancer, cholangiocarcinoma, lung adenocarcinoma, and triple-negative breast cancer. The limited expression of MSLN in normal cells, combined with its high expression in tumor cells, makes it an attractive therapeutic target using monoclonal antibodies (Hassan et al., 2016).

[0012] 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 mature 40 kDa protein, MSLN, remains attached to the plasma 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.

[0013] Membrane-bound mature MSLN is shed from cells as a result of alternative splicing, either by generating mutants lacking the membrane anchor sequence or by protease cleavage by tumor necrosis factor-α-converting enzyme (TACE) (Sapede et al., 2008; Zhang et al., 2011). Soluble shed MSLN is found in the serum of patients and in the tissues of patients with malignant mesothelioma, ovarian cancer, or highly metastatic cancer. It is found in the stroma of tumors, including mesothelioma. Measurement of soluble MSLN levels in the blood and pleural fluid of mesothelioma patients has been approved by the US FDA to monitor patient response to treatment and progression (Hollevoet et al., 2012; Creany et al., 2015).

[0014] Several antibody-based therapies targeting MSLNs have been developed and are being tested in clinical trials, primarily in mesothelioma, pancreatic cancer, and non-small cell lung cancer (Hassan et al., 2016). Strategies include direct tumor cell killing through the use of anti-MSLN antibodies such as amatuximab with antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and antibody-drug conjugates (ADCs) such as SS1P-PE38 and anetumab-ravtansine, which contain antibodies or antibody fragments conjugated to toxins.

[0015] Unconjugated antibodies targeting MSLN have shown favorable safety profiles but their therapeutic efficacy is limited. Meanwhile, ADCs have been shown to be more potent in antitumor activity but are associated with dose-limiting toxicities. Several bispecific molecules aimed at engaging the immune system are also in development, including ABBV-428, which targets MSLN and the costimulatory protein CD40, the MSLN-CD3 bispecific T cell engager (BITE), and the MSLN-CD47 bispecific molecule. Summary of the Invention [Problem to be solved by the invention]

[0016] Description of the invention As discussed in the Background section above, clinical development of CD137 agonist molecules has been hampered due to treatments associated with either dose-limiting high-grade liver inflammation (urelumab) or poor clinical efficacy (utomilumab). [Means for solving the problem]

[0017] The present inventors have recognized that there is a need in the art for CD137 agonist molecules that exhibit high activity but are not associated with dose-limiting liver inflammation.Such molecules can be administered to individuals at doses that optimize the efficacy and thus effectiveness of the molecules, and can be used, for example, as immunotherapeutic agents for the treatment of cancer.

[0018] Without wishing to be bound by theory, it is believed that T cells present in the liver may be activated by anti-CD137 agonist molecules and have the potential to cause liver inflammation. +T cells have been shown to promote liver inflammation and apoptosis after sepsis / viral infection (Wesche-Soldato et al., 2007). However, this effect It was not specific to CD137. Anti-CD137 agonist antibody therapy in mice has been shown to result in CD137-dependent T cell infiltration into the liver (Dubrot J et al., 2010). Taken together, the results of these studies suggest that highly active anti-CD13 antibodies such as urelumab may be effective in reducing the risk of T cell death. 7. Agonist antibodies activate CD8 + It has been shown to cause infiltration of T cells into the liver, which can lead to liver inflammation.

[0019] As discussed in the Background section above, initial ligation of a CD137 ligand to CD137 is thought to initiate a cascade of events that leads to receptor trimerization, subsequent receptor clustering, activation, and subsequent initiation of potent antitumor immune cell activity. Therefore, for a therapeutic agent to efficiently achieve CD137 activation, it would be expected that several receptor monomers would need to be crosslinked together in a manner that mimics crosslinking by a trimeric ligand.

[0020] The present inventors isolated an antibody molecule containing a complementarity-determining region (CDR)-based antigen-binding site of MSLN and a CD137 antigen-binding site located in the constant domain of the antibody molecule, and showed that such an antibody molecule can induce CD137 clustering and signaling when bound to both CD137 and MSLN in vitro.

[0021] Without wishing to be bound by theory, it is believed that antibody molecules bind to MSLN via CDR-based antigen-binding sites, resulting in cross-linking of several antibody molecules on the surface of tumor cells, and then the CD137 antigen-binding sites of the antibody molecules bind to CD137 on the surface of immune cells such as tumor-infiltrating lymphocytes (TILs), resulting in the clustering and activation of CD137, thereby resulting in immune activation. The activated immune cells can then act on tumors and bring about tumor immunotherapy.

[0022] The concentration of MSLN present on the surface of tumor cells is thought to influence the level of CD137 agonism: specifically, higher concentrations of MSLN are thought to increase the binding and cross-linking of antibody molecules across the cell surface, resulting in increased CD137 agonism.

[0023] The CD137 agonist activity of the antibody molecule of the present invention depends on the simultaneous binding of the antibody molecule to MSLN. Therefore, the activation of CD137 by the antibody molecule is expected to be limited to the tumor microenvironment. Therefore, the antibody molecule of the present invention is also referred to herein as a "conditional agonist." In this regard, it should be noted that the conditional agonist activity of the antibody molecule is not an inherent property of an antibody that contains a CD137 antigen-binding site in its constant domain. Rather, many of the molecules isolated during the screening program conducted by the present inventors bound to CD137 but did not require cross-linking for CD137 clustering and activation, and induced limited CD137 clustering and activation in the absence of cross-linking. Due to the conditional agonistic activity of the antibody molecules of the present invention localized in the tumor microenvironment, these molecules are not expected to cause liver inflammation.

[0024] Conventional antibodies specific for TNF receptors, such as CD137, typically have no or very mild intrinsic agonistic activity and require secondary crosslinking of the antibody-TNFRSF member complex using external crosslinkers such as protein A or G or secondary antibodies, or antibody binding to plasma membrane-localized Fcγ receptors, to induce higher levels of TNF receptor clustering and activation (Wajant, 2015). The low or lack of agonistic activity of TNF receptor-specific antibodies in the absence of crosslinking can be explained by the fact that conventional bivalent antibodies can maximally crosslink two monomeric TNF receptors, which is insufficient for TNF receptor activation. Therefore, for in vivo efficacy, monospecific antibodies targeting CD137 require the presence of Fcγ receptor-expressing cells in close proximity to CD137-expressing T cells to achieve crosslinking of the CD137-specific antibody and subsequent clustering and activation of the CD137 receptor. However, Fcγ receptor-mediated crosslinking is believed to be inefficient. Furthermore, because cells expressing Fcγ receptors are present throughout the body, antibody cross-linking and activation of CD137-expressing immune cells is not limited to a specific site, such as the tumor microenvironment. Furthermore, the isotype of such CD137 antibodies must be selected to mediate effective binding to Fcγ receptors for cross-linking. However, this may result in the antibody triggering Fcγ receptor-mediated effector functions, such as ADCC, thereby eliminating the immune cells intended to be activated by the antibody.

[0025] In contrast, the antibody molecules of the present invention can conditionally activate CD137 in the presence of MSLN, for example, without the need for Fcγ receptor cross-linking, as is required by conventional antibody molecules. Furthermore, cross-linking of the antibody molecules of the present invention via binding to MSLN is expected to be more efficient than Fcγ receptor-mediated cross-linking. Mutations that suppress Fcγ receptor binding are known in the art and are preferably included in the antibody molecules of the present invention. Therefore, in the absence of MSLN, the antibody molecules of the present invention do not exhibit CD137 agonist activity and are therefore not expected to induce liver inflammation.

[0026] The present inventors further demonstrated that antibody molecules comprising the MSLN and CD137 antigen-binding sites described above, which have been modified to reduce or eliminate binding to one or more Fcγ receptors, can suppress tumor growth in an in vivo mouse tumor model. Because these antibody molecules have reduced or eliminated ADCC activity, it is expected that the antibody molecules inhibit tumor growth by activating CD137-expressing T cells.

[0027] Antibody molecules have been shown to bind to dimeric CD137 with higher affinity than to monomeric CD137.

[0028] The "affinity" referred to herein is K D As will be readily apparent to one of skill in the art, when an antibody molecule is capable of forming multiple binding interactions with an antigen (e.g., when the antibody molecule is capable of bivalently binding the antigen, and optionally when the antigen is dimeric), K D Affinity, as measured by , can also be affected by avidity, where avidity refers to the overall strength of the antibody-antigen complex.

[0029] Expression of CD137 by T cells is upregulated upon activation. Although it is not our intention to be construed as a misnomer, due to the high expression of CD137 on activated T cells, CD137 is believed to be in the form of dimers, trimers, and higher-order multimers on the surface of such cells. In contrast, naive immune cells, such as naive T cells, express low or negligible levels of CD137 on their cell surface, and therefore the CD137 present may be in a monomeric form. Therefore, antibody molecules that bind with high affinity to CD137 but not to monomeric CD137 are expected to preferentially bind to activated immune cells, such as activated T cells, over naive immune cells present in, for example, the liver.

[0030] Furthermore, the antibody molecules of the present invention have been shown to bind with higher affinity to immobilized MSLN than to MSLN in solution. Specifically, the antibody molecules of the present invention bind to MSLN with high avidity, and therefore, when an antibody can bind to two MSLN molecules, such as when multiple copies of an antigen are immobilized on a surface, it is believed to bind more strongly to MSLN than when MSLN is in a monomeric form, as would be expected for MSLN in solution. Therefore, without wishing to be bound by theory, it is believed that the antibody molecules of the present invention do not remain bound to shed MSLN in solution in vivo due to the antibody's lower affinity for monomeric MSLN, and therefore are not quickly removed from tumor sites, thereby prolonging the therapeutic effect of binding MSLN to the surface of tumor cells.

[0031] The antibody molecules of the present invention bind to different epitopes / regions on MSLN, as evidenced by the fact that some antibody molecules are able to block the binding of the ligand MUC16 to MSLN, while others are not.

[0032] Some antibody molecules of the present invention have been shown to have equal or higher affinity for MSLN than for CD137. This is believed to be beneficial for localizing the antibody molecules to tumors expressing MSLN. Binding of the antibody molecules to MSLN is expected to result in antibody cross-linking, binding to CD137 expressed on the surface of immune cells, subsequent clustering and activation of CD137, and ultimately activation of the immune cells.

[0033] The antibody molecules of the invention have also been shown to be capable of binding with high affinity to both human and cynomolgus monkey MSLN, and human and cynomolgus monkey CD137. This cross-reactivity is advantageous as it allows dosing and safety testing of the antibody molecules in cynomolgus monkeys during preclinical development.

[0034] In vivo studies in mouse syngeneic tumor models demonstrated that antibody molecules containing the Fab-binding site of human MSLN and the mouse CD137-binding site of the CH3 domain had greater antitumor effects than isotype control antibodies or components of bispecific antibody molecules delivered as monotherapy or combination therapy (see Example 13). The antibody molecules demonstrated advantageous characteristics by demonstrating significant reductions in tumor growth and survival benefits, and were able to stimulate antitumor responses in tumors expressing different levels of MSLN. Dose-dependent antitumor responses were also observed after treatment with these molecules. Overall, the antibodies demonstrated advantageous characteristics in vivo in reducing tumor growth and increasing animal survival compared to control molecules.

[0035] Furthermore, no hepatotoxicity was observed in the liver after treatment with the antibody molecule. This is advantageous, as treatment with other anti-CD137 agonist antibodies has been shown in the literature to result in liver toxicity. Mechanistic studies showed that the antibody molecule stimulated T cell activation within the tumor microenvironment, while a control CD137 agonist stimulated greater T cell activation outside the tumor microenvironment, further supporting this advantageous feature.

[0036] A further advantageous feature of the antibody molecule identified by the present inventors is that it binds to MSLN and CD13 The advantage of this approach is that both of the seven antigen-binding sites are contained within the antibody structure itself. Notably, the antibody molecule does not require the fusion of other proteins to the antibody molecule via a linker or other means to result in a molecule capable of bivalently binding both targets. This has many advantages. Specifically, the antibody molecules identified by the inventors can be produced using methods similar to those used for standard antibody production because they do not contain additional fusion moieties. This structure is also expected to improve antibody stability, as linkers can degrade over time, resulting in heterogeneous populations of antibody molecules. Antibodies within a population with only one fused protein may preferentially bind to cell-associated MSLN or clusters and be unable to signal through CD137 as a result of crosslinking by binding to both CD137 and MSLN. Linker cleavage / degradation can occur before or after administration of the therapeutic agent to an individual (e.g., via enzymatic cleavage or the individual's in vivo pH), thereby reducing its efficacy while circulating within the individual. Since the antibody molecule identified by the present inventors does not have a linker, 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 identified by the present inventors is also preferable from the viewpoint of the immunogenicity of the molecule. This is because the introduction of a fusion protein or a linker, or both, can induce immunogenicity when the molecule is administered to an individual, resulting in a decrease in the efficacy of the therapeutic agent.

[0037] Thus, the present invention provides the following:

[0038] [1] (a) a complementarity-determining region (CDR)-based antigen-binding site of MSLN; and (b) CD137 antigen-binding site located in the CH3 domain of the antibody molecule Including, The CDR-based antigen binding site (i) SEQ ID NOs: 42, 33, 44, 20, 22, and 80 [FS28-256-271], respectively; (ii) SEQ ID NOs: 14, 16, 27, 20, 22, and 24 [FS28-024-052], respectively; (iii) SEQ ID NOs: 42, 33, 44, 20, 22, and 40 [FS28-256-021], respectively; (iv) SEQ ID NOs: 42, 33, 44, 20, 22, and 37, respectively [FS28-256-012]; (v) SEQ ID NOs: 50, 33, 52, 20, 22, and 40, respectively [FS28-256-023]; (vi) SEQ ID NOs: 42, 33, 44, 20, 22, and 41 [FS28-256-024], respectively; (vii) SEQ ID NOs: 50, 33, 52, 20, 22, and 41, respectively [FS28-256-026]; (viii) SEQ ID NOs: 42, 33, 44, 20, 22, and 80, respectively [FS28-256-027]; (ix) SEQ ID NOs: 38, 33, 35, 20, 22, and 40, respectively [FS28-256-001]; (x) SEQ ID NOs: 38, 33, 35, 20, 22, and 41, respectively [FS28-256-005]; (xi) SEQ ID NOs: 46, 33, 48, 20, 22, and 37 [FS28-256-014], respectively; (xii) SEQ ID NOs: 50, 33, 52, 20, 22, and 37, respectively [FS28-256-018]; (xiii) SEQ ID NOs: 31, 33, 35, 20, 22, and 37, respectively [FS28-256]; (xiv) SEQ ID NOs: 14, 16, 25, 20, 22, and 24, respectively [FS28-024-051]; (xv) SEQ ID NOs: 14, 16, 29, 20, 22, and 24, respectively [FS28-024-053]; or (xvi) SEQ ID NOs: 14, 16, 18, 20, 22, and 24 [FS28-024], respectively comprising CDR1 to CDR6 described in CDR sequences are defined according to the ImMunoGeneTics (IMGT) numbering scheme; the CD137 antigen-binding site comprises a first sequence and a second sequence located in the AB and EF structural loops of the CH3 domain, respectively, and the first and second sequences have the sequences set forth in SEQ ID NOs: 10 and 11 [FS22-172-003], respectively; An antibody molecule that binds to mesothelin (MSLN) and CD137.

[0039] [2] (a) a complementarity-determining region (CDR)-based antigen-binding site of MSLN; and (b) CD137 antigen-binding site located in the CH3 domain of the antibody molecule Including, The CDR-based antigen binding site (i) SEQ ID NOs: 43, 5, 45, 21, 23, and 80 [FS28-256-271], respectively; (ii) SEQ ID NOs: 15, 17, 28, 21, 23, and 24 [FS28-024-052], respectively; (iii) SEQ ID NOs: 43, 34, 45, 21, 23, and 40, respectively [FS28-256-021]; (iv) SEQ ID NOs: 43, 34, 45, 21, 23, and 37, respectively [FS28-256-012]; (v) SEQ ID NOs: 51, 34, 53, 21, 23, and 40 [FS28-256-023], respectively; (vi) SEQ ID NOs: 43, 34, 45, 21, 23, and 41 [FS28-256-024], respectively; (vii) SEQ ID NOs: 51, 34, 53, 21, 23, and 41, respectively [FS28-256-026]; (viii) SEQ ID NOs: 43, 34, 45, 21, 23, and 80, respectively [FS28-256-027]; (ix) SEQ ID NOs: 39, 34, 36, 21, 23, and 40 [FS28-256-001], respectively; (x) SEQ ID NOs: 39, 34, 36, 21, 23, and 41 [FS28-256-005], respectively; (xi) SEQ ID NOs: 47, 34, 49, 21, 23, and 37 [FS28-256-014], respectively; (xii) SEQ ID NOs: 51, 34, 53, 21, 23, and 37, respectively [FS28-256-018]; (xiii) SEQ ID NOs: 32, 34, 36, 21, 23, and 37, respectively [FS28-256]; (xiv) SEQ ID NOs: 15, 17, 26, 21, 23, and 24, respectively [FS28-024-051]; (xv) SEQ ID NOs: 15, 17, 30, 21, 23, and 24, respectively [FS28-024-053]; or (xvi) SEQ ID NOs: 15, 17, 19, 21, 23, and 24 [FS28-024], respectively comprising CDR1 to CDR6 described in CDR sequences are defined according to Kabat; the CD137 antigen-binding site comprises a first sequence and a second sequence located in the AB and EF structural loops of the CH3 domain, respectively, and the first and second sequences have the sequences set forth in SEQ ID NOs: 10 and 11 [FS22-172-003], respectively; An antibody molecule that binds to mesothelin (MSLN) and CD137.

[0040] [3] The antibody molecule according to [1] or [2], which comprises CDRs 1 to 6 according to (i) of [1] or [2].

[0041] [4] The antibody molecule according to [1] or [2], which comprises CDRs 1 to 6 according to (ii) of [1] or [2].

[0042] [5] The antibody molecule according to any one of [1] to [4], wherein the antibody molecule comprises a heavy chain variable (VH) domain and / or a light chain variable (VL) domain, preferably a VH domain and a VH domain.

[0043] [6] The antibody molecule according to any one of [1] to [5], wherein the antibody molecule comprises an immunoglobulin heavy chain and / or an immunoglobulin light chain, preferably an immunoglobulin heavy chain and an immunoglobulin light chain.

[0044] [7] The antibody molecule comprises a VH domain and / or a VL domain, and preferably the VH domain and the VL domain are: (i) each of the sequence numbers 177 and 76 [FS28-256-271]; (ii) SEQ ID NOs: 58 and 54 [FS28-024-052], respectively; (iii) SEQ ID NOs: 70 and 68 [FS28-256-021], respectively; (iv) SEQ ID NOs: 70 and 64 [FS28-256-012], respectively; (v) SEQ ID NOs: 74 and 68 [FS28-256-023], respectively; (vi) SEQ ID NOs: 70 and 78 [FS28-256-024], respectively; (vii) SEQ ID NOs: 74 and 78 [FS28-256-026], respectively; (viii) SEQ ID NOs: 70 and 76 [FS28-256-027], respectively; (ix) SEQ ID NOs: 66 and 68 [FS28-256-001], respectively; (x) SEQ ID NOs: 66 and 78 [FS28-256-005], respectively; (xi) SEQ ID NOs: 72 and 64 [FS28-256-014], respectively; (xii) SEQ ID NOs: 74 and 64 [FS28-256-018], respectively; (xiii) SEQ ID NOs: 62 and 64 [FS28-256], respectively; (xiv) SEQ ID NOs: 56 and 54 [FS28-024-051], respectively (xv) SEQ ID NOs: 60 and 54 [FS28-024-053], respectively; or (xvi) SEQ ID NOs: 12 and 54 [FS28-024], respectively The antibody molecule according to any one of [5] to [6], which is described in

[0045] [8] The antibody molecules are each represented by SEQ ID NO: 177 and 76 [FS28-256-271].

[0046] [9] The antibody molecule according to [7], wherein the antibody molecule comprises a VH domain and a VL domain set forth in SEQ ID NOs: 58 and 54 [FS28-024-052], respectively.

[0047]

[10] The antibody molecule of any one of [1] to [9], wherein the first sequence is located between positions 14 and 17 of the CH3 domain of the antibody molecule, and the numbering of amino acid residues is according to the IMGT numbering scheme.

[0048]

[11] The antibody molecule of

[10] , wherein the first sequence is located at positions 15, 16, 16.5, 16.4, 16.3, 16.2, and 16.1 of the CH3 domain of the antibody molecule, and the numbering of amino acid residues is according to the IMGT numbering scheme.

[0049]

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

[11] , wherein the second sequence is located at positions 92 to 98 of the CH3 domain of the antibody molecule, and the numbering of amino acid residues is according to the IMGT numbering scheme.

[0050]

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

[12] , further comprising a third sequence located in the CD structure loop of the CH3 domain.

[0051]

[14] The antibody molecule of

[13] , wherein the third sequence is located at positions 43 to 78 of the CH3 domain of the antibody molecule, and the numbering of amino acid residues is according to the IMGT numbering scheme.

[0052]

[15] The antibody molecule of any one of

[13] to

[14] , wherein the third sequence has the sequence set forth in SEQ ID NO: 157.

[0053]

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

[15] , wherein the antibody molecule comprises a CH3 domain sequence set forth in SEQ ID NO: 8 [FS22-172-003].

[0054]

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

[16] , wherein the antibody molecule is a human IgG1 molecule.

[0055]

[18] The antibody molecule is an antibody: (i) FS22-172-003-AA / FS28-256-271 set forth in SEQ ID NOs: 3 and 84, respectively; (ii) FS22-172-003-FS28-024-052 set forth in SEQ ID NOs: 102 and 85, respectively; (iii) FS22-172-003-FS28-256-021 set forth in SEQ ID NOs: 125 and 82, respectively; (iv) FS22-172-003-AA / FS28-256-012 set forth in SEQ ID NOs: 125 and 116, respectively; (v) FS22-172-003-AA / FS28-256-023 as set forth in SEQ ID NOs: 125 and 82, respectively; or (vi) FS22-172-003-AA / FS28-256-024 set forth in SEQ ID NOs: 125 and 83, respectively (vii) FS22-172-003-AA / FS28-256-026 set forth in SEQ ID NOs: 133 and 83, respectively; (viii) FS22-172-003-AA / FS28-256-027 set forth in SEQ ID NOs: 125 and 84, respectively; (ix) FS22-172-003-AA / FS28-256-001 set forth in SEQ ID NOs: 120 and 82, respectively; (x) FS22-172-003-AA / FS28-256-005 set forth in SEQ ID NOs: 120 and 83, respectively; (xi) FS22-172-003-AA / FS28-256-014 set forth in SEQ ID NOs: 129 and 116, respectively; (xii) FS22-172-003-AA / FS28-256-018 set forth in SEQ ID NOs: 133 and 116, respectively; (xiii) FS22-172-003-AA / FS28-256 set forth in SEQ ID NOs: 114 and 116, respectively; (xiv) FS22-172-003-AA / FS28-024-051 set forth in SEQ ID NOs: 98 and 85, respectively; (xv) FS22-172-003-AA / FS28-024-053 set forth in SEQ ID NOs: 106 and 85, respectively; or (xvi) FS22-172-003-AA / FS28-024 set forth in SEQ ID NOs: 94 and 85, respectively. The antibody molecule according to any one of [1] to

[17] , comprising a heavy chain and a light chain of

[0056]

[19] The antibody molecule according to

[18] , wherein the antibody molecule comprises the light chain and heavy chain of FS22-172-003-AA / FS28-256-271 set forth in SEQ ID NOs: 84 and 3, respectively.

[0057]

[20] The antibody molecule according to

[18] , wherein the antibody molecule comprises the light chain and heavy chain of FS22-172-003-AA / FS28-024-052 set forth in SEQ ID NOs: 85 and 102, respectively.

[0058]

[21] An antibody molecule according to any one of

[18] to

[20] , wherein the proline (P) at position 114 in the CH2 domain of the antibody is substituted with alanine (A), and the numbering of amino acid residues is according to the IMGT numbering scheme.

[0059]

[22] The antibody molecule described in any one of [1] to

[21] , wherein the MSLN is MSLN bound to a cell surface.

[0060]

[23] The antibody molecule according to

[22] , wherein the antibody molecule binds to immobilized MSLN with higher affinity than soluble MSLN.

[0061] [twenty four] (i) the antibody molecule binds to the immobilized MSLN with an affinity of 8 nM kD or greater; and / or (ii) The antibody molecule according to

[23] , wherein the antibody molecule binds to soluble MSLN with an affinity of 15 nM kD or lower.

[0062]

[25] The antibody molecule described in any one of [1] to

[24] , wherein the antibody molecule binds to MSLN and human CD137.

[0063]

[26] The antibody molecule according to

[25] , wherein MSLN consists of or comprises the sequence set forth in SEQ ID NO: 375.

[0064]

[27] The antibody molecule according to

[25] or

[26] , wherein the human CD137 consists of or comprises the sequence set forth in SEQ ID NO: 373.

[0065]

[28] An antibody molecule comprising CDR1 to CDR6 according to any one of (ii) or (xiv) to (xvi) of [1] or [2], wherein the antibody blocks binding of MUC16 to MSLN. The antibody molecule according to any one of [1] to

[27] ,

[0066]

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

[27] , wherein the antibody molecule comprises CDRs 1 to 6 of any one of (i) or (iii) to (xiii) of [1] or [2], and the antibody does not block binding of MUC16 to MSLN.

[0067]

[30] The antibody molecule according to

[28] or

[29] , wherein MUC16 is human MUC16.

[0068]

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

[30] , wherein the antibody molecule is capable of activating CD137 on immune cells in the presence of MSLN bound to the surface of tumor cells.

[0069]

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

[31] , wherein binding of the antibody molecule to CD137 on immune cells and to MSLN bound to the surface of tumor cells causes clustering of CD137 on the immune cells.

[0070]

[33] The antibody molecule according to

[31] or

[32] , 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).

[0071]

[34] The antibody molecule of claim

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

[0072]

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

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

[0073]

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

[35] , wherein the antibody molecule does not bind to one or more Fcγ receptors.

[0074]

[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.

[0075]

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

[37] and a biologically active molecule.

[0076]

[39] A conjugate comprising the antibody molecule according to any one of [1] to

[37] and a detectable label.

[0077]

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

[37] .

[0078]

[41] A nucleic acid molecule comprising: (I) FS22-172-003-AA / FS28-256-271 set forth in SEQ ID NOs: 4 and 91, respectively; (ii) FS22-172-003-AA / FS28-024-052 as set forth in SEQ ID NOs: 103 and 86, respectively; (iii) FS22-172-003-AA / FS28-256-021 set forth in SEQ ID NOs: 126 and 122, respectively; (iv) FS22-172-003-AA / FS28-256-012 set forth in SEQ ID NOs: 126 and 117, respectively; (v) FS22-172-003-AA / FS28-256-023 set forth in SEQ ID NOs: 134 and 122, respectively; (vi) FS22-172-003-AA / FS28-256-024 set forth in SEQ ID NOs: 126 and 90, respectively; (vii) FS22-172-003-AA / FS28-256-026 set forth in SEQ ID NOs: 134 and 90, respectively; (viii) FS22-172-003-AA / FS28-256-027 set forth in SEQ ID NOs: 126 and 91, respectively; (ix) FS22-172-003-AA / FS28-256-001 set forth in SEQ ID NOs: 121 and 122, respectively; (x) FS22-172-003-AA / FS28-256-005 set forth in SEQ ID NOs: 121 and 90, respectively; (xi) FS22-172-003-AA / FS28-256-014 set forth in SEQ ID NOs: 130 and 117, respectively; (xii) FS22-172-003-AA / FS28-256-018 set forth in SEQ ID NOs: 134 and 117, respectively; (xiii) FS22-172-003-AA / FS28-256 set forth in SEQ ID NOs: 115 and 117, respectively; (xiv) FS22-172-003-AA / FS28-024-051 set forth in SEQ ID NOs: 99 and 86, respectively; (xv) FS22-172-003-AA / FS28-024-053 set forth in SEQ ID NOs: 107 and 86, respectively; or (xvi) FS22-172-003-AA / FS28-024 set forth in SEQ ID NOs: 95 and 86, respectively.

[0033] One or more nucleic acid molecules encoding the antibody molecule of any one of [1] to

[37] , comprising a heavy chain nucleic acid sequence and / or a light chain nucleic acid sequence.

[0079]

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

[40] or

[41] .

[0080]

[43] A recombinant host cell comprising one or more nucleic acid molecules according to any one of

[40] or

[41] , or one or more vectors according to

[42] .

[0081]

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

[37] , comprising culturing the recombinant host cell of

[43] under conditions for producing the antibody molecule.

[0082]

[45] The method of

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

[0083]

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

[39] and a pharmaceutically acceptable excipient.

[0084]

[47] The antibody molecule or conjugate of any one of [1] to

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

[0085]

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

[39] .

[0086]

[49] Use of the antibody molecule or conjugate according to any one of [1] to

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

[0087]

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

[47] to

[49] , wherein the cancer is ovarian cancer, pancreatic cancer, lung cancer, or mesothelioma.

[0088]

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

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

[0089]

[52] The method of

[48] , wherein the method further comprises administering to the individual a therapeutically effective amount of a second treatment. [Brief explanation of the drawings]

[0090] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1 shows the results of a T cell activation assay in which mAb2 containing Fabs that bind to different regions of human MSLN drive CD137-mediated activation of CD8+ T cells, resulting in the release of human IL-2, when mAb2 is crosslinked by MSLN-positive NCI-H226 cells. These results show that mAb2 containing Fabs from lineage FS28-185 (B) has no functional activity in this assay. mAb2 containing Fabs from lineages FS28-024 (A) and FS28-256 (C) all show increased hIL-2 release in the presence of mAb2 at subnanomolar to 1.5 nM concentrations. [Figure 2] Figure 2 shows the results of a T cell activation assay in the presence of various concentrations of soluble MSLN (sMSLN) (no sMSLN, 2 nM, and 20 nM sMSLN). mAbs 2 (including FS28-024-051, FS28-024-052, FS28-024-053, FS28-256-021, and FS28-256-023) that showed preferential binding to membrane-bound MSLN were less affected by the presence of soluble MSLN (A–E) than FS22-172-003-AA / FS28-256-027 (F), for which a significant shift in EC50 was observed in the presence of 20 nM sMSLN compared to the absence of sMSLN. [Figure 3] Figures 3A and 3B show the results of a T cell activation assay in which mAb2 drives CD137-mediated activation of CD8+ T cells when cross-linked by OVCAR-3 cells. These results indicate that mAb2 can drive CD137-mediated agonism in the context of cells expressing lower MSLN density on the cell membrane. The anti-CD137 Fcab FS22-172-003 in mock mAb2 format (FS22-172-003 / HelD1.3) was also tested in this assay, and the lack of IL-2 release indicated that the anti-human CD137 Fcab was functional only when cross-linked via the Fab arm of the molecule. [Figure 4]Figure 4 shows the results of a CD8+ T cell activation assay in which sequence-optimized mAb2 FS22-172-003-AA / FS28-256-271 (A), FS22-172-003-AA / FS28-256-272 (B), and FS22-172-003-AA / FS28-256-273 (C) were crosslinked with NCI-H226 cells expressing human MSLN in the presence or absence of up to 20 nM sMSLN. For mAb2 FS22-172-003-AA / FS28-256-271 sMSLN, concentrations up to 20 nM resulted in minimal reductions in T cell activation activity, with the EC50 observed. mAb2 FS22-172-003-AA / FS28-256-272 and FS22-172-003-AA / FS28-256-273 showed a more pronounced reduction in T cell activating activity in the presence of 20 nM sMSLN, with a greater than 4-fold reduction in the observed EC50, as did the parental clone FS22-172-003-AA / FS28-256-027(D), which showed a 6.6-fold reduction in EC50. [Figure 5] Figure 5 shows IFNγ release in a T cell activation assay when mAb2 FS22m-063-AA / FS28m-228-010 and FS22m-063-AA / FS28m-228 crosslinked CT26.G10, CT26.B2, or Panc02 cells in the presence or absence of 2 nM soluble murine MSLN (A to D). The mAb2 FS22m-063-AA / FS28m-228 experiment included the following negative controls: an anti-MSLN control antibody in IgG1 format lacking the CD137-targeting Fcab, and mock mAb2 FS22m-063 / HelD1.3 (i.e., a CD137 Fcab without MSLN targeting). The results showed that even relatively high concentrations of soluble MSLN had minimal effect on the potency of mAb2 in this assay. The data also demonstrated subnanomolar potency (EC50) across all MSLN-expressing cell lines tested. [Figure 6]Figure 6 shows individual tumor volume measurements in the CT26.B2 syngeneic mouse tumor model in mice treated with G1 / 4420 (human IgG1 isotype control antibody) (A) or FS22m-063-AA / FS28m-228 (anti-mouse CD137 / MSLN mAb2) (B). Treatment with FS22m-063-AA / FS28m-228 resulted in reduced tumor growth compared to the isotype control. [Figure 7] Figure 7 shows Kaplan-Meier survival plots of mice in the CT26.B2 syngeneic mouse tumor model treated with G1 / 4420 (human IgG1 isotype control antibody) or FS22m-063-AA / FS28m-228 (anti-mouse CD137 / MSLN mAb2). Treatment with FS22m-063-AA / FS28m-228 showed a significant improvement in survival compared to treatment with the human IgG1 isotype control antibody (log-rank analysis, **p<0.01). [Figure 8] Figure 8 shows individual tumor volume measurements in the CT26.G10 syngeneic mouse tumor model in mice treated with G1 / 4420 (human IgG1 isotype control antibody) (A) or FS22m-063-AA / FS28m-228 (anti-mouse CD137 / MSLN mAb2) (B). Mice treated with FS22m-063-AA / FS28m-228 showed reduced tumor growth compared to isotype control, with 4 / 20 (25%) mice having no palpable tumors at the end of the study compared to (1 / 20, 5%) in the isotype control-treated group. [Figure 9] Figure 9 shows Kaplan-Meier survival plots of mice in the CT26.G10 syngeneic mouse tumor model treated with G1 / 4420 (human IgG1 isotype control antibody) or FS22m-063-AA / FS28m-228 (anti-mouse CD137 / MSLN mAb2). Treatment with FS22m-063-AA / FS28m-228 showed a significant improvement in survival compared to treatment with the human IgG1 isotype control antibody (log-rank analysis, **p<0.01). [Figure 10]Figure 10 shows individual tumor volume measurements in the CT26.G10 syngeneic mouse tumor model in mice treated with G1-AA / 4420 (IgG control; 20 μg, equivalent to 1 mg / kg in a 20 g mouse), G1 / Lob12.3 (wild-type human IgG1 anti-CD137 positive control; 20 μg, equivalent to 1 mg / kg in a 20 g mouse), and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2). Treatment with FS22m-063-AA / FS28m-228-010 demonstrated a dose-dependent decrease in tumor growth compared to mice treated with the human IgG1 isotype control. [Figure 11] Figure 11 shows Kaplan-Meier survival plots for mice in the CT26.G10 syngeneic mouse tumor model treated with G1-AA / 4420 (human IgG1 control; 20 μg, equivalent to 1 mg / kg in a 20 g mouse), G1 / Lob12.3 (wild-type human IgG1 anti-CD137 positive control; 20 μg, equivalent to 1 mg / kg in a 20 g mouse), and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN mAb2). Treatment with FS22m-063-AA / FS28m-228-010 at all dose levels tested resulted in significant improvements in survival compared to the IgG control, and the improvement in survival was dose-dependent. (Log-rank pairwise analysis comparing treated groups with G1-AA / 4420 isotype control, p<0.05, **p<0.01, ***p<0.001). [Figure 12]Figure 12 shows the results of (A) G1-AA / HelD1.3 (human IgG1 control), (B) FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in "mock" mAb2 format), (C) FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in mAb2 format), (D) G1-AA / FS28m-228-010 (anti-mouse MSLN antibody), (E) the combination of FS22m-063-AA / HelD1.3 plus G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab in "mock" mAb2 format plus anti-mouse MSLN Fab), and (F) FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN antibody). Figure 1 shows individual tumor volume measurements in the CT26.G10 syngeneic mouse tumor model in mice treated with mAb 2. Results show that 7 / 20 (35%) of mice treated with FS22m-063-AA / FS28m-228-010 had no palpable tumors at the end of the study, while all mice receiving other treatments, except for G1-AA / FS28m-228-010 (1 / 20, 5%), had tumors of 62.5 mm or larger at the end of the study. [Figure 13]Figure 13 shows the results of G1-AA / HelD1.3 (IgG control), FS22m-063-AA / HelD1.3 (anti-mouse CD137 Fcab in "mock" mAb2 format with non-binder Fab, anti-HelD1.3), FS22m-063-AA / 4420 (anti-mouse CD137 Fcab in "mock" mAb2 format with non-binder Fab, anti-4420), G1-AA / FS28m-228-010 (anti-mouse MSLN antibody), the combination of FS22m-063-AA / HelD1.3 plus G1-AA / FS28m-228-010 (anti-mouse CD137 Fcab plus anti-mouse MSLN antibody), and FS22m-063-AA / FS28m-228-010 (anti-mouse CD137 / MSLN antibody). Figure 1 shows Kaplan-Meier survival plots for mice in the CT26.G10 syngeneic mouse tumor model treated with one of two "mock" mAb2-formatted anti-mouse CD137 Fcabs or an anti-mouse MSLN antibody, and mice in the combination treatment group showed no improved survival compared to mice treated with the isotype control, whereas mice treated with FS22m-063-AA / FS28m-228-010-AA showed improved survival compared to mice treated with the isotype control (log-rank pairwise analysis comparing treatment groups to the G1-AAHelD1.3 isotype control, ****p<0.0001). [Figure 14]Figure 14 shows the pharmacokinetic profiles of anti-human CD137 / MSLN mAb2 and anti-mouse CD137 / MSLN mAb2 in a non-tumor-bearing C57BL / 6 mouse model. A: Anti-mouse CD137 / MSLN mAb2 (FS22m-063-AA / FS28m-228-010) at 10 mg / kg compared to a 10 mg / kg human IgG1 isotype control (G1 / 4420) after a single intravenous dose (n=3 per group). Both the human IgG1 isotype control and FS22m-063-AA / FS28m-228-010 mAb2 maintained high levels of exposure at 70.30 μg / ml and 18.11 μg / ml, respectively, 144 hours post-dose. The lower limit of quantitation (LLOQ) is shown at 5.48 ng / mL. B: Levels of anti-human CD137 / MSLN mAb2 (FS22-172-003-AA / FS28-256-271) following a single intravenous dose administered to non-tumor-bearing C57BL / 6 mice are shown, compared to a 6.7 mg / kg human IgG1 isotype control (G1 / 4420). Both the human IgG1 isotype control and anti-human CD137 / MSLN mAb2 maintained high levels of exposure at 28.36 μg / ml and 60.26 μg / ml, respectively, 144 hours post-dose. The lower limit of quantitation (LLOQ) is shown at 1.82 ng / ml. DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description The present invention relates to an antibody molecule that binds to both MSLN and CD137. Specifically, the antibody molecule of the present invention comprises a CDR-based antigen-binding site for MSLN and a CD137 antigen-binding site located in the constant domain of the antibody molecule.

[0092] The antibody molecule preferably specifically binds to MSLN and CD137. The term "specific" can refer to a situation in which the antibody molecule does not exhibit significant binding to molecules other than its specific binding partners (here, MSLN and CD137). The term "specific" can also be applied when the antibody molecule is specific for a particular epitope carried by several antigens, such as epitopes on MSLN and CD137, in which case the antibody molecule can bind to various antigens carrying the epitope. In a preferred embodiment, the antibody molecule of the present invention does not bind to or exhibits significant binding to OX40, GITR, CD40, CEACAM-5, E-cadherin, thrombomodulin, or EpCAM.

[0093] The term "antibody molecule" describes an immunoglobulin, whether natural or partially or wholly synthetically produced. The antibody molecule may be human or humanized, preferably human. The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies are immunoglobulin isotypes such as immunoglobulin G, and their isotypic subclasses such as IgG1, IgG2, IgG3, and IgG4, and fragments thereof. The antibody molecule may be isolated, in the sense that it is free of contaminants, such as antibodies capable of binding to other polypeptides and / or serum components.

[0094] Thus, the term "antibody molecule" as used herein includes antibody fragments, provided that said fragments contain the CDR-based antigen-binding site of MSLN and the CD137 antigen-binding site located in the constant domain.

[0095] The antibody molecule may be natural, or partly or wholly synthetically produced, for example, the antibody molecule may be a recombinant antibody molecule.

[0096] The antibody molecule comprises one or more CDR-based antigen binding sites for MSLN and one or more antigen binding sites for CD137 in one or more constant domains of the antibody molecule, preferably one or more CH3 domains.

[0097] 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, and most preferably an IgG1 molecule, or a fragment thereof. In a preferred embodiment, the antibody molecule is a complete immunoglobulin molecule.

[0098] In other embodiments, the antibody molecule may be an antigen-binding fragment comprising a CDR-based antigen-binding site for MSLN and an antigen-binding site for CD137 located in the constant domain. For example, the antigen-binding fragment may be an scFv-Fc fusion in which the scFv binds to MSLN and the Fc binds to a minibody comprising an scFv bound to an OX40 or CH3 domain (Hu et al. (1996), Cancer Res., 56(13):3055-61).

[0099] Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger and Hudson, 2005. Recombinant monoclonal and other antibodies Using DNA technology techniques, it is possible to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve transferring the CDRs or variable regions of one antibody molecule into a different antibody molecule (EP-A-184187, GB-A-2188638A and EP-A-239400).

[0100] In a preferred embodiment, the antibody molecule is a mAb 2 (TM) bispecific antibodies. The mAbs referred to herein 2 Bispecific antibodies are IgG immunoglobulins that contain a CDR-based antigen-binding site in each of their variable regions and at least one antigen-binding site in the constant domain of the antibody molecule.

[0101] In a preferred embodiment, the antibody is an antibody molecule that binds to MSLN and CD137, and the antibody molecule is (i) two CDR-based antigen-binding sites for MSLN, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain; and (ii) Two antigen-binding sites that bind to CD137 located in the two CH3 domains of the antibody molecule Includes:

[0102] In a more preferred embodiment, the antibody is a complete immunoglobulin molecule, e.g., a complete IgG1 molecule, that binds to MSLN and CD137, and the antibody molecule is (i) two CDR-based antigen-binding sites for MSLN, each formed by an immunoglobulin VH domain and an immunoglobulin VL domain; and (ii) Two antigen-binding sites that bind to CD137 located in the two CH3 domains of the antibody molecule wherein the immunoglobulin molecule further comprises a CH1, a CH2 and a CL domain.

[0103] A CDR-based antigen-binding site is an antigen-binding site of an antibody variable region. A CDR-based antigen-binding site can be formed by three CDRs, such as 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 antigen binding can vary among different antigen-binding sites.

[0104] 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.

[0105] An antibody molecule has one, or preferably more than one, e.g., two CDR-based antigen-binding sites for MSLN. Thus, an antibody molecule may comprise one VH and one VL domain, but preferably comprises two VH and two VL domains, i.e., two VH / VL domain pairs, as in, for example, naturally occurring IgG molecules.

[0106] The CDR-based antigen-binding sites are expressed in antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, and FS22-172-003- AA / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026 , FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, F S22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28- The fragment may comprise the three VH CDRs or three VL CDRs, preferably the three VH CDRs and three VL CDRs, of antibody FS22-172-003-AA / FS28-024-053, or FS22-172-003-AA / FS28-024, preferably antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably antibody FS22-172-003-AA / FS28-256-271.

[0107] The sequences of the CDRs can be readily determined from the VH and VL domain sequences of the antibody molecule using conventional techniques. Antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-1 72-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-A A / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-2 56-026, FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, F The VH and VL domain sequences of S22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, and FS22-172-003-AA / FS28-024 are described herein, and the three VH and three VL domain CDRs of the antibodies can therefore be determined from the 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).

[0108] The sequences of the VH and VL domains of an antibody containing a LALA mutation in the CH2 domain are the same as those of an antibody that does not contain a LALA mutation. For example, the VH and VL sequences of antibody FS22-172-003-AA / FS28-256-271 are the same as the VH and VL sequences of antibody FS22-172-003 / FS28-256-271. Similarly, the VH domain CDR1, CDR2, CDR3 and VL domain CDR1, CDR2, CDR3 of an antibody containing a LALA mutation in the CH2 domain are the same as those of an antibody not containing a LALA mutation. For example, the VH domain CDR1, CDR2, CDR3 and VL domain CDR1, CDR2, CDR3 sequences of antibody FS22-172-003-AA / FS28-256-271 are the same as those of antibody FS22-172-003-AA / FS28-256-271. 3, and the VL domain CDR1, CDR2, and CDR3 sequences are the same.

[0109] The VH domain CDR1, CDR2, and CDR3 sequences of an antibody molecule according to the IMGT numbering system 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.

[0110] The VH domain CDR1, CDR2 and CDR3 sequences of an antibody molecule according to Kabat numbering are , and sequences located at positions 31 to 35, 50 to 65, and 95 to 102 of the VH domain, respectively.

[0111] The VL domain CDR1, CDR2, and CDR3 sequences of an antibody molecule according to the IMGT numbering system may be sequences located at positions 27 to 38, 56 to 65, and 105 to 117 of the VL domain, respectively.

[0112] The VL domain CDR1, CDR2 and CDR3 sequences of the antibody molecule according to Kabat numbering are , and sequences located at positions 24 to 34, 50 to 56, and 89 to 97 of the VL domain, respectively.

[0113] for example, (i) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-271 may be as set forth in SEQ ID NOs: 42, 33, and 44, respectively; (ii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-052 may be as set forth in SEQ ID NOs: 14, 16, and 27, respectively; (iii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-021 may be as set forth in SEQ ID NOs: 42, 33, and 44, respectively; (iv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-012 may be as set forth in SEQ ID NOs: 42, 33, and 44, respectively; (v) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-023 may be as set forth in SEQ ID NOs: 50, 33, and 52, respectively; (vi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-024 may be as set forth in SEQ ID NOs: 42, 33, and 44, respectively; (vii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-026 may be as set forth in SEQ ID NOs: 50, 33, and 52, respectively; (viii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-027 may be as set forth in SEQ ID NOs: 42, 33, and 44, respectively; (ix) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-001 may be as set forth in SEQ ID NOs: 38, 33, and 35, respectively; (x) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-005 may be as set forth in SEQ ID NOs: 38, 33, and 35, respectively; (xi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-014 may be as set forth in SEQ ID NOs: 46, 33, and 48, respectively; (xii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-018 may be as set forth in SEQ ID NOs: 50, 33, and 52, respectively; (xiii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256 may be as set forth in SEQ ID NOs: 31, 33, and 35, respectively; (xiv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-051 may be as set forth in SEQ ID NOs: 14, 16, and 25, respectively; (xv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-053 may be as set forth in SEQ ID NOs: 14, 16, and 29, respectively; (xvi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024 may be as set forth in SEQ ID NOs: 14, 16, and 18, respectively; Here, the CDR sequences are defined according to the IMGT numbering scheme.

[0114] (i) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-271 may be as set forth in SEQ ID NOs: 20, 22, and 80, respectively; (ii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-052 may be as set forth in SEQ ID NOs: 20, 22, and 24, respectively; (iii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-021 may be as set forth in SEQ ID NOs: 20, 22, and 40, respectively; (iv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-012 may be as set forth in SEQ ID NOs: 20, 22, and 37, respectively; (v) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-023 may be as set forth in SEQ ID NOs: 20, 22, and 40, respectively; (vi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-024 may be as set forth in SEQ ID NOs: 20, 22, and 41, respectively; (vii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-026 may be as set forth in SEQ ID NOs: 20, 22, and 41, respectively; (viii) VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-256-027 The sequence of R3 may be as set forth in SEQ ID NOs: 20, 22, and 80, respectively; (ix) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-001 may be as set forth in SEQ ID NOs: 20, 22, and 40, respectively; (x) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-005 may be as set forth in SEQ ID NOs: 20, 22, and 41, respectively; (xi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-014 may be as set forth in SEQ ID NOs: 20, 22, and 37, respectively; (xii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-018 may be as set forth in SEQ ID NOs: 20, 22, and 37, respectively; (xiii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256 may be as set forth in SEQ ID NOs: 20, 22, and 37, respectively; (xiv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-051 may be as set forth in SEQ ID NOs: 20, 22, and 24, respectively; (xv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-053 may be as set forth in SEQ ID NOs: 20, 22, and 24, respectively; (xvi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024 may be as set forth in SEQ ID NOs: 20, 22, and 24, respectively; Here, the CDR sequences are defined according to the IMGT numbering scheme.

[0115] for example, (i) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-271 may be as set forth in SEQ ID NOs: 43, 5, and 45, respectively; (ii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-052 may be as set forth in SEQ ID NOs: 15, 17, and 28, respectively; (iii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-021 may be as set forth in SEQ ID NOs: 43, 34, and 45, respectively; (iv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-012 may be as set forth in SEQ ID NOs: 43, 34, and 45, respectively; (v) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-023 may be as set forth in SEQ ID NOs: 51, 34, and 53, respectively; (vi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-024 may be as set forth in SEQ ID NOs: 43, 34, and 45, respectively; (vii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-026 may be as set forth in SEQ ID NOs: 51, 34, and 53, respectively; (viii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-027 may be as set forth in SEQ ID NOs: 43, 34, and 45, respectively; (ix) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-001 may be as set forth in SEQ ID NOs: 39, 34, and 36, respectively; (x) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-005 may be as set forth in SEQ ID NOs: 39, 34, and 36, respectively; (xi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-014 may be as set forth in SEQ ID NOs: 47, 34, and 49, respectively; (xii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-018 may be as set forth in SEQ ID NOs: 51, 34, and 53, respectively; (xiii) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256 may be as set forth in SEQ ID NOs: 32, 34, and 36, respectively; (xiv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-051 may be as set forth in SEQ ID NOs: 15, 17, and 26, respectively; (xv) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-053 may be as set forth in SEQ ID NOs: 15, 17, and 30, respectively; (xvi) the sequences of the VH domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024 may be as set forth in SEQ ID NOs: 15, 17, and 19, respectively; Herein, the CDR sequences are defined according to the Kabat numbering scheme.

[0116] (i) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-271 may be as set forth in SEQ ID NOs: 21, 23, and 80, respectively; (ii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-052 may be as set forth in SEQ ID NOs: 21, 23, and 24, respectively; (iii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-021 may be as set forth in SEQ ID NOs: 21, 23, and 40, respectively; (iv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-012 may be as set forth in SEQ ID NOs: 21, 23, and 37, respectively; (v) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-023 may be as set forth in SEQ ID NOs: 21, 23, and 40, respectively; (vi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-024 may be as set forth in SEQ ID NOs: 21, 23, and 41, respectively; (vii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-026 may be as set forth in SEQ ID NOs: 21, 23, and 41, respectively; (viii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-027 may be as set forth in SEQ ID NOs: 21, 23, and 80, respectively; (ix) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-001 may be as set forth in SEQ ID NOs: 21, 23, and 40, respectively; (x) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-005 may be as set forth in SEQ ID NOs: 21, 23, and 41, respectively; (xi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-014 may be as set forth in SEQ ID NOs: 21, 23, and 37, respectively; (xii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256-018 may be as set forth in SEQ ID NOs: 21, 23, and 37, respectively; (xiii) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-256 may be as set forth in SEQ ID NOs: 21, 23, and 37, respectively; (xiv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-051 may be as set forth in SEQ ID NOs: 21, 23, and 24, respectively; (xv) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024-053 may be as set forth in SEQ ID NOs: 21, 23, and 24, respectively; (xvi) the sequences of the VL domain CDR1, CDR2, and CDR3 of FS22-172-003-AA / FS28-024 may be as set forth in SEQ ID NOs: 21, 23, and 24, respectively; Herein, the CDR sequences are defined according to the Kabat numbering scheme.

[0117] The CDR-based antigen-binding sites are expressed in antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, and FS22-172-003-AA / FS28 -256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22-172- 003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256-005 , FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-AA / F S28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, or FS22-172- 003-AA / FS28-024, preferably antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably antibody FS22-172-003-AA / FS28-256-271.

[0118] Antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, F S22-172-003-AA / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22-172-003-AA / FS28-256-027, FS22 The VH domains of FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, and FS22-172-003-AA / FS28-024 are set forth in SEQ ID NOs: 177 , 58, 70, 70, 74, 70, 74, 70, 66, 66, 72, 74, 62, 56, 60, and 12.

[0119] Antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-17 2-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256- 026, FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-1 The VL domains of FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, and FS22-172-003-AA / FS28-024 may have the sequences set forth in SEQ ID NOs: 76, 54, 68, 64, 68, 78, 78, 76, 68, 78, 64, 64, 64, 54, 54, and 54, respectively.

[0120] The antibody molecule preferably binds to human MSLN, more preferably human and cynomolgus monkey 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.

[0121] As described in the Background section above, mature MSLN is shed from tumor cells and removed from the tumor site. This shed MSLN can function as a sink for anti-MSLN binding molecules that are bound to the shed MSLN and subsequently removed 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 bind 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 present and expected to be available for bivalent binding by antibody molecules, as opposed to MSLN shed from tumor cells, which is expected to be in a monomeric form. Therefore, without wishing to be bound by theory, it is expected that the antibody molecules of the present invention will be cleared from tumor sites more slowly and therefore have a longer time window to exert their therapeutic effects.

[0122] The antibody molecule preferably binds to immobilized MSLN with higher affinity than MSLN in solution. The immobilized MSLN can be MSLN immobilized on a surface, such as a chip for use in surface plasmon resonance. MSLN in solution is also referred to herein as soluble MSLN and is not immobilized. The soluble MSLN is preferably in a monomeric form, i.e., monomeric mesothelin.

[0123] 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.

[0124] The antibody molecule preferably has an affinity (K D ) or with higher affinity to immobilized MSLN. Preferably, the antibody molecule has a K of 7 nM or 6 nM. D , or lower KD values ​​and bind to immobilized MSLN.

[0125] The antibody molecule preferably has an affinity (K D ) or with lower affinity. More preferably, the antibody molecule binds to MSLN in solution with an affinity of 16 nM, 17 nM, or Affinity (K D ) or with lower affinity to immobilized MSLN.

[0126] In a preferred embodiment, the antibody molecule binds to immobilized MSLN with an affinity (K D ) or higher affinity to MSLN in solution with an affinity of 18 nM (K D ) or with lower affinity.

[0127] The binding affinity of an antibody molecule to cells containing surface-bound MSLN is expressed as the half-maximal binding (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). 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 present Examples (see, e.g., Example 7). As explained above, antibody molecules whose binding to tumor cells containing surface-bound MSLN is unaffected or less affected by the presence of soluble MSLN 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 20 nM soluble MSLN is measured. 50 The concentration of antibody molecules required to achieve half-maximal binding (EC) of antibody to cells in the absence of soluble MSLN is 50 The antibody molecule concentration may be 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 molecule required to achieve a 50% RT-PCR result.

[0128] The binding of antibody molecules that do not block MUC16 binding to MSLN to cells containing cell-bound MSLN has been shown to be less affected by the presence of soluble MSLN, and therefore antibody molecules that cannot or do not block MUC16 binding to MSLN may be preferred.

[0129] The immobilized MSLN may have the sequence set forth in SEQ ID NO: 142. The MSLN in solution may have the sequence set forth in SEQ ID NO: 142.

[0130] The antibody molecules of the present invention have also been shown to bind to cynomolgus monkey MSLN, which may be beneficial in conducting efficacy and toxicity studies with the antibody molecules in cynomolgus monkeys, which may be predictive of the antibody molecules and their efficacy and toxicity in humans.

[0131] The antibody molecule may bind with similar affinity to immobilized human MSLN and immobilized cynomolgus monkey MSLN. Furthermore, the antibody molecule may bind with similar affinity to human MSLN in solution and cynomolgus monkey MSLN in solution. This is believed to be beneficial for ensuring that efficacy and toxicity studies conducted with the antibody molecule in cynomolgus monkeys are predictive of the antibody molecule and its efficacy and toxicity in humans.

[0132] Thus, in a preferred embodiment, the antibody molecule binds to immobilized cynomolgus MSLN with an affinity that is at most 10-fold, preferably at most 5-fold, and more preferably at most 3-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 most 10-fold, preferably at most 5-fold, and more preferably at most 2-fold lower or higher than the affinity with which the antibody molecule binds to human MSLN in solution.

[0133] Antibody molecules have been shown to have varying activities for ligand binding, for example, they may or may not block the binding of MUC16 to MSLN.

[0134] The antibody molecules are antibodies FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-052, FS The antibody molecule may comprise CDRs 1-6, the VH domain and / or the VL domain of FS22-172-003-AA / FS28-024-053, or FS22-172-003-AA / FS28-024, or a variant thereof, wherein the antibody molecule blocks binding of MUC16 to MSLN.

[0135] Alternatively, the antibody molecule may be any of antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22-172-003-AA / FS28-256-02 7, FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, or FS22-172-003-AA / FS28-256, or variants thereof, wherein the antibody molecule does not block binding of MUC16 to MSLN.

[0136] Suitable methods for determining the ability of an antibody molecule to block the binding of MUC16 to MSLN are known in the art and include ELISAs and cell-based assays, e.g., assays in which an antibody competes for binding to MUC16 for binding to cells expressing MSLN, such as NCI-H226 cells.

[0137] The antibody molecule of the present invention comprises a CD137 antigen-binding site. The CD137 antigen-binding site is located in the constant domain of the antibody molecule, preferably in the CH3 domain. The CD137 antigen-binding site comprises one or more modified structural loops in the constant domain of the antibody molecule. Engineering of antibody constant domain structural loops to create antigen-binding sites for target antigens is known in the art and is described, for example, in Wozniak-Knopp G et al. (2010) Protein Eng Des. 23 (4): 289-297; WO 2006 / 072620 and WO 2009 / 132876. The CD137 constant domain antigen-binding site contained in the antibody molecule of the present invention was identified following an extensive selection and affinity maturation program and preferentially binds to dimeric human CD137 over monomeric human CD137.

[0138] The CD137 antigen-binding site of the antibody molecule comprises first and second sequences located in the AB and EF structural loops, respectively, of a constant domain, preferably the CH3 domain, of the antibody molecule. The first and second sequences are preferably the first and second sequences of FS22-172-003 set forth in SEQ ID NOs: 10 and 11, respectively. The first and second sequences are preferably located between positions 14 and 17, and 91 and 99, respectively, of the CH3 domain of the antibody molecule, where residue numbering is according to the IMGT numbering system.

[0139] The CD loop sequence of the antibody molecule is preferably unmodified, i.e., wild-type. Thus, the CD loop sequence preferably has the sequence shown in SEQ ID NO: 157. The CD loop sequence is preferably located at positions 43 to 78 of the CH3 domain of the antibody molecule, where the numbering of residues is according to the IMGT numbering system.

[0140] In a preferred embodiment, the antibody molecule comprises a CH3 domain comprising, having or consisting of the CH3 domain sequence of FS22-172-003 as set forth in SEQ ID NO:8.

[0141] The CH3 domain of the antibody molecule may optionally comprise an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

[0142] With monoclonal and other antibodies, techniques of recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can involve transferring the CDRs or variable regions into a different immunoglobulin. The transfer of CDRs from one immunoglobulin into another is described, for example, in European Patent Application Publication No. A-184. 187, GB 2188638A, and EP-A-239400. Using similar techniques, the constant domain sequence constituting the CD137 antigen-binding site of an antibody molecule according to the present invention can be introduced into the constant domain, e.g., the CH3 domain, of another antibody molecule, thereby generating an antibody molecule comprising a CD137 antigen-binding site in its constant domain. Alternatively, the entire constant domain sequence of an antibody molecule can be replaced with the constant domain sequence of an antibody molecule according to the present invention to prepare an antibody molecule comprising a CD137 antigen-binding site in its constant domain. Similarly, a fragment of the constant domain sequence of an antibody molecule can be replaced with the corresponding fragment of the constant domain sequence of an antibody molecule according to the present invention comprising the CD137 antigen-binding site.

[0143] The antibody molecule preferably binds to human CD137, more preferably human and cynomolgus monkey CD137, even more preferably dimeric human and cynomolgus monkey CD137. The portion of CD137 bound by the antibody molecule is preferably the CD137 extracellular domain. The extracellular domains of human and cynomolgus monkey CD137 may comprise or consist of the sequences set forth in SEQ ID NOs: 149 and 153, respectively. The antibody molecule is preferably capable of binding to CD137 expressed on the surface of a cell. The cell is preferably a CD8 + or CD4 + Immune cells such as T cells or regulatory T (Treg) cells, preferably CD8 +T cells, or B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DCs), or tumor infiltrating lymphocytes (TILs).

[0144] As described in the Background section above, treatment of patients with the anti-CD137 antibody urelumab has been associated with dose-limiting, high-grade liver inflammation. Without wishing to be bound by theory, it is believed that the liver inflammation seen with urelumab treatment may be due to activation of liver-resident T cells or the infiltration and accumulation of activated T cells in the patient's liver. To select molecules with reduced or no liver inflammation, the inventors selected Fcabs with high avidity for CD137. Specifically, the inventors selected Fcabs that bind to dimeric CD137 with higher affinity than monomeric CD137. CD137 expression by T cells is upregulated upon priming and activation. Due to the higher expression of CD137 on activated T cells, CD137 is thought to form dimers, trimers, and higher-order multimers on the surface of such cells. In contrast, CD137 expression by inactive T cells is low or even undetectable. Therefore, CD137 is likely to be in a monomeric form as long as it is expressed on the surface of such T cells. Therefore, CD137 / MSLN mAbs that bind to CD137 with high avidity 2 In contrast to inactive T cells, such as those present in the liver, Specifically, it is believed that the anti-mouse CD137 / MSLN mAb preferentially binds to activated T cells and therefore exhibits reduced or no liver inflammation. 2 Treated with This was confirmed by determining the pharmacology of the liver in mice, which showed that the treatment did not result in hepatotoxicity (Example 13).

[0145] The antibody molecule preferably has an affinity (K D) or higher affinity to dimeric human CD137.

[0146] In preferred embodiments, the antibody molecule binds to dimeric CD137 with higher affinity than to monomeric CD137, hi preferred embodiments, the antibody molecule binds to dimeric CD137 with at least 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 200-fold higher affinity than the antibody molecule for monomeric CD137.

[0147] Monomeric human CD137 can have, for example, the sequence shown in SEQ ID NO:149.

[0148] Antibody molecules of the FS22-172 lineage have also been shown to bind to dimeric cynomolgus monkey CD 137. Binding to cynomolgus monkey CD 137 as well as human CD 137 is advantageous because it allows antibody molecules to be tested in cynomolgus monkeys for efficacy and toxicity prior to administration to humans.

[0149] In preferred embodiments, the antibody molecule has an affinity (K D ) or higher affinity. Preferably, the antibody molecule binds to dimeric cynomolgus CD137 with an affinity (K D ) or higher affinity to cynomolgus CD137.

[0150] The antibody molecule may bind to dimeric human CD137 and dimeric cynomolgus monkey CD137 with similar affinity, which is believed to be beneficial to ensure that efficacy and toxicity studies performed with the antibody molecule in cynomolgus monkeys are predictive of the antibody molecule and its efficacy and toxicity in humans.

[0151] Thus, in a preferred embodiment, the antibody molecule binds to dimeric cynomolgus monkey CD137 with an affinity that is no more than 10-fold, preferably no more than 5-fold, lower or higher than the affinity with which the antibody molecule binds to dimeric human CD137.

[0152] The binding affinity of antibody molecules to a cognate antigen, such as human or cynomolgus CD137, can be determined, for example, by surface plasmon resonance (SPR) such as Biacore.

[0153] The antibody molecule may be capable of blocking the interaction between CD137 and its ligand, CD137L, preferably between human CD137 and human CD137L. The ability of the antibody molecule to block the binding of CD137L to CD137 may be determined using ELISA.

[0154] Furthermore, the antibody molecule may comprise a CH2 domain of an immunoglobulin G molecule, such as the CH2 domain of an IgG1, IgG2, IgG3, or IgG4 molecule. Preferably, the antibody molecule comprises the CH2 domain of an IgG1 molecule. The CH2 domain may have the sequence set forth in SEQ ID NO: 154. 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).

[0155] The CH2 domain of the antibody molecule preferably contains one or more mutations that reduce or inhibit binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγIIa, FcγRIIb, and FcγRIII, and / or complement. The inventors hypothesize that reducing or inhibiting binding to Fcγ receptors reduces or eliminates ADCC mediated by the antibody molecule. Similarly, reducing or inhibiting complement binding is expected to reduce or eliminate CDC mediated by the antibody molecule. Without wishing to be bound by theory, this is expected to reduce or avoid liver inflammation when the antibody molecule is administered to a patient. Mutations that reduce or inhibit 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 involves substituting alanine (L1.3A and L1.2A) for the leucine residues at positions 1.3 and 1.2 of the CH2 domain. Alternatively, the asparagine (N) at position 84.4 of the CH2 domain is substituted with alanine, glycine, or glutamic acid. The generation of α-glycosylated antibodies through mutation of conserved N-linked glycosylation sites by mutating them to N84.4A, N84.4G, or N84.4Q is also known to reduce IgG1 effector function (Wang et al., 2018). Alternatively, complement activation (C1q binding) and ADCC are known to be reduced by mutating proline at position 114 in 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.

[0156] Thus, the antibody molecule may comprise a CH2 domain, wherein the CH2 domain 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; wherein the numbering of amino acid residues is according to the IMGT numbering scheme.

[0157] 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; wherein the numbering of amino acid residues is according to the IMGT numbering scheme.

[0158] For example, the CH2 domain may have the sequence set forth in SEQ ID NO:155.

[0159] In an alternative 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; wherein the numbering of amino acid residues is according to the IMGT numbering scheme.

[0160] For example, the CH2 domain may have the sequence set forth in SEQ ID NO:156.

[0161] In an alternative preferred embodiment, the antibody molecule is an antibody (i) FS22-172-003-AA / FS28-256-271 set forth in SEQ ID NOs: 3 and 84, respectively; (ii) FS22-172-003-AA / FS28-024-052 set forth in SEQ ID NOs: 102 and 85, respectively; (iii) FS22-172-003-AA / FS28-256-021 set forth in SEQ ID NOs: 125 and 82, respectively; (iv) FS22-172-003-AA / FS28-256-012 set forth in SEQ ID NOs: 125 and 116, respectively; (v) FS22-172-003-AA / FS28-256-023 set forth in SEQ ID NOs: 133 and 82, respectively; (vi) FS22-172-003-AA / FS28-256-024 set forth in SEQ ID NOs: 125 and 83, respectively; (vii) FS22-172-003-AA / FS28-256-026 set forth in SEQ ID NOs: 133 and 83, respectively; (viii) FS22-172-003-AA / FS28-256-027 set forth in SEQ ID NOs: 125 and 84, respectively; (ix) FS22-172-003-AA / FS28-256-001 set forth in SEQ ID NOs: 120 and 82, respectively; (x) FS22-172-003-AA / FS28-256-005 as set forth in SEQ ID NOs: 120 and 78, respectively; (xi) FS22-172-003-AA / FS28-256-014 set forth in SEQ ID NOs: 129 and 116, respectively; (xii) FS22-172-003-AA / FS28-256-018 set forth in SEQ ID NOs: 133 and 116, respectively; (xiii) FS22-172-003-AA / FS28-256 set forth in SEQ ID NOs: 114 and 116, respectively; (xiv) FS22-172-003-AA / FS28-024-051 set forth in SEQ ID NOs: 98 and 85, respectively; (xv) FS22-172-003-AA / FS28-024-053 set forth in SEQ ID NOs: 106 and 85, respectively; or (xvi) FS22-172-003-AA / FS28-024 set forth in SEQ ID NOs: 94 and 85, respectively. The antibody may comprise a heavy chain and / or a light chain, preferably a heavy chain and a light chain, of the antibody.

[0162] In a more preferred embodiment, the antibody molecule may comprise the heavy and / or light chains, preferably the heavy and light chains, of antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably antibody FS22-172-003-AA / FS28-256, where the heavy and light chain sequences of these antibodies are as described above.

[0163] The antibody molecules of the present invention may also comprise variants of the first, second, or third sequences, AB, CD, or EF structural loop sequences, CH3 domains, CH2 domains, CDRs, VH domains, VL domains, light chain and / or heavy chain sequences described herein. Suitable variants can be obtained by sequence alteration or mutation and screening methods. In a preferred embodiment, antibody molecules comprising one or more variant sequences retain one or more functional characteristics of the parent antibody molecule, such as binding specificity and / or affinity for MSLN and CD137. For example, antibody molecules comprising one or more variant sequences preferably bind to MSLN and / or CD137 with the same 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 into the variant antibody molecule.

[0164] Antibodies FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA / FS28-256-023, FS22-172-003- AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001 , FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, or FS22-172-003-AA / FS28-256 may comprise an amino acid substitution at position 55 or 57 of the VH domain, wherein the numbering of amino acid residues is according to the IMGT numbering scheme.

[0165] For example, the antibody molecule may comprise CDRs 1-6, a VH domain and / or a heavy chain of antibody FS22-172-003-AA / FS28-256-027, wherein the antibody molecule comprises an amino acid substitution at position 55 of the VH domain, and wherein the numbering of the amino acid residues is according to the IMGT numbering scheme.

[0166] For example, an antibody molecule of the invention may comprise a first, second or third sequence, an AB, CD or EF structural loop sequence, a CH3 domain, a CH2 domain, a CDR, a VH domain, a VL domain, a light chain and / or a heavy chain sequence that has 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 a structural loop, CH3 domain, CH2 domain, CDR, VH domain, VL domain, light chain or heavy chain sequence described herein.

[0167] In preferred embodiments, the antibody molecules of the invention comprise a CH3 domain sequence that has 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 a CH3 domain sequence described herein.

[0168] In a further preferred embodiment, the antibody molecule has a C2 domain sequence that has 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 CH2 domain sequences described herein. It has or contains an H2 domain sequence.

[0169] Sequence identity is generally measured using the algorithm GAP (Wisconsin GCG Package, Accelerys GAP is defined with reference to the Needleman and Wunsch algorithm (http: / / www.ncbi.nlm.nih.gov / pubmed / 2111111). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing 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. While the use of GAP is sometimes preferred, other algorithms, such as BLAST (using the method of Altschul et al., 1990), FASTA (using the method of Pearson and Lipman, 1988), or the Smith-Waterman algorithm (Smith and Waterman, 1981), or the TBLASTN program of Altschul et al. (1990), supra, can also be used, generally using default parameters. In particular, the psi-Blast algorithm (Altschul et al., 1997) can be used.

[0170] The antibody molecules of the present invention may also comprise a first, second, or third sequence, AB, CD, or EF structural loop sequence, CH3 domain, CH2 domain, Fcab, CDR, VH domain, VL domain, light chain, and / or heavy chain having one or more amino acid sequence alterations (additions, deletions, substitutions, and / or insertions of amino acid residues) compared to the first, second, or third sequence, AB, CD, or EF structural loop sequence, CH3 domain, CH2 domain, CDR, VH domain, VL domain, light chain, or heavy chain sequence described herein, preferably no more than 20 alterations, no more than 15 alterations, no more than 10 alterations, no more than 5 alterations, no more than 4 alterations, no more than 3 alterations, no more than 2 alterations, or no more than 1 alteration. In particular, alterations may be made in one or more framework regions of the antibody molecule outside the VH and VL domain sequences and / or in one or more framework regions of the CH3 domain. For example, alterations may occur in the CH3 domain outside of the sequences described herein, as first, second, and third sequences, or as AB, CD, or EF structural loop sequences.

[0171] The antibody molecule may comprise a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and / or a VL CDR3 having one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), preferably no more than three alterations, no more than two alterations, or one alteration, compared to the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 as disclosed herein. It may comprise VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3.

[0172] In a preferred embodiment, the antibody molecule of the present invention may comprise a CH3 domain sequence with one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of amino acid residues) compared to the CH3 domain disclosed herein, preferably no more than 20 alterations, no more than 15 alterations, no more than 10 alterations, no more than 5 alterations, no more than 4 alterations, no more than 3 alterations, no more than 2 alterations, or no more than 1 alteration.

[0173] In preferred embodiments, where one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example, according to the following table: In some embodiments, amino acids in the same category in the middle column are substituted for each other, i.e., a nonpolar amino acid is substituted with another nonpolar amino acid, for example. In some embodiments, amino acids in the same row in the right-most column are substituted for each other.

[0174] [Table 1]

[0175] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or not substantially affect) one or more functional properties (e.g., binding affinity) of an antibody molecule comprising the substitution, compared to a comparable unsubstituted antibody molecule.

[0176] The antibody molecules of the present invention preferably induce increased T cell activation when the antibody molecules are cross-linked, e.g., via binding to MSLN, than when the antibody molecules are not cross-linked.

[0177] The ability of an antibody molecule to activate T cells may be measured using a T cell activation assay. Upon activation, T cells release IL-2. Thus, a T cell activation assay may measure IL-2 release to determine the level of antibody molecule or T cell activation induced by the antibody molecule.

[0178] For example, the ability of an antibody molecule to activate T cells can be determined by measuring the concentration of the antibody molecule, when crosslinked, required to achieve half-maximal release of IL-2 by T cells in a T cell activation assay. This is the EC 50 It is called low EC 50 indicates that a lower concentration of antibody molecule is required to achieve half-maximal IL-2 release by T cells in a T cell activation assay, and therefore the antibody molecule has higher T cell activation activity. The antibody molecule can be cross-linked, for example, using an anti-CH2 antibody.

[0179] In a preferred embodiment, the antibody molecule has an EC value in a T cell activation assay that is higher than the EC value of FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052 in the same assay. 50 EC within 10, 5, 4, 3, or 2 times of 50 It has.

[0180] In an alternative preferred embodiment, the antibody molecule has a T cell activation assay that is more potent than the EC of FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052 in the same assay. 50 EC within 10, 5, 4, 3, or 2 times of 50 and

[0181] For example, the antibody molecule may have an EC50 of 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less in a T cell activation assay. 50 may have:

[0182] Additionally, or alternatively, the ability of an 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 antibody molecule, where the antibody molecule is cross-linked.

[0183] In a preferred embodiment, a T cell activation assay is performed in the presence of an antibody molecule in the presence of cross-linking. In (i), the maximum concentration of IL-2 released by T cells is within 3-fold, 2-fold, or 1.5-fold of the maximum concentration of IL-2 released by T cells in the presence of FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052 in the same assay.

[0184] T cell activation assays can be performed using CD8 T cells, e.g., as described in Example 8. + It may be a T cell assay as described herein, such as a T cell assay.

[0185] For example, T cell activation assays can be performed using CD8 T cells isolated from human peripheral blood mononuclear cells (PBMCs). + The assay may be a T cell-based IL-2 release assay. For example, the T cell activation assay may involve isolating human PBMCs from leukocyte-depleted cones. Methods for isolating PBMCs are known in the art and are described in this example. Next, CD8 + T cells can be isolated from PBMCs. + Methods for isolating T cells are known in the art and are described in this example.

[0186] Next, CD8 +T cells can be added to a multilayer plate coated with anti-human CD3 antibody. Appropriate 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. The supernatant can be collected and assayed to measure 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 this example. The concentration of human IL-2 can be plotted against the logarithmic concentration of the antibody molecule. The resulting curve can be fitted using a log(agonist) vs. response equation.

[0187] The antibody molecule may be conjugated to a biologically active molecule or a detectable label, in which case the antibody molecule may be referred to as a conjugate. Such conjugates find use in the treatment and / or diagnosis of diseases as described herein.

[0188] For example, the bioactive molecule can be an immune system modulator, such as a cytokine, preferably a human cytokine. For example, the cytokine can be a cytokine that stimulates the activation and / or proliferation of T cells. Examples of cytokines for conjugation to antibody molecules include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-gamma.

[0189] Alternatively, the bioactive molecule may be a ligand trap, such as a ligand trap for a cytokine, eg, TGF-beta or IL-6.

[0190] As a further alternative, the bioactive molecule may be a ligand such as CD137L, OX40L, TRAIL, CD40L, CD27L, or GITRL.

[0191] As a further alternative, the bioactive molecule may be a drug such as an inhibitor of tubulin polymerization (e.g., auristatin), a tubulin depolymerizing agent (e.g., maytansine), a DNA strand break inducer (e.g., calicheamicin), a DNA alkylating agent (e.g., duocarmycin), or an RNA polymerase inhibitor (such as alpha-amanitin).

[0192] Suitable detectable labels that can be conjugated to antibody molecules are known in the art and include radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111, technetium-99, and the like; fluorescent dyes such as fluorescein, rhodamine, phycoerythrin, Texas Red, and cyanine dye derivatives (e.g., Cy7 and Alexa750); chromogenic dyes such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; fluorophores or laser dyes with spectrally resolved absorption or emission properties; and via binding to specific cognate detectable moieties (e.g., labeled avidin). The antibody contains a chemical moiety, such as biotin, that can be detected as a marker.

[0193] 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. When the biologically active molecule is a cytokine, the cytokine can be attached to the antibody molecule by a peptide linker. Suitable peptide linkers are known in the art and can be 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 10 to 25 amino acids, 10 to 20 amino acids, or 10 to 15 amino acids in length.

[0194] In some embodiments, the biologically active molecule may be conjugated to the antibody molecule via a cleavable linker. The linker may enable release of the biologically active molecule from the antibody molecule at the therapeutic site. The linker may include an amide bond (e.g., a peptide linker), a disulfide bond, or a hydrazone. For example, a peptide linker may be cleaved by a site-specific protease, a disulfide bond may be cleaved by the reducing environment of the cytosol, and a hydrazone may be cleaved by acid-mediated hydrolysis.

[0195] 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 peptide bond. If the antibody molecule is a multi-chain molecule, for example, if the antibody molecule is or comprises an Fcab, or if the antibody molecule is a mAb, 2 For example, the biologically active molecule may be conjugated to one or more chains of the antibody molecule, such as a mAb. 2 It may be conjugated to one or both of the heavy chains of the molecule. Fusion proteins have the advantage of being easy to produce and purify, facilitating the production of clinical grade material.

[0196] The present invention also provides isolated nucleic acid molecules or 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.

[0197] In an alternative preferred embodiment, the nucleic acid molecule is selected from the group consisting of antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA A / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22 -172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256 -005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-A A / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, or FS22-17 The antibody may encode the heavy and / or light chains, preferably the heavy and light chains, of antibody FS22-172-003-AA / FS28-024, preferably antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably antibody FS22-172-003-AA / FS28-256-271.

[0198] Antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172- 003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA / FS28 -256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS 22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-172-003-A Nucleic acid molecules encoding the heavy chains of FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, and FS22-172-003-AA / FS28-024 are set forth in SEQ ID NOs: 4, 103, 126, 126, 134, 126, 134, 126, 121, 121, 130, 134, 115, 99, 107, and 95, respectively.

[0199] Antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-012, FS22-172-003-AA / FS28-256-023, FS22 -172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-256-001, FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-003-AA / FS28-256-018 Nucleic acid molecules encoding the light chains of FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053, and FS22-172-003-AA / FS28-024 are set forth in SEQ ID NOs: 91, 86, 122, 117, 122, 90, 90, 91, 122, 90, 117, 117, 117, 86, 86, and 86, respectively.

[0200] When a nucleic acid encodes 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.

[0201] Isolated nucleic acid molecules can be used to express the antibody molecules of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Accordingly, another aspect of the present invention provides a vector comprising such a nucleic acid. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, transcription termination fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Preferably, the vector contains appropriate regulatory sequences for driving expression of the nucleic acid in a host cell. The vector can be a plasmid, a viral vector, e.g., a phage, or a phagemid, as appropriate.

[0202] The nucleic acid molecules or vectors described herein can be introduced into host cells. Techniques for introducing nucleic acids or vectors into host cells are well established in the art, and any suitable technique can 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, e.g., HEK293 cells.

[0203] Another aspect of the present invention provides a method for producing an antibody molecule of the present 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 purifying recombinant antibody molecules are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography (e.g., using Protein A or Protein L). In some embodiments, purification may be performed using an affinity tag on the antibody molecule. The method may also comprise formulating the antibody molecule, optionally with a pharmaceutically acceptable excipient or other substance as described below, into a pharmaceutical composition.

[0204] As explained above, MSLN is expressed on the surface of tumor cells, and high expression levels of soluble MSLN correlate with poor prognosis in several cancers. Anti-MSLN antibodies have been investigated as anticancer therapeutics. These antibodies either directly induce cell death via ADCC activity or are used in the form of ADCs.

[0205] It is therefore anticipated that the antibody molecules described herein will find use in the treatment of cancer. Accordingly, a related aspect of the invention is (i) an antibody molecule as described herein for use in a method of treating cancer in an individual; (ii) use of an antibody molecule described herein in the manufacture of a medicament for use in treating cancer in an individual; and (iv) a method for treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody molecule described herein. to provide.

[0206] The individual may be a patient, preferably a human patient.

[0207] The antibody molecules of the present invention have been shown to preferentially bind to MSLN present on the surface of cancer cells compared to soluble MSLN. Therefore, cancers to be treated using the antibody molecules of the present invention preferably express MSLN or have been determined to express MSLN. More preferably, the cancer cells to be treated contain or have been determined to contain MSLN on their cell surface, i.e., have been determined to contain MSLN bound to the cell surface.

[0208] The cancer preferably comprises or is determined to comprise tumor-infiltrating lymphocytes (TILs) that express CD137. Specifically, the TILs preferably comprise or are determined to comprise CD137 on their cell surface.

[0209] Methods for determining the presence of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

[0210] The cancer may be a primary cancer or a secondary cancer. Accordingly, the antibody molecules described herein may be for use in methods of treating cancer in an individual, wherein the cancer is a primary tumor and / or tumor metastasis.

[0211] The cancer to be treated using the antibody molecule of the present invention may be a solid tumor.

[0212] 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 (such as biphasic synovial sarcoma, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, or soft tissue sarcoma), desmoplastic small round cell tumor, leukemia (such as acute lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, hairy cell leukemia, or myeloid leukemia), adrenocortical carcinoma, bladder cancer, brain cancer, cervical cancer, cervical hyperplasia, testicular cholangiocarcinoma, and uterine leukemia. The cancer may be selected from the group consisting of hair carcinoma, essential thrombocytosis, genitourinary cancer, glioma, glioblastoma, lymphoma (such as Hodgkin's disease or non-Hodgkin's lymphoma), malignant carcinoid cancer, malignant hypercalcemia, melanoma (also called malignant melanoma), malignant pancreatic insulinoma, medullary thyroid carcinoma, multiple myeloma, mycosis fungoides, neuroblastoma, polycythemia vera, primary brain cancer, primary macroglobulinemia, prostate cancer, renal cell carcinoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, and Wilms' tumor.

[0213] 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 round cell tumor.

[0214] More preferably, the cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.

[0215] Cancer is characterized by the abnormal proliferation of malignant cancer cells. When referring to a specific type of cancer, 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 not breast cancer as referred to herein, but ovarian cancer.

[0216] In cancer, treatment may include inhibiting the growth of the cancer, including complete cancer remission, and / or inhibiting the metastasis of the cancer, and inhibiting the recurrence of the cancer. Cancer growth generally progresses to more advanced forms within the cancer. "Inhibition of cancer growth" refers to any of a number of indicators that indicate a change in tumor volume or morphology. Thus, indicators for measuring inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound, or other imaging methods), slowing of tumor growth, destruction of tumor vasculature, improved performance in delayed-type hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and a decrease in the level of tumor-specific antigens. Activating or enhancing an individual's immune response against cancerous tumors can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the individual's propensity for cancer growth.

[0217] Although antibody molecules can be administered alone, they are usually administered in the form of a pharmaceutical composition, which 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. Methods are also provided that include formulating the antibody molecule into a pharmaceutical composition.

[0218] In addition to the antibody molecule, the pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" 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, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. 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 other suitable route, as discussed below.

[0219] For parenteral, e.g., subcutaneous or intravenous, administration (e.g., by injection), pharmaceutical compositions containing antibody molecules may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Other additives may be added, such as buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; 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; glycerols such as glycerols, ... Preservatives, stabilizers, buffers, antioxidants and / or other additives may be used as needed, including amino acids such as lysine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, 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).

[0220] In some 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.

[0221] Administration can be in a "therapeutically effective amount," which is sufficient to show benefit to an 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, and the nature and severity of what is being treated. The dosage depends on the type, method of administration, schedule of administration, and other factors known to physicians. Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other physicians and may depend on the severity of the symptoms and / or progression of the disease being treated. Appropriate dosages of antibody molecules are well known in the art (Ledermann et al., 1991; Bagshawe et al., 1991). Specific dosages as set forth herein or in the Physician's Desk Reference (2003) appropriate for the antibody molecule in question can be used. A therapeutically effective amount or appropriate dose of an antibody molecule can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dosage will depend on numerous factors, including the size and location of the area to be treated and the precise nature of the antibody molecule.

[0222] Typical antibody doses range from 100 μg to 1 g for systemic administration and from 1 μg to 1 mg for local administration. An initial higher loading dose may be administered, followed by one or more lower doses. This is the dose for a single treatment of an adult individual and can be adjusted proportionally for children and infants, and for other antibody formats proportionally based on molecular weight.

[0223] Treatments may be repeated daily, twice weekly, once weekly, or once monthly, at the physician's discretion. An individual's treatment schedule may depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

[0224] 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 monthly 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.

[0225] In the treatment of cancer, the antibody molecules described herein may be administered to an individual in combination with another anti-cancer therapy or therapeutic agent, such as an anti-cancer therapy or therapeutic agent shown to be or expected to be suitable for treating the cancer in question. For example, the antibody molecules may be administered to an individual in combination with agents for chemotherapeutic agents, radiation therapy, immunotherapeutic agents, anti-tumor vaccines, oncolytic viruses, adoptive cell transfer (ACT) therapy (such as adoptive NK cell therapy) or chimeric antigen receptors (CARs), autologous tumor-infiltrating lymphocytes (TILs), or gamma / delta T cells, or hormone therapy.

[0226] Without wishing to be bound by theory, it is believed that the antibody molecules described herein contain a second antigen-binding site for an immune cell antigen, such as a TNFRSF receptor, and may act as adjuvants in anti-cancer therapy. Specifically, it is believed that administration of the antibody molecules to an individual in combination with, for example, chemotherapy and / or radiation therapy, or in combination with an anti-tumor vaccine, will elicit a greater immune response against cancer than would be achieved with chemotherapy and / or radiation therapy, or an anti-tumor vaccine alone.

[0227] The one or more chemotherapeutic agents for administration in combination with an antibody molecule as 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, anti-cancer chemotherapeutic agents, and the like. 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, cobemitinib, and trametinib; PARP inhibitors include pilaparib; and B-Raf enzyme inhibitors include bemurafenib.

[0013] Other chemotherapeutic agents suitable for use in the present invention include defactinib, entinostat, eribulin, irinotecan, and vinblastine.

[0014] Other chemotherapeutic agents suitable for use in the present invention include dacarbazine, cyclophosphamide, and temozolomide; platinum analogs include carboplatin, cisplatin, and oxaliplatin; nucleoside analogs include azacitidine, capecitabine, fludarabine, fluorouracil, and gemcitabine; and antifolates include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in the present invention include defactinib, entinostat, eribulin, irinotecan, and vinblastine.

[0228] Preferred therapeutic agents for administration with the antibody molecules described herein are pentostatin, cyclophosphamide, cisplatin, pemetrexed, paclitaxel, carboplatin, gemcitabine, doxorubicin, vinorelbine, docetaxel, or etoposide.

[0229] Radiation therapy for administration in combination with an antibody molecule as 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 therapy or electron beam therapy (EBT), superficial radiation therapy (SRT)), or internal radiation therapy (such as brachytherapy, radioisotope or radionuclide therapy, SIRT). Preferably, the radiation therapy is conventional external beam radiation therapy, external beam radiation therapy (EBRT), stereotactic radiation therapy, or brachytherapy.

[0230] The immunotherapeutic agent for administration in combination with the antibody molecule described herein may be a therapeutic antibody molecule, a nucleic acid, a cytokine, or a cytokine-based therapeutic agent. For example, the therapeutic antibody molecule may bind to an immunomodulatory 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 immunomodulatory 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, GITR, CD27, or ICOS; and other immunomodulatory 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.

[0231] The nucleic acid for administration in combination with an antibody molecule as described herein may be an siRNA.

[0232] The cytokine or cytokine-based therapy may be selected from the group consisting of IL-2, a prodrug of conjugated IL-2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferon.

[0233] Antitumor vaccines for the treatment of cancer have been implemented in clinics and have been extensively discussed in the scientific literature (e.g., Rosenberg, S. 2000). This primarily involves a strategy of stimulating the immune system to respond to various cell markers expressed by autologous or allogeneic cancer cells by using these cells as a vaccination method, both with and without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induces a strong response in antigen presentation and works particularly well when used in this strategy.

[0234] The chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormonal therapy agent is preferably a chemotherapy, radiotherapy, immunotherapy, anti-tumor vaccine, oncolytic virus, ACT therapy, or hormonal therapy agent for the cancer in question, That is, a chemotherapeutic agent, radiation therapy, immunotherapy agent, antitumor vaccine, oncolytic virus, ACT therapy, or hormonal therapy agent that has been shown to be effective in treating the cancer in question. Selection of an appropriate chemotherapeutic agent, radiation therapy, immunotherapy agent, antitumor vaccine, oncolytic virus, ACT therapy, or hormonal therapy agent that has been shown to be effective against the cancer in question is well within the ability of one skilled in the art.

[0235] Further aspects and embodiments of the present invention will be apparent to those skilled in the art given this disclosure, including the experimental exemplification that follows.

[0236] All documents mentioned herein are incorporated by reference in their entirety.

[0237] As used herein, "and / or" should be interpreted as a specific disclosure of each of two particular features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0238] Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0239] Other aspects and embodiments of the present invention provide those aspects and embodiments described above, with the term "comprising" substituted with the term "consisting of" or "consisting essentially of," unless the context dictates otherwise.

[0240] Certain aspects and embodiments of the present invention will now be described, by way of example, with reference to the above-mentioned drawings. [Example]

[0241] Example Example 1: Antigen selection and characterization mAb capable of binding to both MSLN and CD137 and resulting in CD137 agonism 2 The selection and screening methods used to identify these cells involve the use of various MSLN and CD137 antigens, the production of which is described in more detail below.

[0242] 1.1 Recombinant CD137 antigen Tumor necrosis factor receptor superfamily (TNFRSF) members, such as CD137, are known to have a tendency to form clustered multimers when bound to their cognate ligands (Croft, M. 2003). This tendency to aggregate due to their function makes it difficult to produce soluble recombinant proteins that do not aggregate in solution for use in in vitro selections such as phage and yeast display, and for characterization of selected proteins.

[0243] Since the majority of commercially available antigens were deemed unsuitable, the following recombinant dimeric and monomeric CD137 antigens (see Table 1) were produced in-house for use in the selections.

[0244] [Table 2]

[0245] Monomeric antigens were prepared by cloning DNA encoding the extracellular domain of human (as shown in SEQ ID NO: 149) or mouse CD137 (as shown in SEQ ID NO: 150) along with an Avi tag sequence and six C-terminal histidine residues into a modified pFUSE vector (InvivoGen, pfuse-mIgG2A-Fc2) using EcoRI-HF and BamHI-HF restriction enzymes. The vector was transfected into HEK293-6E cells (National Research Council of Canada), and the expressed CD137 was transfected with HisTrap™ excel nickel. Column (GE Healthcare Life Sciences, 29048586) and size exclusion chromatography The antigen was purified using SEC to ensure it was a single species and free of aggregates.

[0246] To produce dimeric antigens, DNA constructs encoding the extracellular domain of human, mouse, or cynomolgus CD137 fused to the mIgG2a Fc domain with an Avi tag sequence were cloned into a modified pFUSE vector and transfected into HEK293-6E cells. Recombinant CD137 was purified using a MabSelect SuRe™ Protein A column (GE Healthcare Life Sciences, 11003494) and size exclusion chromatography (SEC). Purification was performed to ensure that the antigen was single species and free of aggregates.

[0247] The dimeric and monomeric antigens were each biotinylated using a BirA biotin-protein ligase reaction kit (Avidity LLC, BirA500), and monomeric CD4s labeled with a single biotin molecule were synthesized. Dimeric CD137 antigen was produced, labeled with two biotin molecules: one for each of the two monomers and the 137 antigen. 3 mg of antigen was mixed with 7.8 μl of BirA enzyme mix at an enzyme-to-substrate molar ratio of 1:50. Additives were then added according to the manufacturer's recommendations (142 μl Biomix A, 142 μl Biomix B, 142 μl biotin), and the reaction mixture was incubated at room temperature for 2 hours. To maintain the integrity of the biotinylated protein, Amicon The reaction mixture was immediately buffer exchanged into DPBS (ThermoFisher Scientific 14190-169) using a 30 μm filter (Merck, UFC503096).

[0248] The protein was further purified by SEC to ensure the removal of the BirA enzyme and the generation of a final high-quality, monodisperse protein preparation free of high molecular weight aggregates. The material was analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography with multi-angle light scattering detection. The stability and purity of the proteins were analyzed by SEC-MALS. Complete biotinylation of the proteins was confirmed by streptavidin-shift SDS-PAGE gels. The recombinant human and mouse antigens were analyzed in vitro by surface plasmon resonance (SPR) with anti-CD137 positive control antibodies (20H4.9 (U.S. Patent No. 7,288,638) and Lob12.3 (University of Southampton), respectively), and human CD137 by flow cytometry. The binding of this antigen to DO11.10 cells expressing the mouse CD137 ligand was confirmed. After incubating cells with the CD137 antigen for 1 hour, cell binding was detected using a fluorescently labeled anti-mouse Fc fragment antibody. To ensure the highest possible purity for the material used in the selection protocol, thorough protein characterization of the antigen was performed to ensure that the presence of protein aggregates did not exceed 2%.

[0249] 1.2 Cell-expressed CD137 antigen DO11.10 cells (National Jewish Health) expressing full-length murine (SEQ ID NO: 374) or human CD137 (SEQ ID NO: 373), designated "DO11.10.mCD137" and "DO11.10.hCD137," respectively, were produced to present antigen in a membrane-bound conformation most similar to its native form for selection and further characterization of selected Fcabs as listed in Table 2.

[0250] Lentiviral transduction was used to generate these DO11.10 cells overexpressing the human or mouse CD137 receptor using the Lenti-X HTX Packaging System (Clontech, catalog no. 631249). The Lenti-X expression vector (pLVX) (Clontech, catalog no. 631249) containing human or mouse CD137 cDNA was used. No. 631253) is packaged with the Lenti-X 293T Cell Line (Clontech) along with the Lenti-X HTX Packaging Mix. , Cat. No. 632180) to generate virus. The DO11.10 cell line was then transduced with these lentiviral vectors.

[0251] Expression of human or mouse CD137 on these cells was confirmed by flow cytometry by binding of the 20H4.9 and Lob12.3 anti-CD137 positive control antibodies, respectively. After incubating cells with the human or mouse positive control antibodies for 1 hour, cell binding was detected using a fluorescently labeled anti-human Fc detection antibody (Stratech Scientific Ltd, catalog number 109-546-098-JIR).

[0252] [Table 3]

[0253] 1.3 Human, cyno, and mouse mesothelin antigens Recombinant biotinylated human MSLN-His antigen, designated 'hMSLN-His Acro', was obtained from Acrobiosystems (catalog no. MSN-H8223) lacking the C-terminal 18 amino acids. 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 capable of binding to human and cynomolgus MSLN, respectively, and to isolate mouse MSLN binders.

[0254] Briefly, the MSLN antigen is amplified by EcoRI-HF and BamHI-HF restriction enzymes. DNA encoding full-length human (SEQ ID NO: 142) (hMSLN-His-Avi), cynomolgus monkey (SEQ ID NO: 143) (cMSLN-His-Avi), or mouse (SEQ ID NO: 144) (mMSLN-His-Avi) MSLN, along with six C-terminal histidine residues and an Avi tag sequence, was cloned using modified pF Generated by cloning into the USE vector (InvivoGen, pfuse-mIgG2A-Fc2) 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 Healthcare Life Sciences 29048586). Each of the antigens was biotinylated using a BirA biotin-protein ligase reaction kit (Avidity LLC, BirA500). A monomeric MSLN antigen labeled with a single biotin molecule was produced.

[0255] Subsequently, recombinant human cyno Alternatively, mouse MSLN was purified to remove excess free biotin.

[0256] SEC-HPLC of these antigens showed less than 10% aggregation, and PAGE confirmed that the antigens were monomeric. Using ELISA and surface plasmon resonance (SPR), biotinylated MSLN antigens were synthesized as SS1 scFv (US Pat. No. 7,081,518 B1 (Hassan et al. 2002) and MOR6626 (WO 2009 / 068204 A1)). ) could be bound by the MSLN-specific positive control antibody SS1 (VH SEQ ID NO: 140; VL SEQ ID NO: 141), which contains similar CDRs. Based on this data, all antigens were considered suitable for naive selection.

[0257] Example 2: Selection and characterization of anti-human CD137 Fcabs 2.1 Naive selection of anti-human CD137 Fcabs To select Fcabs that bind to human CD137, we employed yeast and phage display selection campaigns to maximize the diversity of identified Fcabs. Cell surfaces displaying both human CD137 and recombinant dimeric human CD137 were used to provide a variety of antigen presentations to exert avidity-driven selection pressure against dimeric or multimeric CD137 proteins. Obtaining Fcabs that avidly bind to the CD137 complex rather than to monomeric CD137 with high affinity was considered beneficial because such Fcabs would preferentially target activated and primed T cells, where CD137 upregulation occurs after T cell stimulation. Without wishing to be bound by theory, we hypothesized that T cells with very low or negligible levels of CD137 membrane expression are more likely to have monomeric CD137, as opposed to activated T cells with highly upregulated CD137, where the majority of the protein is in dimeric, trimeric, or higher multimeric forms. As a result of avidity-driven selection, Fcabs will preferentially bind to activated T cells and will not bind well to naive T cells or other cells that exhibit low expression of CD137. Selecting for avidity CD137 Fcabs reduces potential off-target T cell activation and associated toxicity.

[0258] Naive yeast libraries displaying the CH1 to CH3 domains of human IgG1 were used for yeast display selection. All libraries contained a randomized AB loop (containing residues 14 to 18 according to the IMGT numbering) and a randomized EF loop (containing residues 92 to 101 according to the IMGT numbering) of the CH3 domain. Two of the libraries further contained a five-amino acid residue insertion at position 16 of the AB loop of the CH3 domain (residues 16.5 to 16.1 according to the IMGT numbering).

[0259] Yeast single clones identified from the library selection were analyzed by flow cytometry antigen binding assays, which involve incubating cells with biotinylated recombinant dimeric human antigen or mouse Fc fragment and distinguishing yeast clones that bind to the Fc portion of the recombinant hCD137 antigen. The clones were screened for antigen binding using a ELISA assay. To increase the number of hits, the selection was repeated under various antigen concentrations and conditions, such as increasing the induction temperature, decreasing the selection stringency, or decreasing the number of rounds. Nine Fcab clones with unique sequences were identified.

[0260] 2.2 "Mock" mAb 2 Preparation of anti-human CD137 Fcab in the format "Mock" mAb consisting of IgG1 molecules containing 85 anti-human CD137 Fcab clones 2 Generate antibodies and mAbs 2 This allows for characterization of Fcabs in a mock mAb format. 2 was prepared by substituting a portion of the CH3 domain Fcab, including the AB, CD, and EF loops, for the corresponding region of the CH3 domain of the anti-hen egg lysozyme antibody HelD1.3. The generation of the HelD1.3 antibody is described in Tello et al., 1993. The heavy and light chain sequences of the antibody HelD1.3 are shown in SEQ ID NOs: 138 and 139, respectively. Mock mAb 2The molecules were produced by transient expression in HEK293-6E cells and purified by Protein A affinity chromatography using a mAbSelectSure column. 2 was tested for binding to human recombinant antigen (biotinylated hCD137-mFc-Avi) by biolayer interferometry (BLI).

[0261] 2.3 Selected anti-CD137 mock mAbs in human NF-κB reporter assays 2 Activity of Multimerization and clustering are required for TNFR signaling (Bitra et al., 2017). CD137 is involved in the NF-κB signaling pathway when it interacts with its NF-κB cognate ligand, CD137L. , which clusters and activates the NF-κB signaling pathway. Agonistic molecules mimic ligands that promote CD137 clustering and activation, thereby activating the NF-κB signaling pathway. Some agonistic antibodies, such as urelumab, can intrinsically induce CD137 clustering upon binding, while others, such as utomilumab, require additional cross-linking of the antibody itself to induce CD137 clustering (Fisher et al., 2012). Fc gamma receptors on effector cells are known to induce such cross-linking in vivo, but this is inefficient and may occur away from the site of therapeutic interest. Because dose-limiting toxicities are associated with urelumab but not utomilumab, we chose to select anti-CD137-binding Fcabs that lack intrinsic agonizing capacity but only those that require additional cross-linking to induce CD137 clustering. Therefore, we developed an assay that can detect the activation of the intracellular NF-κB signaling pathway by clustering CD137 expressed on the cell surface with a crosslinking antibody, but shows little activity when the antibody is not crosslinked. This assay was then applied to anti-CD137 Fcab clone mAbs, regardless of whether the Fcab was found to bind to the recombinant antigen by BLI.2 Protein L was used as a cross-linker to cross-link the Fab portion of the mock mAb to test its agonistic functional activity. 2 Cross-linking was promoted and NF-κB activation was measured.

[0262] HEK.FRT.luc.hCD137 cells were cultured by subcloning a cDNA sequence encoding the human CD137 sequence (SEQ ID NO: 149) into the pMSCV-neomycin vector (Takara Clontech, Cat. No. 634401) using EcoRI-HF and XhoI restriction enzymes. Retroviral particles were generated by the RetroPack PT67 cell line (Clontech, Cat. 631510) according to the manufacturer's protocol. This retrovirus was then used to transduce HEK.FRT.luc cells, which had been previously generated by transducing the Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) with Qiagen Cignal Lenti NFkB Reporter (luc) (Qiagen, Cat. No. 336851) lentivirus, which contains an NF-κB-sensitive promoter controlling the expression of luciferase. These HEK.FRT.luc.hCD137 cells were used to generate CD137 binders identified in the selection. Mock mAb with 2 was screened.

[0263] Each mock mAb 2 Prepare a 2 μM dilution of in DPBS (Life Technologies, 14190169) The reporter cells were cultured in a medium containing DMEM (Gibco, Cat. 61965-026); 10% FCS (Gibco, Cat. 10270-106); 1 x PennStrep (Gibco, Cat. 15140-122); 15 μg / mL of blasticidin. The mAbs were further diluted 1:3 in 100 μg / ml (Melford Laboratories Ltd, Cat. B1105); puromycin 5 μg / ml (Life technologies, Cat. A11113803); zeocin 100 μg / ml (InvivoGen, Cat. 11006-33-0); geneticin 500 μg / ml (Life Technologies, Cat. 10131-027). Protein L (Life Technologies, 21189) was used as an artificial crosslinker to crosslink the mAbs. 2 The cells were mixed with the molecule at a 1:4 molar ratio. After 24 hours of incubation, cells were treated with 100 μl of Promega Bio-Glo™ Luciferase Assay Reagent (Promega catalog number G7941) according to the manufacturer's instructions, and luminescence was measured using a plate reader equipped with Gen5 Software, BioTek, with a 0.5-second integration time. Luminescence values ​​are a measure of luciferase produced in response to activation of the NF-κB signaling pathway by crosslinked Fcab-induced CD137 clustering. Luminescence values ​​were plotted against the logarithmic concentration of Fcab, and the resulting curve was fitted using the log(agonist) vs. response equation in GraphPad Prism.

[0264] Hits were identified by at least a 10-fold increase in luciferase signal when cross-linked with protein L compared to uncross-linked, and these clones were determined to be capable of inducing CD137 clustering and subsequent activation of downstream signaling pathways. Of all clones tested, two, including FS22-172, were able to induce this 10-fold increase in luciferase upon cross-linking, but only EC 50Neither of these clones could be determined. Both were selected for further characterization in the DO11.10 T cell activation assay. Surprisingly, despite binding to the CD137 target by BLI, the remaining clones in the cross-linked state showed no observed activity, possibly indicating that they bound to unrelated epitopes on CD137 or that the affinity of such clones was not sufficient to bind CD137 strongly enough to initiate the NF-κB signaling cascade. Overall, two Fcabs (including FS22-172) were identified from the naive selection that, when cross-linked, displayed the desired function in the NF-κB reporter assay but had little activity without cross-linking.

[0265] Example 3: Affinity maturation of anti-human CD137 Fcabs and subsequent characterization 3.1 Affinity maturation of FS22-172 Four yeast display libraries were constructed from the FS22-172Fcab clone. Seven residues (positions 15-16.1 by IMGT) were randomized in the AB loop of the CH3 domain of each clone using the ELLA primer to generate library FS22-172AB. Five residues (positions 92-94 and 97-98 by IMGT) were randomized in the EF loop of the CH3 domain using the ELLA primer to generate library FS22-172EF.

[0266] For libraries FS22-172AB and FS22-172EF, three or four selection rounds were performed on the yeast library to select for affinity-matured clones using dimeric hCD137-mFc-Avi antigen or monomeric hCD137-Avi-His antigen. Monomeric antigen was alternated with dimeric antigen to ensure that clones retained affinity for the antigen and did not bind solely via avidity. The use of monomeric or dimeric antigen and the concentration used were empirically determined during each round by flow cytometry and determined whether enrichment for monomeric or dimeric antigen was observed in the previous round. Whenever possible, a sorting gate above the parent molecule was used to isolate affinity-matured clones relative to the parent molecule. Selection pressure was increased up to 1 nM of dimeric antigen. During each selection round, individual clones were spotted onto agar plates to assess the progress of the selection. Each clone was individually grown and induced, and its binding and structural parameters were then determined by flow cytometry using biotinylated dimeric antigen and anti-CH2 structural markers, as previously described. Following this screening cascade, selection success was determined based on a sample of clones from the selection output, allowing for early screening of individual clones that could then be produced as soluble proteins.

[0267] Yeast single clones were screened for binding to biotinylated recombinant antigen by antigen-binding flow cytometry as previously described. Thirty unique loop sequences were isolated from the FS22-172AB library. The FS22-172EF library did not contain any clones that showed improved binding over the parental clones.

[0268] 3.2 "Mock" mAb 2 Construction of anti-human CD137 Fcab in a 2D format "Mock" mAb containing anti-human CD137 Fcab in HelD1.3 2 The antibody is a mAb 2Affinity-matured Fcabs of this format were prepared for further characterization. 2 was prepared as described in Example 2.2. CD137 / HelD1.3 "Mock" mAb 2 was produced by transient expression in HEK293-6E cells and expressed as mAb Select SuRe protein. The product was purified using a PEG-A column.

[0269] 3.3 Mock mAb in human NF-κB reporter cell assay 2 Human Fcab activity in various formats Enumerated mock mAb 2 The functional activity of the affinity matured anti-human CD137 Fcab in (HelD1.3) format was tested in a similar NF-κB luciferase assay described in Example 2.3. An integration time of 0.5 seconds was used on a plate reader equipped with Gen5 Software, BioTek. Luminescence was measured between 100 and 1000 s. As expected, the Fcabs showed no activity without protein L crosslinking (-XL). All affinity-matured CD137 Fcabs showed significant improvement over the parental CD137 Fcab and were positive in this assay, but not in the EC 50 It was not possible to calculate the value (see Example 2.3). FS22-172-003 had the lowest EC when cross-linked with protein L (+XL). 50 (32.64 nM), showing the highest activity from each family.

[0270] 3.4 Determining the specificity of anti-human CD137 Fcab by surface plasmon resonance (SPR) The specificity of the anti-human CD137 Fcabs for human CD137 compared to other related TNFSFR family members was tested. Eight of the Fcabs were tested against mock mAb 2 (HelD1.3) format and by SPR on a Biacore T200 (GE Healthcare) by testing binding to other human TNFRSF receptors: CD40, OX40, and GITR. Amine coupling (amine coupling kit, GE Healthcare, BR-1000-50) was used to measure the ATP concentration on a Biacore CM5 chip (GE Healthcare, catalog number 291496 Human CD40, GITR, and OX40 were coated with 03) at approximately 1000 RU. Mock mAb starting at 1 μM 2 A dilution of anti-human CD137 Fcab in the format (FS22-172-003 / HelD1.3) was prepared in HBS-EP+ buffer (BR100669) and injected at 30 μl / min for 3 minutes, followed by 4 minutes of dissociation in buffer. The chip was regenerated by injecting 10 mM glycine pH 2.5 at 30 μl / min for 12 seconds. Antibodies specific for different TNFRSF members were used as positive controls to verify the Biacore chip coating. The data were double-reference subtracted and analyzed using BIAevaluation3.2 software. The Fcabs or mAbs containing antigen-binding sites from these Fcabs did not bind to any of the TNFRSF receptors tested, demonstrating specificity for CD137. 2 is not expected to induce binding to anything other than CD137.

[0271] 3.5 Mock mAb against human, cynomolgus monkey, and mouse CD137 by SPR 2 Binding affinity of anti-human CD137 Fcab in various formats Mock mAb against human, cynomolgus monkey (cyno) and mouse CD137 2The affinity of anti-human CD137 Fcabs in this format (see Example 3.2) was measured by SPR to determine whether the Fcabs could be useful for testing in animal studies. Anti-human Fab capture antibodies were immobilized on all four flow cells of a CM5 Series S chip (GE Healthcare #BR-1005-30) according to the manufacturer's recommendations (GE Healthcare, Human Fab Capture Kit, #28958325) to an average surface density of 6000 RU. Immobilization at 25°C and a flow rate of 10 μl / min achieved an average final response of 6000 RU. Each mAb 2 is mAb diluted in HBS-EP+ buffer (GE Healthcare #BR1006-69). 2 30μl / 3μg / ml solution of Up to approximately 150 RU was captured by a 60-second injection at 60 μl / min. Next, different concentrations of human, cyno, or mouse CD137 antigen (non-biotinylated human, cyno, or mouse CD137-mFc-Avi or human CD137-Avi-His) in HBS-EP+ buffer were flowed over the chip at 60 μl / min for 3 minutes, followed by a 10-minute dissociation period. After each antigen concentration, the chip was regenerated by injecting 10 mM glycine pH 2.1 at a flow rate of 30 μl / min for 30 seconds. HBS-EP+ buffer was injected before the highest and after the lowest antigen concentration for reference subtraction, with each random concentration repeated twice. Binding kinetics were fitted to a 1:1 Langmuir model to determine equilibrium binding (K ) for each sample. D ) were generated. Data analysis was performed using BiaEvaluation software version 3.2. The results are shown in Table 3.

[0272] Analysis of the results revealed improved binding to both human and cynomolgus monkey CD137 by all affinity-matured clones compared with their respective parent molecules. The binding affinity for the monomeric human CD137 antigen was weaker (at least 100-fold) than for the dimeric human and cynoFc fusion antigens. As discussed in Example 2.1, Fcabs were selected to preferentially bind to the dimeric over the monomeric form of CD137, and this data confirms the success of the selection strategy. This kinetic behavior reduces the likelihood of binding to monomeric CD137, which is expressed at minimal levels on unstimulated T cells, thereby reducing the risk of liver or systemic toxicity associated with some anti-CD137 monoclonal antibody therapies.

[0273] The data also show that anti-human CD137 Fcab bound to cynomolgus monkey dimeric CD137 with an affinity comparable to that of human dimeric CD137.

[0274] [Table 4]

[0275] The ability of Fcabs to bind to mouse dimeric CD137 was also tested. did not show strong binding to the antigen (as shown in Table 3, N / A indicates K D could not be calculated).

[0276] 3.6 Overview of affinity maturation and characterization of anti-human CD137 Fcabs In summary, we generated affinity-matured anti-CD137 Fcabs and mAbs 2 These mAbs containing the CD137 antigen-binding domain in the CH3 domain were prepared in various formats and subsequently characterized. 2 showed high levels of activity in a human NF-κB reporter cell assay, and this activity was shown to be cross-linking dependent.

[0277] Anti-human CD137 antigen-binding domain-containing mAb 2It was also shown to be specific for CD137 and not to bind to other TNFRSF receptors. 2 were shown to preferentially bind to the human dimeric CD137 antigen over the human monomeric CD137 antigen. 2 was shown to bind to cynomolgus monkey dimeric CD137 with an affinity comparable to that of human dimeric CD137.

[0278] Anti-human CD137 antigen-binding domain-containing mAb 2 demonstrated that CD137 requires cross-linking to cluster and activate. Therefore, our next goal was to identify a mAb that binds human MSLN in addition to CD137. 2 The aim of this study was to prepare a mAb targeting human MSLN via its Fab arm. 2 It was hypothesized that binding of causes cross-linking of antibody molecules, resulting in clustering and activation of CD137, and that this activation should be dependent on the presence of human MSLN expression.

[0279] mAb that binds human MSLN in addition to CD137 2 To prepare the antibody, it was first necessary to generate mAbs capable of binding to MSLN. The generation of these mAbs is described below.

[0280] Example 4: Isolation of anti-human MSLN antibodies: antigen, selection and screening Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesized as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form (termed megakaryocyte-potentiating factor or MPF) and a 40 kDa membrane-bound form of mesothelin (MSLN). Soluble forms of MSLN, generated by shedding from tumor cell surfaces and alternative splicing or by tumor necrosis factor-converting enzyme (TACE) of membrane-bound MSLN, are found in patient sera. This tumor-shed antigen is known to create a "sink" that can function as a decoy for therapeutic antibodies (Lee et al., 2018). This must be overcome to allow antibodies to bind to MSLN on tumors. To circumvent this sink effect, novel anti-mesothelin antibodies were developed that preferentially bind to immobilized MSLN compared with soluble MSLN, meaning that they preferentially bind to membrane-bound MSLN over soluble MSLN in the sink. To this end, different forms of the MSLN antigen were used in phage selection and subsequent screening campaigns. A panel of antibodies was identified that bound to membrane-bound human and cynoMSLN with varying affinities and were able to target different regions of MSLN. A series of screening assays, including ELISA, Biacore blocking assays, and cell binding assays, was performed to compare the binding regions to MSLN.

[0281] 4.1 Phagemid library selection A synthetic naive phagemid library displaying human IgG1 germline Fab domains randomized in CDR1, CDR2 and CDR3 (MSM Technologies) was used for selections with the MSLN antigen as described in Example 1.3.

[0282] The Fab library was constructed using biotinylated human, cyno or mouse MSLN-His-Avi or To isolate phages bound to hMSLN-His Acro, Streptavidin Dynabeads (Thermo Selection campaigns were initially conducted in multiple rounds, with three or four rounds each, using either a neutravidin-binding protein coupled to Dynabeads (Thermo Fisher, 11205D), a neutravidin-binding protein coupled to Dynabeads (Thermo Fisher, 31000), or anti-His Dynabeads (Thermo Fisher, 10103D). Selection campaigns were also conducted using a full-length MSLN antigen produced in-house, alternating human MSLN selection rounds with the cyno MSLN antigen, with the aim of isolating human and cyno cross-reactive clones. Selection of binders to mouse MSLN (R&D mMSLN-His 8604-MS) was also conducted. Standard pharmacokinetics was performed. A phage selection and phage recovery procedure was used.

[0283] To obtain clones binding to different regions of the MSLN antigen, we employed an epitope masking strategy using anti-MSLN antibodies from the initial selection campaign described above. Briefly, the first round of selection of the naive Fab library was performed using 500 nM biotinylated human MSLN-His-Avi. In rounds 2 and 3, phage binding to 500 nM (round 2) or 100 nM (round 3) biotinylated cynoMSLN-His-Avi was tested in the presence of a mixture of naive anti-mesothelin mAb proteins isolated from the initial selection campaign. These epitope masking selections resulted in a decrease in output titer, indicating that the selection strategy was working, with fewer binders identified. This led to the identification of three additional clones targeting additional regions of MSLN compared to clones from the previous selection campaign.

[0284] 4.2 Screening to identify anti-MSLN antibodies Approximately 2,000 clones from the output of rounds 3 and 4 of all selections were screened by phage ELISA for binding to 25 nM immobilized biotinylated hMSLN-His Acro, full-length biotinylated human, or cynoMSLN-His-Avi, matching the antigens used in the selection series. Streptavidin plates or plates immobilized with an irrelevant biotinylated His-tagged antigen were included as negative controls. Clones with MSLN-binding signals at least four-fold higher than the signal for the negative control were selected, and their variable regions were sequenced. 156 unique VH / VL sequence combinations were identified and then selected for soluble expression. Clones were selected from all selection campaigns, including epitope masking selections. For each clone, the VH and VL were individually cloned into pTT5 expression vectors (National Research Council of Canada) containing CH1, CH2 (with the LALA mutation in the CH2 domain (Bruhns et al., 2009; Hezareh et al., 2001) and either the CH3 or CL domain. The resulting pTT5 vectors with the LALA mutation were The pTT5-FS28 VH(AA) and pTT5-FS28 VL vectors were transiently co-transfected into HEK293-6E cells, and the clones were produced as complete IgG1 molecules. Using a similar method, the VH and VL regions of SS1 and the anti-hen egg white lysozyme antibody HelD1.3 were cloned and expressed in the IgG1 LALA format, resulting in G1-AA / SS1 (SEQ ID NO: 1). 140 (heavy chain) and 141 (light chain)) and G1-AA / HelD1.3 (SEQ ID NO: 138 (heavy chain) and 139 (light chain)) were used as positive and negative controls, respectively.

[0285] 4.3 Screening for binding to recombinant MSLN 4.3.1 Binding ELISA Soluble anti-MSLN binding clones or purified clone-containing HEK293 supernatants were analyzed for binding to human MSLN-His Acro, hMSLN-His-Avi, and in some campaigns, cMSLN-His-Avi. The antibodies were screened by ELISA. Briefly, hMSLN-His Acro, hMSLN-His-Avi, cMSLN-His-Avi, or an irrelevant His-tagged antigen was coated onto maxisorp plates at 25 nM overnight at 4°C. The next day, the plates were blocked with 300 μl of PBS containing 0.05% Tween 20 and 2% Marvel dried milk. The supernatant or The purified proteins containing anti-MSLN mAb were incubated at room temperature for 1 hour, and their binding was confirmed with mouse anti-human Fc-IgG conjugated to horseradish peroxidase (HRP). The antibodies were detected. Clones that bound to unrelated antigens or did not cross-react with both human and cynomolgus monkey antigens were discarded. Furthermore, clones that bound to the truncated MSLN antigen, hMSLN-His Acro, but not the full-length MSLN antigen, MSLN-His-Avi, were not included because the full-length antigen was expected to be more representative of the antigen conformation on the cell surface of MSLN-expressing cells.

[0286] To confirm the specificity of the antibodies 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 using PNGase F enzyme (NEB, P0704L) at 37°C for 24 hours, purified using Amicon ultracentrifugal filters (Millipore, UFC901024), and coated onto maxisorp plates at 25 nM. ELISA binding of specific anti-mesothelin mAbs to MSLN was detected using mouse anti-human Fc-IgG-HRP (Sigma, A0170). Clones that showed a 2-fold or greater decrease in binding signal to deglycosylated MSLN antigen compared to glycosylated MSLN antigen were excluded from the panel of native anti-MSLN-binding mAbs.

[0287] 4.3.2 BIAcore Screening Human MSLN-His-Avi was immobilized on flow cell 2 of a CM5 Series S BIAcore sensor chip (GE Healthcare, BR100530) at approximately 200 response units (RU) using an amine coupling kit (GE Healthcare, BR10050). Flow cell 1 was left blank for subtraction. HEK293 supernatant or purified protein was adjusted to approximately 50 nM MSLN mAb per sample in HBS-EP+ (GE Healthcare). Samples were then transferred to the flow cell. The data were injected at 30 μl / min onto channels 1 and 2 for 2.5 min, followed by dissociation in HBS-EP buffer for 2.5 min. Regeneration was achieved by injecting 10 mM glycine pH 1.5 (GE Healthcare, BR100354) at a rate of 30 μl / min for 30 s. The subtracted data ( Flow cell 2 - flow cell 1) were measured using BIAevaluation 3.2 software (GE Healthcare ) were analyzed. Of the 86 clones tested in this assay from the binding ELISA, 39 showed binding responses of greater than 10 RU at 50 nM and were therefore selected for re-expression, purification, and further screening. 4.3.3 Binning of antibodies based on the region of MSLN they bind

[0288] Based on the ELISA and Biacore screening data, clones were then tested in a binning assay, testing the binding of mAbs to human MSLN in the presence of another mAb by BioLayer Interferometry (BLI) in Octet (ForteBio).

[0289] Briefly, biotinylated hMSLN-His-Avi (5 μg / ml) was incubated with streptavidin. The binding was performed on a chip (ForteBIO, 18-5020) for 5 minutes. G1-AA / SS1 was diluted to 200 nM in 1x dynamic buffer (ForteBIO, 18-1092) and allowed to bind to hMSLN-His-Avi for 5 minutes. Binding of a mixture containing 200 nM of test mAb and 200 nM of G1-AA / SS1 was then evaluated for 5 minutes. This was compared to the binding of the test mAb to hMSLN-His-Avi in ​​the absence of conjugated G1-AA / SS1 (i.e., when there was no competition for binding) to determine the maximum extent of binding possible in the absence of SS1. If both antibodies compete for binding to the same region of MSLN, the test antibody will not be able to bind.

[0290] These binning experiments with G1-AA / SS1 demonstrated that the majority of FS28 binders tested ( 19 of 23) were unable to bind to MSLN in the presence of G1-AA / SS1, but This may be due to binding to a region similar to G1-AA / SS1. The MSLN-binding site of the SS1 antibody in the Fab format has been reported (Ma et al., 2012) and defined as the N-terminal region including amino acids 7 to 64, which is also involved in MUC16 binding. An additional clone, F, which showed partial or no competition for binding with SS1, S28-185 and FS28-256 were identified. Binning these clones relative to each other revealed that they represented two additional independent bins, i.e., they were capable of binding to two additional distinct regions of MSLN. FS28-185 was assigned to one bin ("Bin 2"), and 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 binding to multiple regions of MSLN were identified.

[0291] 4.3.4 Affinity For each bin described in Example 4.3.3, binding kinetics were determined using a similar method as described in Example 4.3.2, except that human or cyno MSLN-His-Avi was immobilized at 50 or 100 RU. Clones were tested at concentrations ranging from 81 nM to 0.33 nM in 3-fold dilutions. Binders were ranked, and the best one from each bin was selected: FS28-024 from bin 1; FS28-185 from bin 2; and FS28-256 from bin 3. Although these clones were all cyno cross-reactive (data not shown), affinities were not calculated under these test conditions. As shown in Table 4, the affinity obtained with 50 RU of immobilized MSLN was lower than that obtained with 100 RU of immobilized MSLN antigen, indicating increased binding at higher levels of MSLN.

[0292] [Table 5]

[0293] 4.4 MUC16-MSLN blocking assay FS28-024, FS28-185, and FS28-256 were tested for their ability to block MUC16 binding to mesothelin in a blocking assay. SS1 is known to block MUC16 binding to MSLN (Ma et al., 2012). G1-AA / SS1, which contains similar CDRs to SS1 and is expected to block MUC16 binding, and a control IgG1 antibody (G1AA / HelD1.3) were included as positive and negative controls, respectively.

[0294] Briefly, recombinant human MUC16 (R&D Systems, 5809-MU-050) was coated onto maxisorp plates at 0.65 μg / ml in 1x PBS overnight at 4°C. Plates were washed three times with 1x PBS and then resuspended in 300 μl of PBS containing 2% Tween 20 and 2% Marvel milk. Anti-MSLN mAbs (0.23 nM to 500 nM, 3-fold dilutions) were mixed with biotinylated hMSLN-His-Avi antigen (final concentration 2 μg / ml) in a volume of 100 μl for 1 hour. The mixture was premixed at room temperature for 1 h. After removing the blocking solution, the mAb / MSLN mixture was added to the plate and incubated at room temperature for 1 h. The plate was washed three times with PBST (1x PBS and 0.05% Tween 20) and incubated with streptavidin-HRP (Thermo Scientific, 21126, diluted 1:1000 in 1x PBS) for 1 h at room temperature. Finally, the plate was washed three times with PBST and three times with PBS. MSLN bound to MUC16 was visualized by adding 100 μl of TMB for 15 min followed by the addition of 100 μl of 1 M sulfuric acid solution. The absorbance was read at 450–630 nm (Gen5 software, BioTek). Ta.

[0295] Clone FS28-024 in bin 1 showed a MUC16-MS activity similar to that of G1-AA / SS1. FS28-256 showed no blocking activity, similar to the negative control G1-AA / HelD1.3. On the other hand, FS28-185 inhibited the binding of MUC16 to LN. These results were consistent with the binning data in Example 4.3.3, in that clones that bound to three different regions of MSLN exhibited three different behaviors in the ligand-blocking assay.

[0296] In conclusion, the results show that a panel of clones was selected that bind to three distinct regions (bins) of MSLN: antibodies that bind to one region of MSLN block MUC16 binding to MSLN, but antibodies that bind to the other two regions do not.

[0297] 4.5 Specificity Given the different regions of MSLN bound by the panel of antibodies, their specificity for binding to MSLN was tested. The specificity of FS28-024, 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.

[0298] Using a protocol similar to that described in Example 4.3.1, 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 (in-house production). Binding of anti-MSLN mAbs tested at a concentration range of 1000 nM (3-fold dilutions) was detected using anti-human Fab-HRP (Sigma, A0293). FS28-024, FS28-185, and FS28-256 bound to human MSLN-His-Avi (EC 50Although the maximum binding signal was around 0.5 nM (3), no binding to the cell adhesion molecules tested was observed up to 1000 nM. As expected, the positive control antibodies bound to their respective targets. Thus, the anti-MSLN antibodies demonstrated a high level of specificity.

[0299] 4.6 Cell binding A panel of selected anti-mesothelin mAbs (FS28-024, FS28-185, and FS28-256) was analyzed for binding to endogenous cell surface MSLN of the human lung cancer cell line NCI-H226.

[0300] Briefly, NCI-H226 cells (ATCC CRL-5826) were harvested from T175 cell culture flasks using Accutase (Gibco, A11105-01). The cells were stirred at 1200 rpm. Centrifuge for 3 min and transfer 2 × 10 cells to ice-cold FACS buffer consisting of DPBS (Life Technologies, 14190169) and 1% BSA (Sigma-Aldrich, A7906). 6 Resuspend at 100 cells / ml 50 μl of the solution was plated per well in a 96-well V-bottom plate (Costar, 3894). All tested mAbs 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 resuspended in 100 μl of each mAb dilution and incubated at 4°C for 45 min. The cells were washed twice by centrifugation with 150 μl of FACS buffer and resuspended in 100 μl of goat anti-human IgG (γ-chain specific) F(ab')2 fragment-R-phycoerythrin antibody (Sigma, P8047) diluted 1:1000 in FACS buffer and incubated at 4°C for 45 min. Cells were washed once with 150 μl of FACS buffer, then with 150 μl of DPBS, resuspended 1:10,000 in 150 μl of DPBS containing DAPI (Biotium, 40043), and read on a BD Canto II or iQue (Intellicyt). The data was analyzed using and the geometric mean signal was determined for the PE of live cells in each well.

[0301] mAb 2 FS28-024 inhibited NCI-H226 cells, as did the positive control G1-AA / SS1. In comparison, FS28-185 and FS28-256 showed weaker binding to cell surface MSLN.

[0302] Overview of the naive screening procedure From the 156 mAbs identified by initial screening of the naive phage library, three anti-human MSLN mAb clones (FS28-024, FS28-185, and FS28-256) were selected based on a series of screening assays that first confirmed their ability to bind to full-length, deglycosylated recombinant MSLN and to cynoMSLN. Second, the clones were grouped based on the diversity of the regions (bins) of MSLN they bound and their MUC16-blocking activity, and the highest affinity binders were selected from within these groups. The resulting panel of mAb clones, FS28-024, FS28-185, and FS28-256, bound to three distinct regions of MSLN, and one of them (bin 1, FS28-024) blocked MUC16 binding to MSLN in vitro. A panel of five anti-MSLN antibodies demonstrated specific binding to MSLN, differing affinities for recombinant and cell-surface MSLN, and were selected for further characterization and / or optimization, as described in Example 5 below.

[0303] Example 5: Affinity maturation and sequence optimization of naive anti-MSLN mAb 5.1 Affinity maturation of clone FS28-024 using the NNK-walk strategy FS28-024 bound to human MSLN with subnanomolar affinity, but its affinity to cynoMSLN was approximately 5-fold lower. To improve binding to cynoMSLN, an NNK walk strategy was used against five residues in the VH CDR3 region.

[0304] The FS28-024 VH and VL sequences were optimized. Parsimonious mutagenesis libraries were generated by diversifying one amino acid residue at a time at the RATLF residues (Kabat numbering 95–99) of the VH CDR3. A total of five individual libraries were obtained. The libraries were generated using low-redundancy NNK codons to represent all possible amino acids at the target position. Forward and reverse primers were designed according to the guidelines of the Quickchange Lightning Site-Directed Mutagenesis Kit (Agilent, 200518) used to generate the libraries. Each mutant was expressed at a small scale in HEK293 cells, and the supernatants were screened by BIAcore for retention or improvement of binding to human and cynoMSLN-His-Avi. Screening Of the 84 clones identified, few retained binding, most of which were due to substitution of the T98 residue. Four clones, FS28-024-051, FS28-024-052, FS28-024-053, and FS28-024-060, were re-expressed and purified, and their affinities for human and cynoMSLN were determined. Only one clone, FS28-024-053, retained binding due to a single T98V mutation (Kabat numbering). All four clones were carried forward as they may offer alternative sequences and properties.

[0305] 5.2 Affinity maturation of FS28-185 and FS28-256 Compared to FS28-024, clones FS28-185 and FS28-256 showed weaker affinity for both recombinant and cell surface MSLN and were therefore subjected to affinity maturation.

[0306] The VH and VL CDR3 regions were cloned using NNK primers, with 5 to 6 amino acids The overlapping cassettes were randomized to perform parallel affinity maturation in an scFv format. The randomized regions for FS28-185 were VHG95-M100F and VLS91-A95, and for FS28-256 they were VHY95-L100B and VLS91-I96 (Kabat numbering). Prior to library generation, CDR1 and CDR2 were randomized. Parsimonious mutagenesis was performed at potential methionine oxidation and deamidation sites in three regions (excluding the FS28-256VL CDR3 library). Two rounds of selection were performed as described for the naive campaign, using 20 nM biotinylated human MSLN-His-Avi in ​​the first round and either 20 or 2 nM cynoMSLN-His-Avi in ​​the second round. 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 using StemPro Accustase (Gibco, A11105-01) and stirred at 1200 rpm. Centrifuge for 3 minutes and place 2x10 cells in FACS buffer (2% BSA in DPS). 6 Cells were resuspended 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. The parental clone FS28-185 and 256 ScFvs were included as controls. After washing, cells were resuspended in 100 μl of Penta His Alexa-Fluor 647 (Qiagen, 109-546-098), washed, and then resuspended in 100 μl of DPBS containing Sytox Green Nucleic Acid Stain (Invitrogen S7020, 1:10000 dilution). Samples were run on an iQue (Intellicyt Corporation, IQue Plus) and analyzed. The geometric mean of the APC was recorded.

[0307] 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 selection and 9 clones 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 from the VL CDR3 selection. The selected clones were mAbs 2 It was further characterized in a bispecific antibody format.

[0308] 5.3 mAb 2 Generation of FS28-185 and FS28-256 based on For further characterization of anti-MSLN binders, the affinity-matured VH or VL regions of FS28-024, FS28-185, or 256, as well as the parent clones, were used as mAbs. 2 The resulting mAb was produced in the following format: 2 is an IgG1 antibody containing the LALA mutation in the CH2 domain and the CDRs of clones FS28-024, FS28-185, or FS28-256, or affinity matured variants derived therefrom, containing the human CD137 receptor binding site in the CH3 domain. 2 The heavy and light chain sequences of the molecule are shown in the SEQ ID NOs: FS22-172-003-AA / FS28-024mAb 2 : SEQ ID NOs: 94 and 85 FS22-172-003-AA / FS28-024-051mAb 2 : SEQ ID NOs: 98 and 85 FS22-172-003-AA / FS28-024-052mAb 2 : SEQ ID NOs: 102 and 85 FS22-172-003-AA / FS28-024-053mAb 2 : SEQ ID NOs: 106 and 85 FS22-172-003-AA / FS28-024-060mAb 2 : SEQ ID NOs: 108 and 85 FS22-172-003-AA / FS28-026mAb 2 :270 SEQ ID NOs: 109 and 87 FS22-172-003-AA / FS28-091mAb 2 : SEQ ID NOs: 110 and 88 FS22-172-003-AA / FS28-185mAb 2 : SEQ ID NOs: 111 and 89 FS22-172-003-AA / FS28-256mAb 2 : SEQ ID NOs: 114 and 116 FS22-172-003-AA / FS28-256-001mAb 2 : SEQ ID NOs: 120 and 82 FS22-172-003-AA / FS28-256-005mAb 2 : SEQ ID NOs: 120 and 83 FS22-172-003-AA / FS28-256-012mAb 2 : SEQ ID NOs: 125 and 116 FS22-172-003-AA / FS28-256-014mAb 2 : SEQ ID NOs: 129 and 116 FS22-172-003-AA / FS28-256-018mAb 2 : SEQ ID NOs: 133 and 116 FS22-172-003-AA / FS28-256-021mAb 2 : SEQ ID NOs: 125 and 82 FS22-172-003-AA / FS28-256-023mAb 2 : SEQ ID NOs: 133 and 82 FS22-172-003-AA / FS28-256-024mAb 2 : SEQ ID NOs: 125 and 83 FS22-172-003-AA / FS28-256-026mAb 2 : SEQ ID NOs: 133 and 83 FS22-172-003-AA / FS28-256-027mAb 2 : SEQ ID NOs: 125 and 84 FS22-172-003-AA / FS28-256-271mAb 2:3 and 84 FS22-172-003-AA / FS28-256-272mAb 2 :158 and 84 FS22-172-003-AA / FS28-256-273mAb 2 :163 and 84

[0309] These mAbs 2 was produced by transient expression in HEK293-6E cells and, as shown here, purified using a mAb Select SuRe Protein A column.

[0310] 5.4 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). Four variants of clone FS28-256-027 were obtained by substituting N55 of VH CDR2 with alanine, histidine, serine, and threonine. These clones were designated FS28-256-271, FS28-256-272, FS28-256-273, and FS28-256-274, respectively. The clones were characterized by SPR for binding to immobilized and solution-based MSLN. Table 5 shows the SPR results. FS28-256-274 had a much weaker affinity for immobilized MSLN than the other clones and therefore did not progress. FS28-256-272 and FSFS28-256-273 bound more strongly to soluble MSLN or similarly to immobilized MSLN. Consequently, the binding of these clones to cell surface-expressed MSLN is likely affected by the presence of soluble MSLN. In contrast, FS28-256-271 bound most strongly to immobilized MSLN and weaker to soluble MSLN, suggesting that it preferentially targeted immobilized MSLN over MSLN in solution.

[0311] [Table 6]

[0312] Overview of Fcab and mAb selection and screening The anti-CD137 Fcabs identified in the previous examples showed no activity in NF-κB reporter assays when cross-linked with either an external cross-linker such as protein L (Example 2). Among the panel of anti-human CD137 Fcabs identified, FS22-172-003 was selected for pairing with the MSLN-targeting Fab because this clone exhibited the most favorable functional and biophysical properties.

[0313] To localize this CD137-driven agonist activity to the tumor microenvironment, we developed Fabs and mAbs that specifically target the tumor-associated antigen mesothelin (MSLN). 2 The expression of MSLN on tumor cells was determined by pairing with mAb 2 This may be beneficial as it is predicted that this will result in cross-linking of Fcabs, which may then be able to induce agonist activity when Fcabs bind to CD137.

[0314] As identified in the previous examples, a panel of MSLN-binding Fabs was selected that bind to different regions of MSLN and that preferentially bind to immobilized MSLN over soluble mesothelin.

[0315] Example 6: mAbs 2 Targets CD137 and mesothelin (MSLN) production CD137 / MSLN mAb consisting of an IgG1 molecule containing anti-CD137 FcabFS 22-172-003 2 , and a panel of anti-MSLN Fabs (listed in Table 6) were produced, and mAbs 2These CD137 / MSLNmAbs were prepared by substituting portions of the CH3 domain of Fcab, including the AB, CD, and EF loops, for the corresponding regions of the CH3 domain of an MSLN-binding antibody. 2 contained a LALA mutation in the CH2 domain (AA). 2 The isotype of CD137 / MSLNmAb is human IgG1, and it can induce ADCC against cells to which it binds. 2 Because this mAb binds to CD137-expressing immune cells, the inclusion of the LALA mutation induces ADCC against these immune cells. 2 Furthermore, Fcγ receptors on effector cells are known to induce antibody cross-linking in vivo, but this is inefficient and can occur at sites distant from the target treatment site. Because CD137 antibodies in clinical settings are associated with dose-limiting toxicities, we decided to use CD137 / MSLN mAbs. 2 It was decided to include the LALA mutation so that all mAbs exert their agonist activity only through binding and cross-linking MSLN. 2 and control antibodies were produced by transient expression in HEK293-6E cells and cloned into mAbSelect Sure Protein A capsids. The mAb was then purified using 2 was assessed for purity by SEC-HPLC, which confirmed that the molecule was greater than 98% monomeric.

[0316] [Table 7]

[0317] Example 7: CD137 / MSLN mAb 2 Characterization The binding strength of CD137 and CD137 / MSLNmAb2 to MSLN was measured by surface plasmon resonance using a BIAcore instrument, as well as that of related endogenously expressing MSLN. Binding to cell surface MSLN was tested by flow cytometry on related cell lines. A soluble form of MSLN, generated by alternative splicing of membrane-bound MSLN or tumor necrosis factor-α converting enzyme (TACE), has been found in the serum of MSLN-positive cancer patients, and it has been hypothesized that this could act as a decoy for therapeutic antibodies (Lee et al., 2018). b 2 The binding properties and avidity of MSLN were investigated, particularly the difference in binding affinity to soluble and immobilized MSLN. This was measured in BIAcore binding experiments set up in two ways. First, mAbs that bind to antigens present on cell surfaces 2 To reproduce the kinetic profile of mAb, recombinant antigen was immobilized at moderate density on the SPR chip, followed by mAb 2 Next, mAb was injected into the MSLN cells bound to the cell surface. 2 Cycles for joins To simulate the potential sink effect caused by soluble MSLN, mAb 2 On the chip The cells were captured on a plate and loaded with recombinant antigens at various concentrations (see Example 7.2). Typically found in patient sera (Onda et al., 2006, 10.1158 / 1078-0432.CCR-05-1477) The effect of soluble recombinant MSLN added at a representative level was also investigated by binding assay to MSLN expressed on the cell surface and CD8 + This was thoroughly investigated in T cell assays.

[0318] 7.1 CD137 / MSLN mAb to human MSLN under avidity conditions 2 Binding-antigen capture method All mAbs against MSLN immobilized on a BIAcore chip 2 Testing the binding of Avidity conditions allow for high concentrations of immobilized antigen to be used to allow strong antibody binding. The binding affinity to human MSLN was determined under the following conditions: CM5 chips (GE Healthcare BR-1005-30) were coated with hMSLN-His-Avi at approximately 100 RU according to the manufacturer's instructions. CD137 / MSLN mAbs listed in Table 6 2 The panel of antibodies, as well as the control antibodies G1-AA / HelD1.3 and G1-AA / SS1 (Hassan et al. 2002), were run at a concentration range of 3-fold dilutions starting from 300 nM. The flow cells were regenerated by injecting glycine chloride, pH 1.5, at a flow rate of 30 μl / min for 30 seconds. Data were analyzed by double referencing against a flow cell (without antigen coating) that was intentionally left blank. Binding kinetics were fitted to a 1:1 Langmuir model to calculate the binding association (k a ) and dissociation (k d ) rate was generated. The equilibrium binding constant (K D ) was calculated by dividing the dissociation rate by the association rate for each sample. Data analysis was performed using BiaEvaluation software. The results are shown in Table 7.

[0319] FS28-185 and FS28-256 parent MSLN Fab arm containing FS22-172-003-AA / FS28-185 and FS22-172-003-AA / FS28-256 mAbs 2 All other mAbs containing affinity-matured progeny of FS28-256 showed binding affinities between 36 and 50 nM. 2 has an MSLN binding affinity (K D ) was shown. In fact, the K D MSLNFab arm-containing mAb from line FS28-024 2 It was decided that:

[0320] mAb 2For FS22-172-003-AA / FS28-256-271, cytochrome c cross-reactivity was determined 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. 2 was injected at a flow rate of 10 μl / min over a concentration range of 3-fold dilutions starting from 243 nM. The association time to steady state was 1000 s and the dissociation time was 30 s. The running buffer was HBS-EP (GE Healthcare BR100188). The flow cell was regenerated by injecting glycine chloride, pH 1.5, for 30 s at a flow rate of 30 μl / min. Data were analyzed by double referencing against a flow cell (without antigen coating) that was intentionally left blank. The steady-state affinity model was generated using the BiaEvaluation software version. Section 3.2 was used to analyze the kinetic data. Binding to cMSLN-Avi-His was within 3-fold of binding to hMSLN-Avi-His.

[0321] 7.2 CD137 / MSLNmAb against soluble human MSLN 2 Binding-antibody capture method CD137 / MSLN mAbs listed in Table 7 2 and control antibodies G1-AA / HelD1.3 and G1-AA / SS1 were also tested for binding to human MSLN and cynomolgus monkey MSLN by SPR, where the antibodies were captured and binding to MSLN in solution was assessed (K D Protein G (GE Healthcare 29179315) was used to inject mAb or mAb at approximately 100 RU. 2After sample capture, hMSLN-His-Avi or cMSLN-His-Avi were injected at a flow rate of 70 μl / min in a concentration range of 3-fold dilutions starting at 1000 nM. The association time was 5 min, and the dissociation time was 5 min. The running buffer was HBS-EP (GE Healthcare BR100188). The flow cell was regenerated by injecting glycine chloride, pH 2.1, for 15 s at a flow rate of 30 μl / min. Data were analyzed as described in Example 7.1. The results are shown in Table 7. From the antibody capture method, mAb 2 The majority of the antibodies significantly reduced binding affinity, supporting the screening strategy we undertook to generate avid anti-MSLN antibodies with differential binding to immobilized versus soluble MSLN. D The highest value is 2 4.27 nM for FS22-172-003-AA / FS28-024-060, the mAb with the lowest affinity 2 FS22-172-003-AA / FS28-256 were between 1.2 μM.

[0322] All mAbs tested 2 mAb 2 All but FS22-172-003-AA / FS28-024-060 showed cyno cross-reactivity within 10-fold of affinity with human MSLN, but this showed 54-fold less cross-reactivity compared to human MSLN and was not pursued further.

[0323] [Table 8]

[0324] All mAbs 2 The affinity of the MSLN-binding Fab arm (K D The K in solution (as measured by K) has been confirmed by both antigen capture (immobilized MSLN) and antibody capture (MSLN in solution) methods. D Value vs. fixed K D These K values ​​were different for each antibody. DThe ratio was calculated from the values ​​to assess the behavior of each mAb with respect to whether its binding was altered by the presence of soluble MSLN (Table 7). 2 Without wishing to be bound by theory, mAb 2 We hypothesize that there is a threshold of binding affinity that needs to be overcome in order for MSLN to be able to crosslink efficiently, but differences in binding affinity to immobilized versus solution MSLN play an important role in how this function may be affected by the shed-MSLN-driven sink effect.

[0325] 7.3 Biacore binding to CD137:mAb 2 Retained binding affinity in the format In addition to the binding strength to MSLN, the K D We also tested to prove that the binding properties of Fcabs are retained when paired with new Fabs. Biacore CM5 chips were coated with anti-human Fab using the Human Fab Capture Kit (GE Healthcare 28958325) according to the manufacturer's specifications, and the surface density was adjusted to 100 μg / cm. The total elution volume was approximately 9000 RU. Samples of FS22-172-003-AA / FS28-024-052 and FS22-172-003-AA / FS28-256-271 were captured to approximately 100 RU, followed by the flow of human CD137 antigen (hCD137-mFc-Avi) at a 3-fold dilution range starting at 81 nM at a flow rate of 70 μl / min. The association time was 5 min, and the dissociation time was 5 min. The running buffer was HBS-EP. The flow cell was regenerated with two 30-second injections of 10 mM glycine chloride, pH 2.1, at a flow rate of 30 μl / min. Data analysis was performed as described in previous examples (see, e.g., Example 7.1). The results in Table 8 show that the elution volume of mAbs containing the same Fcab but different Fabs was significantly higher than that of mAbs containing the same Fcab but different Fabs. 2These Fcabs show consistent affinity for CD137 among their respective CD137 / MSLN subunits. Anti-CD137 Fcabs were selected to preferentially bind dimeric or multimeric CD137 over monomeric CD137 via avidity interactions. This avidity mechanism of action is hypothesized to be beneficial for preferentially targeting cells with upregulated CD137 expression, such as activated T cells, over other cells that express CD137 at much lower levels. This may be beneficial for minimizing extratumoral T cell activation, which can result in undesirable effects such as toxicity. Furthermore, the Fcabs were designed to be active only when crosslinked, so that they cannot induce agonistic effects by binding to CD137 alone. These CD137 / MSLN mAbs 2 In relation to the dynamic mode of action of both specificities of the molecule, mAb 2 It is believed that mAb preferentially binds to upregulated MSLN, which is expressed at high levels on tumor cells, and subsequently binds to activated T cells with upregulated CD137 expression. Due to the binding kinetics of the Fcab domain of the molecule, mAb 2 If the molecule first binds to CD137 on T cells, the lack of MSLN-mediated cross-linking ensures that T cell agonism is not elicited.

[0326] [Table 9]

[0327] 7.4 Binding to cell surface-expressed MSLN and interference by soluble MSLN To confirm binding to cell surface MSLN, the mAbs listed in Table 9 were tested for binding to endogenous cell surface MSLN of the human lung cancer cell line NCI-H226. 2 In addition, MSLN can be found in the blood in a shed soluble form, and this circulating MSLN was analyzed by our mAb. 2 There is a risk that the presence of soluble MSLN may affect the exposure and ultimately efficacy of To confirm that the effect of soluble MSLN on the IL-1 signaling pathway was minimal, we performed NCI-H22 in the presence of 20 nM soluble MSLN, a level 10–20 times higher than the level of soluble MSLN found to be diagnostically valuable for defining malignant mesothelioma and lung cancer patients as MSLN-positive (Cui et al., 2014). Additional binding experiments were performed on 6 cells. Briefly, NCI-H226 cells (ATCC CRL-5826) from a T175 cell culture flask using Accutase (Gibco, A11105-01). The antibody preparation protocol was slightly modified when supplementing with soluble MSLN: the antibody was diluted in FACS buffer in a 96-well V-bottom plate to obtain a 2x final concentration, and then 60 μl from each well was added to either FACS buffer alone or 60 μl of 40 nM recombinant hMSLN-His (R&D systems, 3265-MS-050) (final concentration 20 nM hMSLN). The antibodies were added to either FACS buffer containing 100 μl of IgG (giving 100 μl of IgG) and pre-incubated for 1 hour at room temperature before adding 100 μl to the cells.

[0328] In both cases, the cells were centrifuged at 1200 rpm for 3 min and resuspended in DPBS (Life Technology). Ice-cold FACS buffer consisting of 1% BSA (Sigma-Aldrich, A7906) and 1% BSA (Sigma-Aldrich, A7906). 2x10 to 6Cells were resuspended at 100 μl / ml and seeded in 50 μl per well in a 96-well V-bottom plate (Costar, 3894). All tested antibodies 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 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 and resuspended in 100 μl of goat anti-human IgG (γ-chain specific) F(ab')2 fragment-R-phycoerythrin antibody (Sigma, P8047) diluted 1:1000 in FACS buffer and incubated at 4°C for 45 min. Cells were washed once with 150 μl of FACS buffer and then with 150 μl of DPBS and resuspended in 100 μl of goat anti-human IgG (γ-chain specific) F(ab')2 fragment-R-phycoerythrin antibody (Sigma, P8047) diluted 1:1000 in FACS buffer. The cells were then incubated at 4°C for 45 min. Cells were washed once with 150 μl of FACS buffer and then with 150 μl of DPBS and resuspended in DAPI (Biotium Ionophosphate). Cells were resuspended 1:10,000 in 150 μl of DPBS containing 40043 (Biotin, 40043) and read on a BD Canto II or iQue (Intellicyt). Data were analyzed using FlowJo v10 to determine the geometric mean signal for live cell PE in each well.

[0329] According to the cell binding results in Table 9, binding to cell surface MSLN on the endogenously expressing cell line NCI-H226 was observed in EC 50 All mAbs with values ​​ranging from 0.3 to over 47 nM 2 The effect of the presence of soluble recombinant MSLN on cell binding affinity was generally low, with ECs observed in most clones. 50 There was a small (less than three-fold) increase in

[0330] FS22-172-003-AA / FS28-256 exhibited lower EC than most of the other clones tested, both in the presence and absence of soluble MSLN. 50The binding of the parent FS28-256 antibody was high, suggesting weak binding of the parent FS28-256 antibody. However, affinity-matured variants of this parent antibody showed stronger binding. In particular, FS28-256-derived clones containing mAbs 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 2 The cell binding affinity of the mAbs was much better than that of the parent mAb and was not affected by the presence of soluble MSLN. This indicates that most mAbs 2 The results show that mAbs containing FS28-024-060 and FS28-256-027 preferentially bound to membrane-bound MSLN. 2 The parental FS28-185 clone (i.e., FS22-172-003-AA / FS28-185)-containing mAb was most affected when binding to cells in the presence of soluble MSLN, suggesting that higher affinity binders to MSLN in solution are likely affected by the presence of shed MSLN. 2 Also, high EC 50 The mAb had a 5'-fold increase in binding activity and showed weak binding in the presence and absence of soluble recombinant MSLN. Affinity matured variants of the parent clone were also tested. The improved binding in the absence of soluble MSLN was due to the mAb 2 This was observed for these affinity matured clones.

[0331] [Table 10]

[0332] 7.5 mAbs with a wide range of endogenous mesothelin expression levels 2 combined cell lines To demonstrate the ability of anti-MSLN Fab clone FS28-256-271 to bind to cells expressing a range of MSLN cell densities, the mAb was tested in a cell-binding flow cytometry assay similar to that described in Example 7.3 using the following human cancer cells: 2The following cells were tested: NCI-H226 [H226] (ATCC® CRL-5826), OVCAR-3 [OVCAR3] (ATCC® HTB-161), and AsPC-1 [AsPC-1] (ATCC® CRL-1682™). The MSLN-negative cell line HEK.FRT described in Example 2 was also used as a negative control. To determine the relative expression of MSLN in each cell line, antibody binding assay (ABC) was used according to the manufacturer's recommended protocol (Quantum™ Simply Cellular® #816 Bangs Labs). The cell lines were H226 (ABC 315,478), which had the highest MSLN among the cells tested, and OVCAR3 (ABC 103,444) with high levels, and AsPC-1 (ABC 20,999) with low levels were ranked in order of MSLN expression levels.

[0333] The results of the cell binding assay are shown in Table 10. Regarding expression levels, FS28-256-271 binds strongly to H226, which exhibits the highest MSLN expression among the cell lines tested. 2 has comparable binding to OVCAR-3 and AsPC-1 cells. As expected, FS28-256-271 selectively binds to cells expressing MSLN but not to the negative HEK.FRT cell line. Consequently, the CD137 / MSLN mAb 2 would have the potential to act on cells that express a range of membrane MSLN expression.

[0334] [Table 11]

[0335] Example 8: CD137 / MSLN mAb 2 Functional activity of Cell lines with endogenous levels of MSLN were identified using CD137 / MSLNmAb. 2Cytotoxic CD8+ / IL-2 or TNFγ cytokine release is used as the endpoint of this assay to demonstrate that CD137 crosslinking leads to CD137 agonism and subsequent T cell activation. + A T cell assay was developed. NCI-H226, OVCAR-3, AsPC-1, and HEK.FRT, as described in Example 7, were used in this T cell assay to identify CD137 / MSLN mAb. 2 was used to test.

[0336] To isolate T cells, peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte-depleted cones, a by-product of platelet donation. Briefly, the contents of the leukocyte cones were rinsed with PBS and layered on a Ficoll (Sigma-Aldrich, 1440-02) gradient. PBMCs were separated by centrifugation, and cells that did not pass through the Ficoll gradient were collected. PBMCs were further washed with PBS, and remaining red blood cells were lysed by adding 10 ml 1X red blood cell lysis buffer (eBioscience, 00-4300-54) according to the manufacturer's instructions. CD8 + T cells, CD8 + T cell isolation PBMCs present in the eluate were isolated using Kit II (Miltenyi Biotec Ltd, 130-096-495) according to the manufacturer's instructions.

[0337] Incubation with anti-CD3 antibody was used as an initial signal to promote early T cell activation. 96-well flat-bottom tissue culture plates were coated with 8 μg / ml 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.

[0338] 8.1 CD8 using NCI-H226 for MSLN crosslinking + T cell assay Next, cytotoxic CD8 + T cells were isolated from PBMCs as described above. NCI-H226 cells were cultured at 2 × 10 4Cells / well were plated onto a 96-well flat-bottom plate coated with anti-CD3 antibody (8 μg / ml) in 100 μl of T cell culture medium (10% FBS (Life Technologies), 1X 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). After 4 hours of incubation to allow cells to adhere, all T cell culture medium was removed and 4.0 × 10 T cells were added. 5 Replace with 50 μl of T cell culture medium containing 2.0 x 10 cells / ml 4 Cells / well obtained. mAb 2 was diluted in T cell medium at 2x the final concentration starting at 60 nM, and serial dilutions of 1:3 or 1:7 were performed. 2 The titration was added to the cells for a total assay volume of 200 μl and an antibody concentration of 1. Details of the molecules tested in this assay are provided in Table 11; in each assay, G1-AA / 20H4.9 was used as a positive control (data not shown).

[0339] The assay was incubated at 37°C, 5% CO for 72 hours. Supernatants were collected and assayed using Meso Scale Discovery's V-PLEX IL-2 kit (K151QQD-4) according to the manufacturer's instructions. Human IL-2 (hIL-2) concentrations were plotted against the log concentration of antibody, and the resulting curves were fitted using the log(agonist) vs. response equation in GraphPad Prism.

[0340] Table 11 shows the EC values ​​of IL-2 release observed in the T cell activation assay across different repeats of the assay. 50Values ​​and maximum responses are shown. Due to the number of assays performed, T cells from multiple donors were used. In each case, positive and negative controls were used in each assay to ensure consistent data sets across assays and donors. The positive control anti-human CD137 antibody, 20H4.9, had an EC 50 The lack of off-target CD137-mediated T cell agonism was observed in a similar study to that described above, but using HEK-FRT cells that were not transduced to express MSLN instead of MSLN-positive NCI-H226 cells. + This was confirmed in T cell assays. Avoidance of off-target activation is highly desirable, especially for CD137 antibodies, due to the dose-limiting toxicity cases observed with some other CD137 molecules in the clinic; therefore, this factor was used to determine the efficacy of selected mAbs. 2 The following combinations of mAbs were determined: 2 showed increased IL-2 release, each with a subnanomolar EC50: FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-052, FS22-172-003-AA / FS28-024-053, FS22-172-003-AA / FS28-024-060, FS22-172-003-AA / FS28-256-021, FS22-172-003-AA / FS28-256-023, FS22-172-003-AA / FS28-256-026, and FS22-172-003-AA / FS28-256-027. Figure 1 shows a representative plot of IL-2 release for a T cell activation assay. These results are consistent with the results of mAbs containing Fabs from the FS28-185 lineage (Figure 1B). 2 Surprisingly, all mAbs paired with Fabs from the FS28-185 lineage showed no functional activity in this particular assay. 2 showed very limited and reduced or no IL-2 release, suggesting that while Fabs of that lineage can bind to MSLN, binding to this specific region of MSLN inhibits CD137 / MSLN mAbs in a manner that enhances the efficacy of MSLN cross-linking in this assay. 2All mAbs, including the Fabs of line FS28-024 (Fig. 1A) and line FS28-256-021, FS28-256-023, FS28-256-023, FS28-256-026, and FS28-256-027 (Fig. 1C), were cross-linked. 2 is a subnanomolar EC 50 of hIL-2 release Indicates an increase.

[0341] [Table 12]

[0342] CD8 using NCI-H226 in the presence of soluble MSLN + T cell assay CD8 as above + T cell functional assays were repeated in the presence of 20 mM hMSLN-His, a 10–20-fold higher level of soluble MSLN that was found to be of diagnostic value for defining malignant mesothelioma and lung cancer patients as MSLN-positive ( Cui et al., 2014 ).

[0343] Figure 2 shows the effect of mAb in the presence or absence of soluble MSLN. 2 As expected, soluble MSLN exhibits activity against a subset of mAbs paired with Fabs that preferentially bind to immobilized MSLN versus soluble MSLN. 2 Specifically, it does not alter the efficacy of MSL mAbs including N Fab FS28-024-051, FS28-024-052, FS28-024-053, FS28-256-021, and FS28-256-023 2 is unaffected by the presence of up to 20 nM soluble MSLN, which is consistent with the affinity and cell binding data described in Examples 7.1 and 7.2.

[0344] Interestingly, mAb 2 FS22-172-003-AA / FS28-256-027 is EC50 The greatest loss of potency occurred when incubated with 20 nM soluble MSLN, which resulted in a significant shift in This is consistent with the affinity results in Examples 7.1 and 7.2, where FS28-256-027 showed high affinity for both immobilized and soluble MSLN, supporting the theory that high affinity for soluble MSLN is undesirable. The results are consistent with the aforementioned hypothesis that clones that preferentially bind to membrane MSLN are less likely to be disrupted by the presence of soluble MSLN. In this context, clone FS28-256-027 showed high affinity binding to both immobilized MSLN and MSLN in solution, indicating that mAb 2 Functional data for FS22-172-003-AA / FS28-256-027 showed an EC 50 The remaining mAbs, including MSLN Fabs FS28-024-051, FS28-024-052, FS28-024-053, FS28-256-021, and FS28-256-023, reveal significant shifts in the 2 is unaffected in the presence of up to 20 nM, which is consistent with the affinity and cell binding data above.

[0345] 8.2 CD8 using OVCAR-3 for cross-linking + T cell assay Because mesothelin-positive cancers have a wide range of expression levels, several mAbs identified in Example 8.1 as having subnanomolar potency were tested using cells with low cell surface MSLN density. 2 To accomplish this, it is desirable to evaluate the function of T cell activation assays were performed as described in Example 8.1, but this time using the OVCAR-3 cell line (ATCC® HTB-161). These are MSLN-positive ovarian cancer cells that endogenously express lower levels of MSLN than NCI-H226 cells. The same protocol as described in Example 8.1 was followed, with the following modification: due to differences in cell size and morphology, OVCAR-3 cells were plated at 1 x 10 per well in 100 μl of T cell culture medium onto anti-CD3 antibody-coated (8 μg / ml) 96-well flat-bottom plates. 4 After 4 hours of incubation to allow cells to adhere, all T cell culture medium was removed and 8.0 × 10 T cells were added. 5 Replace with 50 μl of T cell culture medium containing 4.0 x 10 cells / ml 4 Cells / well were obtained. Results showed that all tested mAbs drove IL-2 release when crosslinked with OVCAR-3 cells. 2 These data confirm the ability of these mAbs 2 These results suggest that HelD1.3 Mock mAb may function across a wide range of MSLN densities across different tumor cell lines. 2 The Fcab FS22-172-003 in this format (FS22-172-003 / HelD1.3) was also tested in this assay because it does not bind to MSLN, and the lack of IL-2 release indicated that the anti-human CD137 Fcab was functional only when cross-linked via the Fab arm (in this case MSLN).

[0346] 8.3 Functional screening of sequence-optimized FS28-256 affinity-matured clones All mAbs, including the anti-MSLN Fab of the FS28-256 lineage described in Example 8.2 2 contains a potential N-linked glycosylation site in VH CDR2 removed as described in Example 5.4. Three mAbs with the following substitutions: 2Mutants were generated: FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-256-272, and FS22-172-003-AA / FS28-256-273, which contain the amino acid substitutions N55A, N55H, or H55S, respectively, according to the IMGT nomenclature. 2 To demonstrate the efficacy of + T cell activation assays were performed in co-culture with MSLN+NCI-H226 cells as described in Example 8.1. 2 was also tested in the presence of soluble MSLN.

[0347] [Table 13]

[0348] Figure 4 and Table 12 show the results of these mAbs 2 The effect of soluble MSLN on the potency of mAb and the overall potency of the molecule is shown. 2 FS22-172-003-AA / FS28-256-271 are the three mAbs tested. 2 Among the mAbs, the MSLN Fabs showed the least decrease in potency. Consistent with the data described in Examples 8.1 and 7.2, the MSLN Fabs, which bind with higher affinity to immobilized MSLN compared to MSLN in solution, were less affected by the presence of soluble MSLN. 2 FS22-172-003-AA / FS28-256-271 significantly increased EC in the presence of soluble MSLN compared to the absence of soluble MSLN. 50 shows minimal change in EC 50 A three-fold decrease in β-glucan was observed. Consistent with the affinity measurements reported in Example 7.1, this mAb 2 showed a stronger affinity for immobilized MSLN compared to MSLN in solution. Also consistent with Example 7.1, mAb 2FS22-172-003-AA / FS28-256-272 and FS22-172-003-AA / FS28-256-273 bind to MSLN in solution with stronger affinity, which is reflected in the EC of these clones in this assay in the presence of soluble MSLN compared to the absence of soluble MSLN, as shown in Figure 4. 50 This translated into a significant effect on the mAb 2 FS22-172-003-AA / FS28-256-271 was selected for further characterization.

[0349] 8.4 Expression-Dependent CD137 / MSLN mAbs Across Endogenous Cell Lines and Multiple PBMC Donors Expressing a Wide Range of MSLN Levels 2 Effect of As noted in Examples 8.1 and 8.2, cancer patients express heterogeneous levels of MSLN. Therefore, the FS22-172-003-AA / FS28-256-271 mAb was administered across a range of cells expressing various levels of MSLN. 2 It is desirable to determine the efficacy of CD8 + T cell assays , performed as described in Examples 8.1 and 8.2. In addition to measuring IL-2 after 72 hours, IFNγ production was also measured after 96 hours using the V-PLEX human IFNγ MSD kit (Meso Scale Discovery, K151QOD-4) according to the manufacturer's instructions.

[0350] [Table 14]

[0351] In all assays reported in Table 13, mAb 2FS22-172-003-AA / FS28-256-271 was able to induce potent T cell activation when crosslinked with cells expressing different levels of MSLN, as evidenced by subnanomolar potency on IL-2 (72 hours) and IFNγ (96 hours) production (see Table 13). Interestingly, IL-2 and IFNγ production decreased with the amount of MSLN present in the crosslinked cells, for example, when crosslinked with cells expressing low levels of MSLN (AsPC-1). This decrease was less pronounced when IFNγ was measured relative to IL-2 production, but in all cases, the mAb 2 demonstrated subnanomolar potency. mAb 2 When tested in co-culture with MSLN-negative HEK.FRT cells, the mAb 2 Overall, these results suggest that mAb 2 The results showed that even low levels of MSLN can induce T cell activation, and that mAb 2 The level of T cell activation induced by mAb 2 This suggests that the level of MSLN present to crosslink mAbs is dependent on the level of MSLN present. 2 This shows that the efficacy of is correlated with the MSLN expression density on cells.

[0352] Example 9: Production of anti-mouse CD137 Fcab Due to the low sequence homology between mouse and human CD137 sequences in vivo, mAbs containing the CD137 antigen-binding region in their constant domains will be tested in an in vivo mouse model. 2 To enable the activity of Fcabs, we generated and characterized Fcabs that specifically bind to mouse CD137.

[0353] 9.1 Naive selection of anti-mouse CD137 Fcab To select Fcabs that bind to human CD137, a yeast display selection campaign similar to that described above for the selection of Fcabs that bind to human CD137 was employed (see Example 2.1). Recombinant mouse dimers were used as antigens (see Example 1).

[0354] Four naive yeast libraries displaying the CH1 to CH3 domains of human IgG1, previously used to select Fcabs that bind to human CD137, were used to select Fcabs that bind to mouse CD137. A total of 53 separate rounds of selection were performed to identify anti-mouse CD137 binders. Binders were selected from the yeast naive libraries using recombinant dimeric biotinylated mouse CD137 (mCD137-mFc-Avi) antigen produced in-house.

[0355] 9.2 Anti-mouse CD137 Fcab characterization from naive selection The specificity of anti-mouse CD137 Fcab for mouse CD137 was confirmed by the HelD1.3 "mock" mAb 2 In addition, Fcabs were tested in a 3D format and the binding of Fcabs to other mouse TNFRSF receptors (CD40, OX40, GITR) was tested for BLI on the Octet QKe system. Streptavidin biosensors (PALL ForteBio 18-5021) were used to coat 10 ng / μl of mouse CD40, GITR, and OX40 receptors (all from R&D Systems and biotinylated using the EZ-Link Sulfo-NHS-SS-Biotin kit from Thermoscientific #21328). 2 Anti-mouse CD137 Fcab in the format was diluted 1:1 in dynamic buffer (PALL 18-1092) to a final concentration of at least 1 μM. The antigen-coated sensor was then incubated with mAb 2The cells were immersed in the solution for 180 seconds, followed by immersion in 1x dynamic buffer for 180 seconds. Antibodies for each TNFRSF receptor were used as positive controls. Fcab clones FS22m-055, FS22m-063, FS22m-066, FS22m-075, FS22m-135, FS22m-055, FS22m-063, and FS22m-066 did not bind to any of the TNFRSF receptors tested, thus demonstrating their specificity for mouse CD137.

[0356] HEK.FRT.luc cells expressing the mouse CD137 sequence (SEQ ID NO: 150) were cultured as described in Example 2. mAb containing the previously selected anti-mouse CD137 Fcab was produced according to the same method as previously described in 3. 2 were screened using this cell line, HEK.FRT.luc.mCD137, according to the method described in Example 2.3. 2 were tested, of which 29 were positive for NF-κB activity. Lob12.3, which contains a human IgG1 Fc with the LALA mutation (G1AA / Lob12.3), was used as a positive control anti-mouse CD137 mAb and showed increased luminescence, confirming the validity of the assay. HelD1.3, which also contains a human IgG1 Fc with the LALA mutation, was used as a negative control human IgG isotype to rule out interference from a human IgG mock Fab in this assay. When possible, EC 50 mAbs that did not reach a plateau in activity were calculated. 2 was a mAb that exhibited classic sigmoidal activity kinetics. 2 was ignored in favor of mAb 2 is the EC of activity upon cross-linking of protein L 50 The results were ranked in order of the fold change and the fold change. Best e-commerce 50 (1.44 nM) and was selected based on the largest fold change in activity upon crosslinking (27-fold).

[0357] Example 10: Selection and characterization of anti-mouse MSLN antibodies 10.1 Naive selection of anti-mouse MSLN mAb 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 properties similar to those of the anti-human MSLN mAb described in Examples 4 and 5.

[0358] Phage selections using a synthetic naive phagemid library displaying human IgG1 germline Fab domains randomized at CDR1, CDR2, and CDR3 (MSM Technologies) were used followed by selections with biotinylated mouse MSLN-His-Avi (SEQ ID NO: 143, see Section 1.1) as described in Section 1.2. Four rounds of selection were performed with decreasing concentrations of biotinylated mMSLN-His-Avi, followed by selections with anti-human M Similar to SLN selection, an epitope masking strategy was implemented in subsequent campaigns. Furthermore, after the first round using recombinant antigens, HEK293-mMSLN cells were generated and used in rounds 2, 3, and 4.

[0359] Briefly, the cDNA encoding the mouse MSLN (SEQ ID NO: 145) sequence was subcloned into the pcDNA5 / FRT / TO vector (Life Technologies, V652020) and then The Flp-In TREx 293 cell line (Life Technologies, R78007) was co-transfected with the Flp recombinase expression plasmid pOG44 (Life Technologies, V600520). 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 amplified in the presence of 1 μg / ml doxycycline (Sigma-Aldrich, D9891) and tested for MSLN expression using an anti-mouse MSLN antibody (LS Bio, LS-C179484).

[0360] A total of 47 individual mAbs from the enriched population were screened for antigen binding, and 45 unique positive binders were subcloned and purified to IgG1 as described above in Example 1.3. The mAb was expressed as a soluble mAb in the LALA format. The mAb was characterized for specific binding to immobilized mMSLN-His-Avi by ELISA and Biacore analysis. In a kinetics experiment using mAbs, the mAbs were ranked based on their affinity to approximately 50 or 200 RU of immobilized mMSLN-His-Avi. This identified a panel of mAbs, including FS28m-228, with affinities ranging from 1 to 25 nM. Further, as described in Section 2 Binding to different regions of MSLN was tested as described in .1.3. The mouse cross-reactive mAb G1-AA / MOR6626, generated by cloning the VH and VL of the mouse mAb G1-AA / MOR6626 (WO 2009 / 068204 A1), was used as a positive control. Most clones, including FS28m-228, were unable to bind to MSLN already bound to MOR6626, whereas other clones, such as FS28-194 and FS28-261, showed partial or complete binding, respectively. Thus, clones binding to distinct regions (bins) were isolated.

[0361] Overview: Selection and screening of anti-mouse Fcabs and mAbs The anti-mouse CD137 Fcabs identified in the previous examples were shown to have agonist activity in NF-κB reporter assays when cross-linked with either an external cross-linker such as protein L (Example 9). Of the panel of anti-human CD137 Fcabs identified, FS22m-066 was selected for pairing with the mouse MSLN-targeting Fab because this clone exhibited the most favorable functional and biophysical properties.

[0362] Phage selection and antibody screening strategies identified a panel of anti-mouse mesothelin-binding clones with a range of affinities that bound to distinct 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 for in vivo proof-of-concept studies in mice.

[0363] Example 11: Anti-mouse CD137 / MSLN mAb 2 Production of As described in Example 10, a panel of naive anti-mouse MSLN antibodies was discovered and screened for beneficial binding and targeting properties. As described in Example 9, an anti-mouse CD137 Fcab was selected. Anti-mouse CD137 Fab (FS22m-063) was used. ) and anti-mouse MSLN Fab (FS28m-228 and FS28m-228-010) were used for complete in vitro characterization and in vivo proof-of-concept of MSLN-targeted CD137 agonism in syngeneic mouse tumor models. 2The Fab was selected to investigate the relationship between binding affinity, avidity, and functional activity. mAb with LALA mutation 2 were constructed as described in Example 6 with the aim of minimizing the contribution of Fc gamma receptor-driven cross-linking and effector function, and were assigned the identifiers FS22m-063-AA / FS28m-228 (SEQ ID NOs: 136 (light chain), 137 (heavy chain)) and FS22m-063-AA / FS28m-228-010 (SEQ ID NOs: 136 (light chain) and 166 (heavy chain), respectively). 2 was produced by transient expression in HEK293-6E cells and purified using the mAb Select Sure primer. Purification was performed using a protein A column.

[0364] 11.1 Binding kinetics mAbs for binding to immobilized and soluble MSLN as well as anti-human MSLN binders 2 The affinity of was tested by SPR using a Biacore instrument.

[0365] The procedure for binding to immobilized MSLN was similar to that described in Examples 7.1 and 7.2 using 50 RU of immobilized mMSLN-His-Avi. To determine the affinity of soluble MSLN, mAb 2 Briefly, 25 μg / ml of anti-human IgG (Fc) antibody (GE Healthcare, Human Antibody Capture Kit, BR100839) was applied to a Biacore sensor chip CM5 (GE Healthcare, BR100530) and captured via anti-human Fc. Immobilization was performed to achieve a final response of approximately 750 RU. mAb was diluted to 50 nM in HBS-EP buffer (GE Healthcare, BR100188). 2 The molecules were injected individually at 30 μl / min. A response of approximately 100 RU was achieved. Recombinant mMSLN-His-Avi antigen diluted in HBS-EP buffer was diluted 3-fold in the concentration range of 243 nM to 0.11 nM and injected at 70 μl / min for 5 min, followed by dissociation in buffer for 5 min. Regeneration was performed with 3 M magnesium chloride (GE Healthcare, Human Antibody Capture Kit, BR100839) at 30 μl / min. This was achieved by injecting at this rate for 30 seconds.

[0366] Kinetic data are shown in Table 14. FS22m-063-AA / FS28m-228-010 showed stronger binding to membrane-bound MSLN than to soluble shed MSLN, likely due to enhanced binding interactions of avidity. Affinity matured mAbs 2 FS22m-063-AA / FS28m-228-010 showed improved binding to both immobilized MSLN and MSLN in solution. FS28m-228-010 was selected because it preferentially binds to immobilized MSLN, which may be beneficial for avoiding the sink effect of circulating MSLN in the blood. This clone, like human Fab FS28-256-271, binds immobilized MSLN in the single-digit nanomolar range and to MSLN in solution. The immobilized MSLNs were preferentially targeted over the inactive MSLNs.

[0367] [Table 15]

[0368] 11.2 Mouse CD137 / MSLN mAb using mouse MSLN-positive cells 2 Functional activity of Activated cytotoxic CD8 + T cells are involved in the direct killing of cancer cells and express CD137 on their cell surface (Ye et al., 2014). CD137 clustering promotes downstream signaling and further CD8 + It is known to be essential for the induction of T cell activation.+ T cell activation assay using mAb 2 We assessed the ability of CD8 to drive CD137 clustering and downstream signaling. + T cell activation was achieved by antigen stimulation of genetically modified OT-I T cells isolated from C57 BL / 6-Tg(TcraTcrb)1100Mjb / Crl OT-I mice (Jackson Laboratory, catalog no. 003831) bearing T cell receptors specific for ovalbumin peptide 257-264, as determined by IFNγ release.

[0369] To isolate T cells, splenocytes were isolated from fresh OT-1 mouse spleens. Briefly, each spleen from a C57BL / 6 OT-1 mouse was collected and stored in PBS before being transferred to a well of a 6-well tissue culture plate and mechanically disrupted with two needles. The disrupted spleen was passed through a 70 μm cell strainer, which was then rinsed with PBS. The cell suspension was then pelleted by centrifugation, the supernatant removed, and red blood cells were lysed by adding 10 ml of 1x red blood cell lysis buffer (eBioscience, 00-4300-54) according to the manufacturer's instructions. Spleen Cells were plated at 2 x 10 per well for T cell activation. 6 10 x 10 cells per well 6 Cells were plated in 6-well plates in medium (IMDM, 5% FCS, 50 μM 2-mercaptoethanol, 1× Penstrep) containing 10 nM SIINFEKL peptide. Plates were incubated at 37°C with 5% CO for 48 hours. After 48 hours, CD8 T cells were isolated by CD8 T cell cloning according to the manufacturer's instructions. + CD8 T cells were isolated using a T cell isolation kit (Milentyi Biotec, 130-104-075). Isolated and activated CD8 T cells were plated in medium (IMDM, 5% FCS, 50 μM 2-mercaptoethanol, 1× Penstrep) supplemented with 30 U / ml IL-2 (Peprotech, AF-200-02) at 1 × 10 per ml in each daily split for an additional 3 days.6 After 3 days of expansion, the cells were used in the following assay.

[0370] CD8 used in this example + Because T cells were derived from two separate animals and splenocyte expansion was performed 18 months apart, variability in T cell activation between specimens A and B is expected.

[0371] CT26 colon carcinoma cells (ATCC, CRL-2638) expressing full-length murine mesothelin (SEQ ID NO: 145) were generated to present the antigen in a membrane-bound conformation. These cells were generated using the pcDNA3.1 vector (+) (Thermo Fisher Scientific, Cat. No. V79020) and Lipofection (Lipofectamine 3000, Thermo Fisher Scientific, Cat. No. V79020). The log number L3000008 was used. CT26 cells were transfected with the pcDNA3.1 vector containing the mouse MSLN cDNA according to the manufacturer's protocol. Stable transfection was then achieved using geneticin as the selection antibiotic (600 μg / ml) in complete medium (RPMI, 10% FBS).

[0372] Expression of mouse MSLN in CT26 cells was confirmed by flow cytometry using the positive control antibody MOR6626. Cell binding was confirmed by flow cytometry. After incubation of cells with a positive control antibody for 1 hour, cell binding was detected using a fluorescently labeled anti-human IgG detection antibody (Stratech Scientific Ltd, catalog number 109-546-098-JIR). Clonal populations were expanded and subsequently analyzed to determine relative expression levels using the same flow cytometry procedure. Two clones expressing mouse MSLN at different levels were subsequently used as tools for studying the anti-mouse MSLN Fabs: CT26.B2 (high MSLN expression) and CT26.G10 (intermediate / low MSLN expression). Panc02 cells (NIC / NIH, Maryland, USA), which endogenously express MSLN, were also used to provide a range of MSLN expression cells. These cells expressed lower levels of MSLN in vitro and ex vivo, demonstrating cytosolic expression as determined by IHC (data not shown). Mesothelin expression was subsequently confirmed ex vivo by IHC (data not shown).

[0373] CT26.B2, CT26.G10, and Panc02 cells were incubated with SIINFEKL peptide (500 nM) and plated at 2 × 10 cells per well in 50 μl of medium. 4 of OT-1 cells in MSLN + Test antibodies were prepared in a 1:4 titration starting at 60 nM (4x the final concentration), and 50 μl of antibody mix was added to each well, bringing the final assay volume to 200 μl accordingly. The assay was incubated at 37°C with 5% CO2 for 3 days. After 3 days, supernatants were collected and analyzed by mIFNγ (eBioscience, Catalog No. 100010001). ELISA was performed according to the manufacturer's instructions for the antibody (antibody product code 88-7314-88).

[0374] In this T cell assay, murine CD137 / MSLN mAb was used in the absence and presence of soluble murine MSLN. 2 The commercially available mouse mMSLN-His Biolegend (#594008), C Serum analysis of mice bearing T26.G10 tumors determined that the median concentration of MSLN in the blood was 100 pM (data not shown). Therefore, to study interference with soluble MSLN, up to 2 nM was used in functional assays.

[0375] As shown in Table 15 and Figure 5, mAb 2 FS22m-063-AA / FS28m-228-010 showed greater potency than the parental FS22m-063-AA / FS28m-228 when cross-linked with the CT26.G10 cell line. The following negative controls were also tested in one assay and, as expected, did not produce a cytokine readout: mock mAb 2 Format FS22m-063 Fcab(HelD1.3), and MSLN Targeting positive control (G1 / MOR6626). As shown in Figure 5, the presence of 2 nM soluble MSLN had minimal effect on IFNγ release, and all EC 50 The value is the manipulated MSLN + The concentration was in the low picomolar range, except for Panc02 cells, which expressed less membrane MSLN than CT26 cells. As with human mAb2, the level of T cell activation induced by mAb2 depends on the level of MSLN present in crosslinked cells.

[0376] [Table 16]

[0377] Example 12: In vivo proof of concept mAb 2 The mAb was shown to be functional in T cell assays and to be effective in vivo in syngeneic immunocompetent tumor models. 2 It was desirable to test the functionality of FS22m-063-AA / FS28m-228.

[0378] 12.1 Efficacy of FS22m-063-AA / FS28m-228 In Vivo in the CT26.B2 Syngeneic Tumor Model To determine the antitumor effect of FS22m-063-AA / FS28m-228 in the high MSLN-expressing tumor model, 9- to 10-week-old Balb / C female mice (Charles River) were euthanized for 1 week before the start of the study. All animals were microchipped and given a unique identifier. Each cohort contained 15 or 20 mice. CT26.B2 cells were expanded to generate a cell bank, which was then prescreened by IDEXX Bioresearch for pathogens using the IMPACT I protocol and shown to be pathogen-free. Each animal was 1 x 10 mice in 100 µl serum-free medium on the left flank 5 On day 17 after tumor cell inoculation, tumor-free mice were removed from the study.

[0379] FS22m-063-AA / FS28-228 mAb 2 (SEQ ID NOs: 360 and 361) or human IgG1 isoforms The control type (G1-AA / 4420) was injected with 200 μl of antibody in DPBS + 1 mM arginine + 0.05% Tween 80 at a fixed concentration of 200 μg per dose (approximately 10 mg / kg for a 20 g mouse). 2 The molecules were administered to mice by intraperitoneal injection on days 17, 19, and 21 post-inoculation, while a control human IgG1 antibody was administered to mice on days 7, 9, and 11 post-inoculation. Tumor 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: LX(S 2 ) / 2 (where L = longest axis, S = shortest axis)

[0380] The study endpoint was determined by a humane endpoint based on tumor volume and status.

[0381] As shown in Figure 6, FS22m-063-AA / FS28m-228 mAb 2Treatment with showed a visible delay in tumor growth compared to mice treated with the isotype control (G1 / 4420).

[0382] Time-to-endpoint (survival) analysis was performed using GraphPad Prism 8.0 software. The data shown in Figure 7 and Table 15 were analyzed using the log-rank (Mantel-Cox) test. The data were analyzed by log-rank analysis for FS22m-063-AA / FS28m-228 mAb. 2 Isotope It was shown that IgG1 induces a significant survival benefit compared to the IgG1 control (G1 / 4420). The median survival time for the group was 33.5 days, but the median survival time for FS22m-063-AA / FS28m-228 was not reached.

[0383] [Table 17]

[0384] 12.2 Efficacy of FS22m-063-AA / FS28m-228 in vivo in the CT26.G10 syngeneic tumor model FS22m-063-AA / FS28m-228 mAb 2 Efficacy was also tested in the CT26.G10 syngeneic tumor model CT26.G10 cells express lower levels of MSLN compared to CT26.B2 cells. The same procedure as described in Example 12.1 was followed, except that the CT26.G10 cell line was used to inoculate mice. Mice were injected with FS22m-063-AA / FS28m-228 mAb on days 12, 14, and 16 post-inoculation. 2 and / or G1 / 4420 control antibody was administered intraperitoneally in 200 μl injections on days 7, 9, and 11 post-inoculation. Mice were administered a fixed dose of 200 μg / mouse (equivalent to approximately 10 mg / kg in a 20 g mouse).

[0385] As shown in Figure 8, FS22m-063-AA / FS28m-228 mAb 2Mice treated with isotype Compared to control-treated mice, tumor growth was reduced, and a significant delay in tumor volume increase was observed in FS22m-063-AA / FS28m-228-treated mice compared to the G1 / 4420-treated group. Finally, 1 / 20 (5%) of the mice were tumor-free after treatment with G1 / 4420, whereas 1 / 20 (5%) of the mice were tumor-free after treatment with G1 / 4420. Four out of 20 mice (20%) had tumors at the end of the study after treatment with FS22m-063-AA / FS28m-228. Furthermore, as shown in Figure 9 and Table 16, this translated into a significant improvement in tumor-free survival when assessed using log-rank pair analysis. Specifically, median survival increased from 25 days (G1 / 4420) to 29 days with FS22m-063-AA / FS28m-228.

[0386] [Table 18]

[0387] mAb 2 The and isotype controls contained the LALA mutation, which significantly reduced the ability of the antibody to bind to Fcγ receptors, so the observed tumor growth inhibition may not have been the result of ADCC activity. Therefore, it is expected that no anti-tumor activity will be observed in mice treated with only the anti-MSLN mAb containing the LALA mutation. However, the mAb containing the LALA mutation 2 showed significant tumor growth inhibition even in the presence of LALA mutations. This was due to the Fab arm of the mAb that binds to MSLN on the cell surface. 2 Cross-linking of mAb drives the clustering and activation of CD137 on immune cells. 2 This is thought to be because it provides antitumor activity.

[0388] Example 13: In vivo affinity maturation of anti-mouse CD137 / MSLN (FS22m-063-AA / FS28m-228-010) mAb 2 Characterization 13.1 Affinity-matured CD137 / MSLN mAb in the CT26.G10 syngeneic mouse tumor model 2 Dose-response activity of FS22m-063-AA / FS28m-228 mAb 2 When treated with , high levels of mouse MSLN (CT26 Significant antitumor effects and improved survival were observed in syngeneic tumor models expressing murine MSLN (CT26.B2) and tumors expressing low levels of murine MSLN (CT26.G10) (Example 12). Therefore, it was desirable to investigate the antitumor effects of affinity-matured FS22m-063-AA / FS28m-228-010 in vivo at a range of doses (6, 20, 60, and 200 μg / mouse, equivalent to approximately 0.3, 1, 3, and 10 mg / kg in a 20 g mouse).

[0389] Balb / c female mice (Charles River) aged 9-11 weeks and weighing 17.0-25.2 g were acclimated for 1 week before the start of the study. All animals were microchipped and secured in place. Each animal was given a unique identifier. There were 20 mice in each cohort. The CT26.G10 colon cancer cell line described in Example 11.2 was expanded to generate a cell bank. Each animal received 1 x 10 cells in 100 μl of serum-free medium in the left flank. 5 The mice received subcutaneous injections of tumor cells. On day 12 after tumor cell inoculation, tumor-free mice were removed from the study.

[0390] FS22m-063-AA / FS28m-228-010 mAb 2 and a fixed final dose was prepared according to the dose ranges above. The human IgG1 isotype control (G1-AA / 4420) and the anti-CD137 positive control antibody Lob12.3 (G1 / Lob12.3), both of which have a human IgG1 backbone and contain LALA, were injected intraperitoneally (IP) into mice at a concentration of 20 μg (approximately 1 mg / kg for a 20 g mouse). All antibodies were prepared in DPBS + 1 mM arginine + 0.05% Tween 80. Each mouse Mice received 200 μl IP injection of mAb 12, 14, and 16 days after tumor inoculation. 2 Mice were administered either a medicament or a control antibody. Tumor volume measurements were taken three times a week using calipers as described in Example 12.1, and the mice were closely monitored. The study endpoint was determined by a humane endpoint based on tumor volume and condition.

[0391] Mixed models for the analysis of tumor growth rates were performed using STATA / IC 15.1 software. Statistical significance was shown for pairwise growth rates over the course of the study using a mixed model analysis comparing all groups. A separate model was fitted for each pair of treatments of interest. The model was as follows:

[0392] log10(volume) = A + B × (number of days - start date) + ε A and B are the intercept and slope, respectively, which vary for each mouse and include a fixed effect for group and a random effect for animal. A=A0+A1T+εA B=B0+B1T+εB T is a dummy variable representing the treatment group with a value of 0 for one group and a value of 1 for the other group. The random effects are normally distributed. εA ~ (0, σA), εB ~N (0, σB) where σA and σB are the standard deviations of the intercept and slope variability between animals, respectively. The interanimal variability is also usually distributed with a standard deviation σ:ε ~ (0,σ).

[0393] For each pair of treatments, the above model was fitted to the data. For A1 and B1, a (two-tailed) p-value of the difference from zero was calculated. A p-value of less than 0.05 is statistically significant evidence of a difference between the treatment groups.

[0394] As shown in Figure 10, FS22m-063-AA / FS28m-228-010 mAb 2treated with isotype control Compared with mice treated with 62.5 mm β-glucan, tumor growth was reduced at all dose levels. 3 All animals bearing the following tumors were counted as complete responders at the end of the study (see Table 17): FS22m-063-AA / FS28m-228-010 mAb 2 10, 3, 1, and 0.3 mg / kg, respectively 30%, 20%, 20%, and 10% of animals treated with the anti-CD137 antibody, G1 / Lob12.3 (15%), and G1-AA / 4420 isotype control (0%) were considered tumor-free.

[0395] Additionally, Table 17 shows pairwise comparisons of growth rates over the course of the study using mixed model analysis, comparing all groups to the human IgG1 isotype control treatment group. None of the mice showed signs of overt toxicity or adverse effects, and all treatments were well tolerated by the mice.

[0396] Survival analysis (Figure 11 and Table 18) showed that FS22m-063-AA / FS28m-228-010 at all dose levels mAb 2 showed significant antitumor activity compared to mice treated with the isotype control (G1-AA / 4420). The results showed that the mouse CD137 / MSLN mAb induced a response that appeared to be dose-dependent. Furthermore, Table 18 summarizes the median survival times for each group, where 10 mg / kg, 3 mg / kg, 1 mg / kg, and 0.3 mg / kg of the murine CD137 / MSLN mAb 2 Treatment with The values ​​increased from 29.5 days (G1-AA / 4420) to 36.5, 37.5, 35 and 31 days, respectively.

[0397] [Table 19]

[0398] [Table 20]

[0399] CD137 / MSLN mAb 2 CD137 / MSLN mAb, similar to FS28m-228 MSLN Fab in the format 2 The FS28m-228-010 Fab in this format also resulted in a significant reduction in tumor growth compared to the isotype control. , mAb 2 A significant dose-dependent improvement in survival was observed with the Fab arm of the mAb 2 These results suggest that cross-linking of CD137 to its MSLN target drives the agonistic action of CD137 and thus the antitumor effects observed in vivo.

[0400] 13.2 Antitumor Efficacy of FS22m-063-AA / FS28m-228-010 Compared to Its Constituent Parts in the CT26.G10 Syngeneic Tumor Model FS22m-063-AA / FS28m-228-010 showed significant antitumor effects and improved survival rates in tumor-bearing mice. 2 has superior antitumor activity in vivo compared to its individual components It is desirable to determine whether the mouse MSLN antibody (G1-AA / FS28m-228-010), "mock" mAb 2 CD137 Fcabs (FS22m-063-AA / HelD1.3 and FS22m-063-AA / 4420), mouse MSLN antibody, and mock mAb 2 CD137 Fcab combinations in the format (i.e., G1-AA / FS28m-228-010 plus FS22m-063-AA / HelD1.3), as well as FS22m-063-AA / FS28m-228-010 mAb 2 Treatment with a human isotype control antibody (G1-AA / HelD1.3 ) treatment was compared.

[0401] Mice were prepared according to Example 13.1 and inoculated with the CT26.G10 colon cancer cell line. All cohorts contained 20 mice, except for FS22m-063-AA / 4420, which contained 10 mice. there was.

[0402] FS22m-063-AA / FS28-228-010 mAb 2 , G1-AA / FS28m-228-010, FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420 and G1-AA / HelD1.3 antibodies were all administered at 200 μg per dose (for a 20 g mouse). The CD137 mAb was formulated at approximately 10 mg / kg and injected intraperitoneally (IP) into mice at a fixed dose. 2 Both the and MSLN antibodies were prepared at 200 μg per dose (approximately 10 mg / kg for a 20 g mouse) for the combination group. All antibodies were prepared in DPBS + 1 mM arginine + 0.05% Tween 80. Similar to the dosing regimen in Example 13.1, each mouse received antibody via intraperitoneal injection of 200 μl on days 12, 14, and 16 (q2dx3) after tumor inoculation. Tumor volume measurements were performed three times a week using calipers as described in Example 12.1, and mice were closely monitored. The study endpoint was determined by a humane endpoint based on tumor volume and condition.

[0403] As shown in Figure 12, FS22m-063-AA / FS28m-228-010 mAb 2 Treatment with G1-AA / HelD1.3 significantly reduced tumor growth compared to mice treated with the G1-AA / HelD1.3 isotype control. 3 All animals bearing the following tumors were counted as complete responders at the end of the study (see Table 19): 1 / 20 (5%) and 1 / 20 (6%) of mice treated with G1-AA / FS28m-228-010. and G1-AA / HelD1.3 isotype control, 0 / 20 (0%), FS22m-063-AA / HelD1.3, FS22m-063-AA / 4420, and the combination of FS22m-063-AA / HelD1.3 plus G1-AA / FS28m-228-010. Compared with mice treated with FS22m-063-AA / FS28-228-010 mAb 2 7 / 20 (35%) of the animals treated with were considered complete responders to treatment at the end of the study.

[0404] Additionally, Table 19 shows pairwise comparisons of proliferation rates over the course of the study using mixed model analysis, comparing all groups to the G1-AA / HelD1.3 isotype control.

[0405] Survival analysis (Figure 13 and Table 20) showed that the FS22m-063-AA / FS28m-228-010 mAb significantly outperformed the G1-AA / HelD1.3 antibody. 2 Furthermore, we demonstrated that FS22m-063-AA / FS28m-228-010 mAb induces a significant survival benefit, whereas its constituent components do not confer a survival advantage. 2 Treatments 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 a combination of FS22m-063-AA / HelD1.3 and G1-AA / FS28m-228-010. This resulted in an improved median survival of 42.5 days compared with 29 days.

[0406] [Table 21]

[0407] [Table 22]

[0408] These data demonstrate that mAb 2 exerts its protective effects at levels not observed when the individual components of mAb 2 are administered alone or in combination. 2 We show that bispecific molecules targeting both CD137 and MSLN are required for in vivo activity, as this results in tumor growth inhibition.

[0409] 13.3 Anti-mouse CD137 / MSLN mAb in the CT26.G10 syngeneic mouse tumor model 2 Hepatic pharmacology of Anti-CD137 mAb treatment of solid tumor patients with urelumab in clinical trials resulted in severe treatment-related immune events that were shown to be related to the dose of urelumab administered. The effects of these immune events manifested in the liver as severe hepatotoxicity (Segal, NH, et al., 2017).

[0410] Preclinical mechanistic studies conducted in mice using CD137 agonistic tool antibodies administered to animals have shown similar hepatotoxicity. These studies demonstrated the requirement of T cells and CD137 for the resulting hepatotoxicity (Niu, L., et al. 2007 and Dubrot J, et al. al. 2010). Although not fully understood, interactions between the bone marrow and T cell compartments This action has been shown to be important in initiating the inflammatory cascade that leads to liver injury and hepatotoxicity (Bartkowiak, T et al., 2018). Therefore, these animal models are suitable for the treatment of liver damage with other CD137 agonists, such as CD137 / MSLN mAbs. 2 This has clinical translational relevance in predicting the risk of hepatotoxicity in human patients following administration of

[0411] Mice from the CT26.G10 syngeneic tumor study described in Examples 13.1 and 13.2 were treated with FS22m-063-AA / FS28m-228-010 mAb 2 showed no obvious signs of toxicity after repeated administration of To determine whether immune activation correlated with hepatotoxicity in these animals administered according to a dosing regimen similar to that shown in Examples 13.1 and 13.2, mice were culled at four time points post-dose and liver samples were taken at necropsy for histological evaluation.

[0412] Mice were prepared as described in Example 13.1 and inoculated with the CT26.G10 colon cancer cell line. Each cohort consisted of 24 mice. FS22m-063-AA / FS28-228-010 mAb 2 The (CD137 / MSLN) and G1-AA / 4420 (human IgG1 isotype control) antibodies were prepared in DPBS + 1 mM arginine + 0.05% Tween 80 at 200 μg per dose (approximately 10 mg / kg for a 20 g mouse) and injected intraperitoneally (IP) into mice at fixed doses. Each mouse received the antibody via 200 μl IP injection on days 12, 14, and 16 after tumor inoculation. Tumor volume measurements were performed three times a week using calipers as described in Example 12.1, and the mice were closely monitored. Six mice per group were necropsied 2, 5, 8, and 11 days after the last dose, and liver samples were formalin-fixed and paraffin-embedded. Liver sections were then cut and subjected to histopathological evaluation by hematoxylin and eosin staining and liver inflammation and damage scoring by an independent, board-certified veterinary pathologist.

[0413] Liver pathology was assessed on hematoxylin and eosin-stained sections using a scoring system. Livers were scored for pathology corresponding to multiple mixed inflammatory cells, multiple degenerated hepatocytes, increased hepatocyte mitosis, and portal vein mixed inflammatory cell infiltration. The frequency of mice with minimal, mild, and moderate effects within each group is shown in Table 21.

[0414] FS22m-063-AA / FS28-228-010 mAb 2 Animals treated with IFN-γ-α showed minimal liver lesions, specifically, minimal multiple mixed inflammatory cells (mainly granulocytes) located throughout the parenchyma. Minimal degenerated hepatocytes scattered throughout the parenchyma Minimal degenerated hepatocytes Minimal mixed inflammatory cells in the portal tract showed.

[0415] [Table 23]

[0416] These findings do not represent hepatotoxicity, as has been observed with other anti-CD137 agonist antibodies. 2 agonizes CD137 by cross-linking mediated through MSLN binding, and given that MSLN is overexpressed primarily on the cell surface of tumor cells but not in the liver (Ordonez 2003, 14576474), the agonistic effects of CD137 are expected to be restricted to the tumor microenvironment.

[0417] 13.4 Anti-mouse CD137 / MSLN mAb in the CT26.G10 syngeneic mouse tumor model 2 of Mechanism of action CD137 / MSLN mAb 2 The limited hepatic pharmacology observed after repeated administration of (Example 13.3) To further understand the pharmacology of the antitumor responses observed with FS22m-063-AA / FS28m-228-010, we performed a CD137 / MSLN mAb in an MSLN-positive syngeneic tumor model. 2 We investigated the mechanism of action of .

[0418] Mice were prepared as described in Example 13.1 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 , human IgG1 isotype control (G1-AA / 4420) and anti-CD137 agonist A control antibody (clone 3H3; G1 / 3H3) was 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 received antibody at a fixed dose of 134 μg via a single 200 μl IP injection on day 20 post-tumor inoculation. Tumor volume measurements were performed three times a week using calipers as described in Example 12.1, and mice were closely monitored.

[0419] Six mice per group were administered at 24, 72, 144, and 192 hours after administration on the 20th day after tumor inoculation. Mice were necropsied. 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 collected for flow cytometry, as T cell activation and proliferation markers are known to be downstream effects of CD137 agonism (Fisher et al., 2012). T cell abundance and proliferation were examined by cytometry. Additionally, serum from the blood was 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 with red blood cell lysis buffer (Miltenyi Biotec Ltd., 130-094-183). Blood was then collected. Whole blood was collected by peripheral cardiac bleeding, half of which was collected in EDTA-containing tubes for single-cell analysis by flow cytometry, and half of the blood was collected in clot activator / serum tubes for analysis of soluble MSLNs. Whole blood collected in EDTA-containing tubes 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 the serum was removed for analysis of soluble MSLN.

[0420] Next, single cells from spleen, tumor, and blood were treated in the same way, the cells were washed once with PBS, and the 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 22 (all except intracellular markers Ki67 and FoxP3) in the presence of Fc block (eBioscience, 16-0161-85, 1:25) for 45 minutes at 4°C. Cells were then fixed and permeabilized with the eBioscience FoxP3 staining kit (eBioscience, 00-5523-00) according to the manufacturer's instructions. Cells were resuspended in 100 μl of permeabilization buffer containing antibodies for the intracellular markers Ki67 and FoxP3 and incubated overnight at 4°C in the dark. BD Fortessa flow cytometer was used. Cells were washed once with permeabilization buffer and resuspended in 120 μl of PBS containing 0.5% BSA before acquisition with a chromatograph. Data were acquired using BD FACS Diva software and analyzed with FlowJo (V10) and Microsoft Excel. Data were analyzed for CD8+ cells at 144 hours post-injection. + T cell abundance and proliferation are shown as a percentage of the parental population.

[0421] [Table 24]

[0422] As shown in Table 23, G1 / 3H3 and FS22m-063-AA / FS28-228-010 mAb 2 At 144 hours after administration, the CD8 + An increase in the percentage of T cells was observed. + The mean percentage of T cells at 144 hours post-dose ranged from 32.1% (G1-AA / 4420) to 56.1% (G1 / 3H3) and 5% (FS22m-063-AA / FS28m-228-010). It increased to 8.4%.

[0423] 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%). Similarly, in the spleen, CD8 + 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%).

[0424] This is FS22m-063-AA / FS28m-228-010 mAb 2 However, specific tumors expressing MSLN CD8 + While increasing T cells, G1 / 3H3, an antibody targeting CD137, also increases CD8 + This suggests that there is an increase in T cells in the periphery (blood and spleen).

[0425] CD8 after administration + To determine whether there were differences in T cell proliferation, CD8 + The proliferation marker Ki67 was analyzed in T cells. As shown in Table 24, a high percentage of CD8 + T cells were Ki67 in the control group + (mean expression 75.1%) and high levels of proliferative CD8 + This suggests a significant difference in CD8 T cells between tumor dose groups. + This may be one reason for the unclear differences in Ki67 expression on T cells.

[0426] In comparison, blood and spleen CD8 were significantly increased at 144 hours after administration of G1 / 3H3 compared to the IgG1 control. + Ki67 in T cells +A clear increase in CD8 expression was observed in the blood. + Mean Ki67 of 10.4% in T cells + While the expression of CD8 + The mean Ki67 expression on T cells was shown to be 86.3% at 144 hours post-injection. In comparison, this increase was not observed with FS22m-063-AA / FS28m-228-010, while it was observed with CD8 + Mean Ki67 in T cells + Expression is measured in the blood by mAb 2 Similarly, in the spleen, CD8+ cells were detected in 13.1% of patients after treatment with G1 / 3H3 compared with 8.1% after treatment with the isotype control and 11.4% after treatment with FS22m-063-AA / FS28m-228-010. + Average Ki67 in 36.1% of T cells + Expression was observed.

[0427] [Table 25]

[0428] [Table 26]

[0429] Six patients per group received either FS22m-063-AA / FS28-228-010, G1-AA / 4420, or G1 / 3H3. Serum collected from mice was purified using Mesothelin Mouse Immunosorbent Assay (MMA) according to the manufacturer's instructions for serum. Soluble MSLN levels were analyzed using a SimpleStep ELISA Kit (Abcam, ab204528). Data were plotted in Prism to show the concentration of serum MSLN levels over time. As shown in Table 25, at 144 hours post-dose, serum levels of MSLN were increased with G1 / 3H3 and FS22m-063-AA / FS28m-228-010 compared to the isotype control G1-AA / 4420. This similar increase was observed with the G1 / 3H3 and FS22m-063-AA / FS28m-228-010 mAbs. 2 It was observed in both was expressed in tumor cells in the CT26.G10 syngeneic mouse model, suggesting that CD137 agonism in tumors may increase soluble MSLN detected in serum.

[0430] [Table 27]

[0431] Taken together, these data suggest that FS22m-063-AA / FS28m-228-010 inhibits intratumoral cytotoxic CD8 + This was also observed with G1 / 3H3, but not with FS22m-063-AA / FS28m-228-010 mAbs. 2 Unlike G1 / 3H3, CD8 + T cells Furthermore, these CD8 + T cells also showed increased proliferation after administration of G1 / 3H3. + The primary role of T cells (also called cytotoxic lymphocytes) is the killing of infected or malignant cells through three main mechanisms: 1) the release of cytokines, such as TNFα and IFNγ, 2) the production and release of cytotoxic granules, and 3) the expression of FasL. Thus, the CD8 +Increased T cells may result in cytotoxic activity against tumors via these mechanisms, potentially leading to increased mesothelin release as a result of tumor cell killing, as observed in this example. Because tumors do not express CD137, it is hypothesized that mesothelin release is an indirect PD response triggered by T cell-mediated cytotoxicity.

[0432] Example 14: Anti-mouse CD137 / MSLN mAb in mice 2 and anti-human CD137 / MSLN mAb 2 Pharmacokinetics of 14.1 Anti-mouse CD137 / MSLN mAb in non-tumor-bearing mice 2 Pharmacokinetics of (FS22m-063-AA / FS28-228-010) Anti-mouse CD137 / MSLN mAb in mice 2 To determine the pharmacokinetics of BL / 6 female mice were treated with 10 mg / kg anti-mouse CD137 / MSLN mAb 2 (FS22m-063-AA / FS28m-228-010) or a human IgG1 control antibody (G1 / 4420) was administered intravenously once and monitored for up to 144 hours.

[0433] Approximately 20 μl of whole blood was microsampled at 0.5, 1, 6, 24, 48, 96, and 144 hours and processed to isolate approximately 5 μl of serum for analysis. The amount of antibody present at each time point was determined using a Gyrolab xPlore system (system name XPS1055) from Gyros Protein Technologies. Biotinylated goat anti-human IgG (heavy and light chains) was used as the capture reagent. ) monkey adsorbed antibody (Cambridge Biosciences, A80-319B), and goat anti-human as the detection antibody. IgG-AlexaFluor® 647 (Cambridge Biosciences, 2040-31) was used for sandwich immunoprecipitation using a Gyrolab Bioaffy 200 CD (Gyros Protein Technologies, P0004180). A standard curve ranging from 4000 ng / mL to 0.0677 ng / mL for each compound was prepared in 0.1% mouse serum (Sigma-Aldrich M5905) in Rexxip AN buffer (Gyros Protein Technologies, P0004994) and diluted 1:1000 in Rexxip AN (Gyros Protein Technologies, P0004994). Concentrations were determined and the mean sample concentrations from individual mice per time point (3 mice per time point) were plotted.

[0434] Figure 14A shows anti-mouse CD137 / MSLN mAb 2 pharmacokinetics of mAb 2 Non-MSLN results The results show that systemic exposure during administration was slightly lower than that of the human IgG1 antibody, which may be explained by a target-mediated clearance mechanism.

[0435] 14.2 Anti-human CD137 / MSLN mAb in non-tumor-bearing mice 2 Pharmacokinetics of For comparison, anti-human CD137 / MSLN mAb in non-tumor-bearing C57BL / 6 female mice 2 The pharmacokinetic profile of anti-human CD137 / MSLN (FS22-172-003-AA / FS28-256-271) mAb was determined in mice at 6.7 mg / kg. 2 Or a human IgG1 control antibody (G1-AA / 4420) was administered intravenously and monitored for up to 144 hours. Microsampling of approximately 20 μl of whole blood was performed at 0.5, 1, 6, 24, 48, 96, and 144 hours and processed to isolate approximately 5 μl of serum for analysis. Analysis was performed as described in Example 14.1.

[0436] Figure 14B shows anti-human CD137 / MSLN mAb 2 pharmacokinetics of mAb 2 binds to MSLN The results show that the antibody had a blood exposure equivalent to that of a standard human IgG1 antibody without IgG1 (Bergman et al., 1998).

[0437] Sequence Listing Heavy chain annotation i.mAb 2 In the amino acid sequence of the heavy chain of Figure 1, the variable domains are shown in italics, the CDRs according to IMGT are shown in bold italics, and the CDRs according to Kabat are shown in italic underline (hence Overlapping IMGT and Kabat CDR sequences are shown in bold, italics, and underlined), CH1 Domains are underlined, the hinge region is double underlined, the CH2 domain is shown in bold (where applicable, the location of the LALA mutation is shown in bold underline), the CH3 domain is shown in normal font, and the altered region of the CH3 structural loop is underlined (no underline if the loop is unchanged). ii. In the amino acid sequences of the variable domains, CDRs according to IMGT are shown in bold italics, and CDRs according to Kabat are shown in italic and underlined (thus the overlapping IMGT and Kabat CDR sequences are shown in bold, italics, and underlined). iii. CDR amino acid sequences according to both IMGT and Kabat are provided.

[0438] Light chain annotation i.mAb 2 In the amino acid sequence of the light chain of , the variable domain is shown in italics, the CDRs according to IMGT are shown in bold italics, and the CDRs according to Kabat are shown in italic and underlined (thus the overlapping IMGT and Kabat CDR sequences are shown in bold, italics, and underlined). ii. In the amino acid sequences of the variable domains, CDRs according to IMGT are shown in bold italics, and CDRs according to Kabat are shown in italic and underlined (thus the overlapping IMGT and Kabat CDR sequences are shown in bold, italics, and underlined). iii. CDR amino acid sequences according to both IMGT and Kabat are provided. Amino acid and cDNA sequences of the CH3 domain and FS22-172-003 Fcab-containing mAb 2 Amino acid sequences of the modified regions of the CH3 AB and EF structural loops of all clones and FS22-172-003 Fcab

[0439] CH3 SEQ ID NO:8 AA [ka] CH3 SEQ ID NO:9 DNA [ka] SEQ ID NO: 10 Loop AB(AA)PYIIPPY SEQ ID NO: 11 Loop EF(AA)GADRWLE FS22-172-003-AA / FS28-024 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 92 Heavy chain AA (no LALA) [ka] SEQ ID NO: 93 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 94 Heavy chain AA (with LALA) [ka] SEQ ID NO: 95 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 12 VH domain AA [ka] SEQ ID NO: 13 VH domain DNA [ka] SEQ ID NO: 14 HCDR1(AA)(IMGT)GFTLSYSS SEQ ID NO: 15 HCDR1(AA)(Kabat)YSSMS SEQ ID NO: 16 HCDR2(AA)(IMGT)ITPSTGYT SEQ ID NO: 17 HCDR2 (AA) Kabat) FITPSTGYTHYADSVKG SEQ ID NO: 18 HCDR3(AA)(IMGT)ARRALTFDY SEQ ID NO: 19 HCDR3(AA)(Kabat)RALTFDY FS22-172-003-AA / FS28-024 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 85 Light chain AA [ka] SEQ ID NO: 86 Light chain DNA [ka] SEQ ID NO: 54 VL domain AA [ka] SEQ ID NO: 55 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 24 LCDR3(AA)(IMGT)QQASSYPLT SEQ ID NO: 24 LCDR3(AA)(Kabat)QQASSYPLT FS22-172-003-AA / FS28-024-051 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 96 Heavy chain AA (no LALA) [ka] SEQ ID NO: 97 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 98 Heavy chain AA (with LALA) [ka] SEQ ID NO: 99 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 56 VH domain AA [ka] SEQ ID NO: 57 VH domain DNA [ka] SEQ ID NO: 14 HCDR1(AA)(IMGT)GFTLSYSS SEQ ID NO: 15 HCDR1(AA)(Kabat)YSSMS SEQ ID NO: 16 HCDR2(AA)(IMGT)ITPSTGYT SEQ ID NO: 17 HCDR2 (AA) Kabat)FITPSTGYTHYADSVKG SEQ ID NO: 25 HCDR3(AA)(IMGT)ARRALIFDY SEQ ID NO: 26 HCDR3(AA)(Kabat)RALIFDY FS22-172-003-AA / FS28-024-051 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 85 Light chain AA [ka] SEQ ID NO: 86 Light chain DNA [ka] SEQ ID NO: 54 VL domain AA [ka] SEQ ID NO: 55 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 24 LCDR3(AA)(IMGT)QQASSYPLT SEQ ID NO: 24 LCDR3(AA)(Kabat)QQASSYPLT FS22-172-003-AA / FS28-024-052 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 100 Heavy chain AA (no LALA) [ka] SEQ ID NO: 101 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 102 Heavy chain AA (with LALA) [ka] SEQ ID NO: 103 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 58 VH domain AA [ka] SEQ ID NO: 59 VH domain DNA [ka] SEQ ID NO: 14 HCDR1(AA)(IMGT)GFTLSYSS SEQ ID NO: 15 HCDR1(AA)(Kabat)YSSMS SEQ ID NO: 16 HCDR2(AA)(IMGT)ITPSTGYT SEQ ID NO: 17 HCDR2 (AA) Kabat)FITPSTGYTHYADSVKG SEQ ID NO: 27 HCDR3(AA)(IMGT)ARRALLFDY SEQ ID NO: 28 HCDR3(AA)(Kabat)RALLFDY FS22-172-003-AA / FS28-024-052 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 85 Light chain AA [ka] SEQ ID NO: 86 Light chain DNA [ka] SEQ ID NO: 54 VL domain AA [ka] SEQ ID NO: 55 VL DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 24 LCDR3(AA)(IMGT)QQASSYPLT SEQ ID NO: 24 LCDR3(AA)(Kabat)QQASSYPLT FS22-172-003-AA / FS28-024-053 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 104 Heavy chain AA (no LALA) [ka] SEQ ID NO: 105 Heavy chain DNA (No LALA) [ka] SEQ ID NO: 106 Heavy chain AA (with LALA) [ka] SEQ ID NO: 107 Heavy chain DNA (LALA available) [ka] SEQ ID NO: 60 VH domain AA [ka] SEQ ID NO: 61 VH domain DNA [ka] SEQ ID NO: 14 HCDR1(AA)(IMGT)GFTLSYSS SEQ ID NO: 15 HCDR1(AA)(Kabat)YSSMS SEQ ID NO: 16 HCDR2(AA)(IMGT)ITPSTGYT SEQ ID NO: 17 HCDR2 (AA) Kabat)FITPSTGYTHYADSVKG SEQ ID NO: 29 HCDR3(AA)(IMGT)ARRALVFDY SEQ ID NO: 30 HCDR3(AA)(Kabat)RALVFDY FS22-172-003-AA / FS28-024-053 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 85 Light chain AA [ka] SEQ ID NO: 86 Light chain DNA [ka] SEQ ID NO: 54 VL domain AA [ka] SEQ ID NO: 55 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 24 LCDR3(AA)(IMGT)QQASSYPLT SEQ ID NO: 24 LCDR3(AA)(Kabat)QQASSYPLT FS22-172-003-AA / FS28-024-060 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 108 Heavy chain AA (with LALA) [ka] SEQ ID NO: 85 Light chain AA [ka] FS22-172-003-AA / FS28-026 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 109 Heavy chain AA (with LALA) [ka] SEQ ID NO: 87 Light chain AA [ka] FS22-172-003-AA / FS28-091 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 110 Heavy chain AA (with LALA) [ka] SEQ ID NO: 88 Light chain AA [ka] FS22-172-003-AA / FS28-185 mAb 2 Amino acid sequences of the heavy and light chains of SEQ ID NO: 111 Heavy chain AA (with LALA) [ka] SEQ ID NO: 89 Light chain AA [ka] FS22-172-003-AA / FS28-256 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 112 Heavy chain AA (no LALA) [ka] SEQ ID NO: 113 Heavy chain DNA (no lala) [ka] SEQ ID NO: 114 Heavy chain AA (with LALA) [ka] SEQ ID NO: 115 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 62 VH domain AA [ka] SEQ ID NO: 63 VH domain DNA [ka] SEQ ID NO: 31 HCDR1(AA)(IMGT)GFTFTNTY SEQ ID NO: 32 HCDR1(AA)(Kabat)NTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 35 HCDR3(AA)(IMGT)ARYNSYQGGLDY SEQ ID NO: 36 HCDR3(AA)(Kabat)YNSYQGGLDY FS22-172-003-AA / FS28-256 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 116 Light chain AA [ka] SEQ ID NO: 117 Light chain DNA [ka] SEQ ID NO: 64 VL domain AA [ka] SEQ ID NO: 65 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 37 LCDR3(AA)(IMGT)QQSYYYPIT SEQ ID NO: 37 LCDR3(AA)(Kabat)QQSYYYPIT FS22-172-003-AA / FS28-256-001 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 118 Heavy chain AA (no LALA) [ka] SEQ ID NO: 119 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 120 Heavy chain AA (with LALA) [ka] SEQ ID NO: 121 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 66 VH domain AA [ka] SEQ ID NO: 67 VH domain DNA [ka] SEQ ID NO: 38 HCDR1(AA)(IMGT)GFTFTETY SEQ ID NO: 39 HCDR1(AA)(Kabat)ETYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 35 HCDR3(AA)(IMGT)ARYNSYQGGLDY SEQ ID NO: 36 HCDR3(AA)(Kabat)YNSYQGGLDY FS22-172-003-AA / FS28-256-001 mAb 2The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 82 Light chain AA [ka] SEQ ID NO: 122 Light chain DNA [ka] SEQ ID NO: 68 VL domain AA [ka] SEQ ID NO: 69 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 40 LCDR3(AA)(IMGT)QQHNQYPNT SEQ ID NO: 40 LCDR3(AA)(Kabat)QQHNQYPNT FS22-172-003-AA / FS28-256-005 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 118 Heavy chain AA (no LALA) [ka] SEQ ID NO: 119 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 120 Heavy chain AA (with LALA) [ka] SEQ ID NO: 121 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 66 VH domain AA [ka] SEQ ID NO: 67 VH domain DNA [ka] SEQ ID NO: 38 HCDR1(AA)(IMGT)GFTFTETY SEQ ID NO: 39 HCDR1(AA)(Kabat)ETYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 35 HCDR3(AA)(IMGT)ARYNSYQGGLDY SEQ ID NO: 36 HCDR3(AA)(Kabat)YNSYQGGLDY FS22-172-003-AA / FS28-256-005 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 83 Light chain AA [ka] SEQ ID NO: 90 Light chain DNA [ka] SEQ ID NO: 78 VL domain AA [ka] SEQ ID NO: 79 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 41 LCDR3(AA)(IMGT)QQALGYPHT SEQ ID NO: 41 LCDR3(AA)(Kabat)QQALGYPHT FS22-172-003-AA / FS28-256-012 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 123 Heavy chain AA (no LALA) [ka] SEQ ID NO: 124 Heavy chain DNA (no 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: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-012 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 116 Light chain AA [ka] SEQ ID NO: 117 Light chain DNA [ka] SEQ ID NO: 64 VL domain AA [ka] SEQ ID NO: 65 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 37 LCDR3(AA)(IMGT)QQSYYYPIT SEQ ID NO: 37 LCDR3(AA)(Kabat)QQSYYYPIT FS22-172-003-AA / FS28-256-014 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 127 Heavy chain AA (no LALA) [ka] SEQ ID NO: 128 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 129 Heavy chain AA (with LALA) [ka] SEQ ID NO: 130 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 72 VH domain AA [ka] SEQ ID NO: 73 VH domain DNA [ka] SEQ ID NO: 46 HCDR1(AA)(IMGT)GFTFTDTY SEQ ID NO: 47 HCDR1(AA)(Kabat)DTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 48 HCDR3(AA)(IMGT)ARYNAYAAGLDY SEQ ID NO: 49 HCDR3(AA)(Kabat)YNAYAAGLDY FS22-172-003-AA / FS28-256-014 mAb 2The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 116 Light chain AA [ka] SEQ ID NO: 117 Light chain DNA [ka] SEQ ID NO: 64 VL domain AA [ka] SEQ ID NO: 65 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 37 LCDR3(AA)(IMGT)QQSYYYPIT SEQ ID NO: 37 LCDR3(AA)(Kabat)QQSYYYPIT FS22-172-003-AA / FS28-256-018 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 131 Heavy chain AA (no LALA) [ka] SEQ ID NO: 132 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 133 Heavy chain AA (with LALA) [ka] SEQ ID NO: 134 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 74 VH domain AA [ka] SEQ ID NO: 75 VH domain DNA [ka] SEQ ID NO: 50 HCDR1(AA)(IMGT)GFTFTQTY SEQ ID NO: 51 HCDR1(AA)(Kabat)QTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 52 HCDR3(AA)(IMGT)ARYNAYQIGLDY SEQ ID NO: 53 HCDR3(AA)(Kabat)YNAYQIGLDY FS22-172-003-AA / FS28-256-018 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 116 Light chain AA [ka] SEQ ID NO: 117 Light chain DNA [ka] SEQ ID NO: 64 VL domain AA [ka] SEQ ID NO: 65 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 37 LCDR3(AA)(IMGT)QQSYYYPIT SEQ ID NO: 37 LCDR3(AA)(Kabat)QQSYYYPIT FS22-172-003-AA / FS28-256-021 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 123 Heavy chain AA (no LALA) [ka] SEQ ID NO: 124 Heavy chain DNA (no 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: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-021 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 82 Light chain AA [ka] SEQ ID NO: 122 Light chain DNA [ka] SEQ ID NO: 68 VL domain AA [ka] SEQ ID NO: 69 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 40 LCDR3(AA)(IMGT)QQHNQYPNT SEQ ID NO: 40 LCDR3(AA)(Kabat)QQHNQYPNT FS22-172-003-AA / FS28-256-023 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 131 Heavy chain AA (no LALA) [ka] SEQ ID NO: 132 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 133 Heavy chain AA (with LALA) [ka] SEQ ID NO: 134 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 74 VH domain AA [ka] SEQ ID NO: 75 VH domain DNA [ka] [ka] FS22-172-003-AA / FS28-256-023 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 82 Light chain AA [ka] SEQ ID NO: 122 Light chain DNA [ka] SEQ ID NO: 68 VL domain AA [ka] SEQ ID NO: 69 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 40 LCDR3(AA)(IMGT)QQHNQYPNT SEQ ID NO: 40 LCDR3(AA)(Kabat)QQHNQYPNT FS22-172-003-AA / FS28-256-024 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 123 Heavy chain AA (no LALA) [ka] SEQ ID NO: 124 Heavy chain DNA (no 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: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-024 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 83 Light chain AA [ka] SEQ ID NO: 90 Light chain DNA [ka] SEQ ID NO: 78 VL domain AA [ka] SEQ ID NO: 79 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 41 LCDR3(AA)(IMGT)QQALGYPHT SEQ ID NO: 41 LCDR3(AA)(Kabat)QQALGYPHT FS22-172-003-AA / FS28-256-026 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 131 Heavy chain AA (no LALA) [ka] SEQ ID NO: 132 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 133 Heavy chain AA (with LALA) [ka] SEQ ID NO: 134 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 74 VH domain AA [ka] SEQ ID NO: 75 VH domain DNA [ka] SEQ ID NO: 50 HCDR1(AA)(IMGT)GFTFTQTY SEQ ID NO: 51 HCDR1(AA)(Kabat)QTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 52 HCDR3(AA)(IMGT)ARYNAYQIGLDY SEQ ID NO: 53 HCDR3(AA)(Kabat)YNAYQIGLDY FS22-172-003-AA / FS28-256-026 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 83 Light chain AA [ka] SEQ ID NO: 90 Light chain DNA [ka] SEQ ID NO: 78 VL domain AA [ka] SEQ ID NO: 79 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 41 LCDR3(AA)(IMGT)QQALGYPHT SEQ ID NO: 41 LCDR3(AA)(Kabat)QQALGYPHT FS22-172-003-AA / FS28-256-027 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 123 Heavy chain AA (no LALA) [ka] SEQ ID NO: 124 Heavy chain DNA (no 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: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 34 HCDR2(AA)Kabat)NISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-027 mAb 2 The amino acid sequence and cDNA sequence of the light chain of SEQ ID NO: 84 Light chain AA [ka] SEQ ID NO: 91 Light chain DNA [ka] SEQ ID NO: 76 VL domain AA [ka] SEQ ID NO: 77 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 80 LCDR3(AA)(IMGT)QQTVPYPYT SEQ ID NO: 80 LCDR3(AA)(Kabat)QQTVPYPYT Mouse mAb and mAb 2 Amino acid sequence of the heavy chain of FS28m-228 mAb SEQ ID NO: 135 Heavy chain AA (with LALA) [ka] Amino acid sequence of the light chain of FS28m-228 mAb SEQ ID NO: 136 Light chain AA [ka] FS22m-063-AA / FS28m-228 mAb 2 The amino acid sequence of the heavy chain of SEQ ID NO: 137 Heavy chain AA (with LALA) [ka] FS22m-063-AA / FS28m-228 mAb 2The amino acid sequence of the light chain of SEQ ID NO: 136 Light chain AA [ka] Amino acid sequence of the heavy chain of G1AA / HelD1.3 mAb SEQ ID NO: 138 Heavy chain AA (with LALA) [ka] Amino acid sequence of the light chain of G1AA / HelD1.3 mAb SEQ ID NO: 139 Light chain AA [ka] G1AA / SS1 mAb SEQ ID NO: 140 Heavy chain (with LALA) [ka] SEQ ID NO: 141 Light chain [ka] MSLN-His-Avi Mesothelin (without MPF and C-terminus) (shown); His and Avi tags (not shown) SEQ ID NO: 142 Human [ka] SEQ ID NO: 143 Cynomolgus monkey [ka] SEQ ID NO: 144 Mouse [ka] CD137-mFc-Avi and CD137-Avi-His (Extracellular domain CD137 (shown); mFc, Avi-tag, His-tag (not shown) SEQ ID NO: 146 Human [ka] SEQ ID NO: 147 Cynomolgus monkey [ka] SEQ ID NO: 148 Mouse [ka] Cell-expressed antigen (CD137) (Extracellular domain (italic); transmembrane and intracellular domain (bold)) SEQ ID NO: 149 Human [ka] SEQ ID NO: 150 Mouse [ka] SEQ ID NO: 153 Cynomolgus monkey [ka] Overexpressing cell lines - membrane-bound mature form of mesothelin (shown in bold italics) [NBMMPF and the propeptide are shown in normal font before and after the mesothelin sequence. Neither is present in the membrane-bound mature form of mesothelin.] Human MPF+MSLN SEQ ID NO: 151 [ka] Mouse MPF+MSLN SEQ ID NO: 145 [ka] Cynomolgus monkey MPF+MSLN SEQ ID NO: 152 [ka] Amino acid sequence of wild-type CH2 domain SEQ ID NO: 154 CH2 (WT) [ka] Amino acid sequence of the CH2 domain containing the LALA mutation (the LALA mutation is bold and underlined) SEQ ID NO: 155 CH2 (LALA) [ka] Amino acid sequence of the CH2 domain containing the LALA-PA mutation (the LALA-PA mutation is bold and underlined) SEQ ID NO: 156 CH2 (LALA-PA) [ka] Amino acid and cDNA sequences of the light chains of FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-256-272, and FS22-172-003-AA / FS28-256-273 mAb2 SEQ ID NO: 84 Light chain AA [ka] SEQ ID NO: 91 Light chain DNA [ka] SEQ ID NO: 76 VL domain AA [ka] SEQ ID NO: 77 VL domain DNA [ka] SEQ ID NO: 20 LCDR1(AA)(IMGT)QSVSSSY SEQ ID NO: 21 LCDR1(AA)(Kabat)RASQSVSSSYLA SEQ ID NO: 22 LCDR2(AA)(IMGT)GAS SEQ ID NO: 23 LCDR2(AA)(Kabat)GASSRAT SEQ ID NO: 80 LCDR3(AA)(IMGT)QQTVPYPYT SEQ ID NO: 80 LCDR3(AA)(Kabat)QQTVPYPYT FS22-172-003-AA / FS28-256-271 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 1 Heavy chain AA (no LALA) [ka] SEQ ID NO: 2 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 3 Heavy chain AA (with LALA) [ka] SEQ ID NO: 4 Heavy chain DNA (with LALA) [ka] Sequence number 177 VH domain AA [ka] Sequence number 178 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 5 HCDR2(AA)Kabat)AISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-272 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 6 Heavy chain AA (no LALA) [ka] SEQ ID NO: 7 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 158 Heavy chain AA (with LALA) [ka] SEQ ID NO: 159 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 160 HCDR2 (AA) Kabat)HISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22-172-003-AA / FS28-256-273 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 161 Heavy chain AA (no LALA) [ka] SEQ ID NO: 162 Heavy chain DNA (no LALA) [ka] SEQ ID NO: 163 Heavy chain AA (with LALA) [ka] SEQ ID NO: 164 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 70 VH domain AA [ka] SEQ ID NO: 71 VH domain DNA [ka] SEQ ID NO: 42 HCDR1(AA)(IMGT)GFTFTHTY SEQ ID NO: 43 HCDR1(AA)(Kabat)HTYMS SEQ ID NO: 33 HCDR2(AA)(IMGT)ISPTYSTT SEQ ID NO: 165 HCDR2(AA)Kabat)SISPTYSTTNYADSVKG SEQ ID NO: 44 HCDR3(AA)(IMGT)ARYNAYHAALDY SEQ ID NO: 45 HCDR3(AA)(Kabat)YNAYHAALDY FS22m-063-AA / FS28m-228-010 Amino acid sequence of the heavy chain of mAb2 SEQ ID NO: 166 Heavy chain AA (with LALA) [ka] FS22m-063-AA / FS28m-228-010 Amino acid sequence of the light chain of mAb2 SEQ ID NO: 136 Light chain AA [ka] Amino acid sequences of the heavy and light chains of FS22m-063-AA / HelD1.3 mAb2 SEQ ID NO: 167 Heavy chain AA (with LALA) [ka] SEQ ID NO: 168 Light chain AA [ka] FS22-172-003-AA / FS28-185-002 mAb 2 Amino acid sequences and cDNA sequences of the heavy and light chains of SEQ ID NO: 169 Heavy chain AA (with LALA) [ka] SEQ ID NO: 170 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 171 Light chain AA [ka] SEQ ID NO: 172 Light chain DNA [ka] FS22-172-003-AA / FS28-185-003 mAb 2 Amino acid sequence and cDNA sequence of the heavy chain of SEQ ID NO: 173 Heavy chain AA (with LALA) [ka] SEQ ID NO: 174 Heavy chain DNA (with LALA) [ka] SEQ ID NO: 175 Light chain AA [ka] SEQ ID NO: 176 Light chain DNA [ka] Amino acid sequence of WT Fcab CH3 domain (SEQ ID NO: 81) The AB, CD, and EF loops are underlined. [ka] WT CD loop sequence SEQ ID NO: 157 WT Fcab CD Loop - SNGQPENNY

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Claims

1. An antibody molecule that binds to mesothelin (MSLN) and CD137, (a) a complementarity-determining region (CDR)-based antigen-binding site of MSLN; and (b) a CD137 antigen-binding site located in the CH3 domain of the antibody molecule; Including, the CDR-based antigen-binding site comprises: (i) SEQ ID NOs: 43, 5, 45, 21, 23, and 80 [FS28-256-271], respectively; (ii) SEQ ID NOs: 15, 17, 28, 21, 23, and 24 [FS28-024-052], respectively; (iii) SEQ ID NOs: 43, 34, 45, 21, 23, and 40, respectively [FS28-256-021]; (iv) SEQ ID NOs: 43, 34, 45, 21, 23, and 37, respectively [FS28-256-012]; (v) SEQ ID NOs: 51, 34, 53, 21, 23, and 40, respectively [FS28-256-023]; (vi) SEQ ID NOs: 43, 34, 45, 21, 23 and 41 [FS28-256-024], respectively; (vii) SEQ ID NOs: 51, 34, 53, 21, 23, and 41 [FS28-256-026], respectively; (viii) SEQ ID NOs: 43, 34, 45, 21, 23, and 80, respectively [FS28-256-027]; (ix) SEQ ID NOs: 39, 34, 36, 21, 23, and 40 [FS28-256-001], respectively; (x) SEQ ID NOs: 39, 34, 36, 21, 23, and 41 [FS28-256-005], respectively; (xi) SEQ ID NOs: 47, 34, 49, 21, 23, and 37 [FS28-256-014], respectively; (xii) SEQ ID NOs: 51, 34, 53, 21, 23, and 37, respectively [FS28-256-018]; (xiii) SEQ ID NOs: 32, 34, 36, 21, 23, and 37 [FS28-256], respectively; (xiv) SEQ ID NOs: 15, 17, 26, 21, 23, and 24, respectively [FS28-024-051]; (xv) SEQ ID NOs: 15, 17, 30, 21, 23, and 24, respectively [FS28-024-053]; or (xvi) SEQ ID NOs: 15, 17, 19, 21, 23 and 24 [FS28-024], respectively. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 as set forth in the CDR sequences are defined according to Kabat; the CD137 antigen-binding site comprises a first sequence and a second sequence located in the AB and EF structural loops of the CH3 domain, respectively, and the first and second sequences have the sequences set forth in SEQ ID NOs: 10 and 11 [FS22-172-003], respectively; the first sequence is located between positions 14 and 17 of the CH3 domain, and the second sequence is located between positions 91 and 99 of the CH3 domain; The amino acid residue positions of the CH3 domain are numbered according to the ImMunoGeneTics (IMGT) numbering scheme. An antibody molecule that binds to MSLN and CD137.

2. The antibody molecule of claim 1, wherein the antibody molecule comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 [FS28-256-271] set forth in SEQ ID NOs: 43, 5, 45, 21, 23, and 80, respectively.

3. The antibody molecule of claim 1, wherein the antibody molecule comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 [FS28-024-052] set forth in SEQ ID NOs: 15, 17, 28, 21, 23, and 24, respectively.

4. The antibody molecule of claim 1 , wherein the antibody molecule comprises a CH3 domain sequence [FS22-172-003] set forth in SEQ ID NO:

8.

5. The antibody molecule of claim 4, wherein the CH3 domain sequence comprises an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

6. The antibody molecule of claim 1 , wherein the antibody molecule does not bind to an Fcγ receptor.

7. The antibody molecule of claim 1 , wherein the antibody molecule binds to immobilized MSLN with higher affinity than soluble MSLN.

8. The antibody molecule of claim 1 , wherein the antibody molecule is capable of activating CD137 on immune cells in the presence of MSLN bound to the surface of tumor cells.

9. 9. The antibody molecule of claim 1, wherein binding of the antibody molecule to CD137 on immune cells and to MSLN bound to the surface of tumor cells causes clustering of CD137 on the immune cells.

10. 10. One or more nucleic acid molecules encoding the antibody molecule of any one of claims 1 to 9.

11. 11. One or more vectors comprising one or more nucleic acid molecules of claim 10.

12. 12. A recombinant host cell comprising one or more nucleic acid molecules according to claim 10 or one or more vectors according to claim 11.

13. 13. A method for producing an antibody molecule according to any one of claims 1 to 9, comprising culturing the recombinant host cell of claim 12 under conditions for the production of said antibody molecule.

14. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

15. A pharmaceutical composition for the treatment of cancer in an individual, comprising an antibody molecule according to any one of claims 1 to 9.

16. 16. The pharmaceutical composition of claim 15, further comprising a second therapeutic agent.

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