Cancer therapy comprising anti-MSLN / CD137 antibody and chemotherapeutic agent

Abstract

The present application relates to the use of antibody molecules that bind to MSLN and CD137 and chemotherapeutic agents for the treatment of cancer in a patient.

Description

Technical Field

[0001] The present invention relates to the use of bispecific antibody molecules that bind to MSLN and CD137 and chemotherapeutic agents for treating cancer in a patient. Background Art

[0002] Co-stimulatory pathways, such as the CD137 / 4-1BB pathway, are critical in driving effective anti-cancer immunity, and strong genetic evidence supports their role in mediating anti-cancer immune responses 1-5 Therefore, a growing number of studies are aimed at modifying the signaling to enhance antitumor T-cell responses by using agonistic antibodies targeting co-stimulatory molecules.

[0003] CD137 (also known as 4-1BB or TNFRSF9) is an inducible T-cell surface receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily that activates multiple cellular functions, including the production of type 1 interferons and the regulation of antigen-activated T-cell survival. 6 CD137 in activated CD4 + and CD8 + CD137 is expressed on the surface of T cells, monocytes and B lymphocytes. CD137 expression can be induced by T cell receptor (TCR) stimulation. 7 , which is called "signal 1" (TCR / CD3 / MHC interaction between human T cells and target cells). Activation of the CD137 pathway promotes T cell differentiation and survival 8-10 , providing strong protection against activation-induced T cell death and increasing cytotoxicity 11-13 .

[0004] The efficacy of anti-CD137 therapy has been demonstrated in multiple nonclinical tumor models 14-18 Agonistic antibodies against CD137 have been shown to induce + T cells release effector molecules, increase proliferation, and prevent cytotoxic T lymphocyte (CTL) anergy, thereby breaking T cell tolerance to tumor antigens 19 and increase the persistence of tumor-specific T cells 20 Based on promising nonclinical antitumor effects, two first-generation CD137 agonists, utomilumab (PF-05082566) and urelumab (BMS-663513), have been developed and are undergoing clinical studies. However, due to the low efficacy of utomilumab and the hepatotoxicity of urelumab, 21,22 , clinical studies of both urerulumab and urerulumab monotherapy were suspended. Further structural analysis showed that these results were mediated by known epitopes on CD137 and Fcγ receptor (FcγR) ligand-dependent aggregation.23 .

[0005] To overcome the low antitumor efficacy or hepatotoxicity mediated by FcγR ligand-dependent aggregation of first-generation CD137 agonists, strategies to deliver CD137 agonists to tumor sites are needed that reduce systemic toxicity while allowing clinical administration. 24 These second-generation CD137 agonists are either monospecific antibodies that purportedly bind to CD137 epitopes not associated with hepatotoxicity, or CD137 / tumor-associated antigen (TAA) bispecific antibodies that target the tumor microenvironment (TME), do not bind FcγRs, and are linked to antibodies that target tumor antigens or tumor tissue. 15,25,26 .

[0006] Mesothelin (MSLN) is a 40 kilodalton (kD) membrane-bound protein that is overexpressed in various cancers, including mesothelioma, ovarian cancer, lung cancer, and pancreatic cancer. 27-38 The limited expression of MSLN in normal human tissues and its high expression in many common cancers make it an attractive candidate for cancer therapy. Several agents are in various stages of development for the treatment of patients with tumors expressing MSLN, including monoclonal antibodies, immunotoxins, tumor vaccines, and antibody-drug conjugates. 39 .

[0007] M9657 (FS22-172-003-AA / FS28-256-271 of WO 2020 / 011976) is a first-in-class tumor-targeting conditional agonist antibody developed to enhance anti-tumor immune responses in the TME. The bispecific antibody M9657 is engineered as a tetravalent bispecific antibody (mAb 2 ) format, in which the Fab portion binds to the tumor antigen MSLN, while the modified CH3 domain binds to CD137 as the Fc antigen binding (Fcab) portion. M9657 has a human IgG1-LALA main chain that blocks binding to Fcγ receptors but retains FcRn binding for IgG-like pharmacokinetics (PK). High expression of MSLN on tumor cells should lead to increased binding and cross-linking of antibody molecules and interactions with CD137 trimers, thereby increasing CD137 excitation. Therefore, aggregated M9657 can act as a bridge connecting CD137 trimers and tumor cells. Since M9657 promotes CD137 activation signaling within the TME, which avoids systemic immune activation, it is expected that M9657 will have advantages over monospecific CD137 antibodies. In preclinical studies, M9657 exhibited MSLN target-dependent and dose-dependent anti-tumor immunity.

[0008] Nonetheless, there remains a need in the art for additional anti-cancer therapies. Summary of the invention

[0009] There is increasing evidence that chemotherapeutic drugs can induce the immunogenic death of tumor cells, which releases or exposes these immunogenic tumor antigens, making them available for interaction with innate immune cells such as monocytes, macrophages, and dendritic cells (DCs). 40,41 This leads to the activation and maturation of these immune cells, which migrate to draining lymph nodes loaded with cancer-derived antigen-specific cargo. The cancer antigens are then presented to T cells, which enable a powerful anti-cancer adaptive immune response. Conventional chemotherapeutic agents induce immunogenic cell death by directly interfering with DNA or targeting key proteins required for cell division. 42 Immunogenically dying tumor cells can release tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs), both of which actively recruit immune cells in the TME. 43 Some chemotherapeutic agents have been reported to deplete myeloid-derived suppressor cells (MDSCs), cancer-associated neutrophils, and macrophages. 44,45 Optimal doses of certain chemotherapeutic agents promote effector T cell proliferation and Treg exhaustion 46 The optimal combination of immunotherapy with standard of care (SOC) chemotherapy is actively being investigated in both nonclinical and clinical trials to achieve additive or synergistic clinical activity.

[0010] As mentioned above, chemotherapy may promote the use of immunotherapy in cancer treatment. However, immune cell activation will not be limited to the TME where an increased cancer antigen load occurs, raising concerns about its efficacy and specificity. In particular, CD137 agonist molecules have been hampered in the past by concerns about liver inflammation and clinical efficacy.

[0011] The inventors recognized that target-specific anti-tumor activity can be enhanced by combining MSLN expression-dependent CD137 co-stimulation of T cells with chemotherapy. Unexpectedly, the inventors were able to demonstrate that the combination of an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent produced an in vivo anti-tumor effect in a mouse tumor model that was greater than the sum of the increased anti-tumor effects observed when mice were treated with antibody molecules that bind to MSLN and CD137 or chemotherapeutic agents alone. In other words, the anti-tumor effect of the combination therapy is not only additive, but also synergistic. This is unexpected. If the effect achieved by the combination of two agents is greater than the sum of the individual effects of the two agents, the effect is synergistic 47 Thus, the present inventors have found that the combination of an antibody molecule that binds to MLSN and CD137 and a chemotherapeutic agent synergistically enhances the in vivo anti-tumor effect in a mouse tumor model. Similar synergistic anti-tumor effects are expected when human patients are treated with a combination of an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent.

[0012] Due to the lack of cross-reactivity between M9657 (SEQ ID NO: 2 and SEQ ID NO: 10) and mouse MSLN and CD137 proteins, anti-mMSLN-mCD137-huIgG1-LALA (FS122m) (SEQ ID NO: 84 and SEQ ID NO: 85) was developed as an alternative antibody to M9657 for in vivo studies in mouse tumor models. Like M9657, FS122m was engineered as a tetravalent bispecific antibody (mAb 2 ) format, in which the Fab portion targets the tumor antigen mouse MSLN, and the modified CH3 domain as the Fcab portion targets mouse CD137. FS122m has a human IgG1 backbone with a LALA mutation to eliminate binding to Fcγ receptors. The binding affinity of FS122m to mouse MSLN / CD137 is similar to that of M9657 to human MSLN / CD137.

[0013] As outlined above, the inventors have demonstrated that the combination of FS122m and either of the two chemotherapeutic agents cisplatin or gemcitabine can slow down tumor growth in ST26 and JC mouse tumor models or reduce tumor volume therein to a greater extent than the sum of the slowed tumor growth or reduced tumor volume observed when mice were treated with FS122m or cisplatin or gemcitabine alone. The inventors have also demonstrated that the combined treatment of FS122m and cisplatin or gemcitabine increased the median survival and the percentage of mice with complete tumor regression in the same mouse tumor model compared to the sum of the median survival and the percentage of mice with complete tumor regression observed when mice were treated with FS122m or cisplatin or gemcitabine alone. Thus, the inventors demonstrated that the combination of FS122m with cisplatin or gemcitabine synergistically enhanced antitumor activity in both ST26 and JC mouse tumor models as measured by tumor growth reduction / tumor volume reduction, median survival, and percentage of mice showing complete tumor regression.

[0014] These nonclinical studies conducted in mouse tumor models support the expectation that the combination of antibody molecules that bind to MSLN and CD137 with chemotherapeutic agents enhances anti-tumor activity and acts in a synergistic manner in human patients. These findings suggest that combination therapy with antibody molecules that bind to MSLN and CD137 with chemotherapeutic agents is a new therapeutic strategy to improve cancer treatment.

[0015] Thus, the present invention provides an antibody molecule that binds to MSLN and CD137 for use in a method of treating cancer in a patient, wherein the method comprises administering the antibody in combination with a chemotherapeutic agent. The present invention also provides a chemotherapeutic agent for use in a method of treating cancer in a patient, wherein the method comprises administering a chemotherapeutic agent in combination with an antibody molecule that binds to MSLN and CD137.

[0016] The antibody molecule that binds to MSLN and CD137 can be an immunoglobulin or an antigen-binding fragment thereof. For example, the antibody molecule can be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule, more preferably an IgG1 or IgG2 molecule, most preferably an IgG1 molecule, or a fragment thereof, and in a preferred embodiment, the antibody molecule is a complete immunoglobulin molecule.

[0017] The antibody molecule may include at least one, preferably more than one, MSLN binding site based on the complementarity determining region (CDR), and at least one, preferably more than one, CD137 binding site in the constant domain of the bispecific antibody molecule, preferably in the CH3 domain.

[0018] The CD137 binding site may include a first sequence and a second sequence located in the AB and EF structural loops of the CH3 domain of the antibody molecule. Preferably, the first sequence has the sequence shown in SEQ ID NO:87. Preferably, the second sequence has the sequence shown in SEQ ID NO:88. More preferably, the first sequence has the sequence shown in SEQ ID NO:87, and the second sequence has the sequence shown in SEQ ID NO:88. According to the IMGT numbering scheme, the first sequence may be located between the 14th and 17th positions of the CH3 domain of the antibody molecule. According to the IMGT numbering scheme, the second sequence may be located between the 91st and 99th positions of the CH3 domain of the antibody molecule. Preferably, the sequence of the CH3 domain of the antibody molecule has the sequence shown in SEQ ID NO:86.

[0019] In a preferred embodiment, the bispecific antibody molecule comprises a CH3 domain comprising, having or consisting of the CH3 domain sequence of FS22-172-003 shown in SEQ ID NO: 86. The CH3 domain of the bispecific antibody molecule may optionally comprise an additional lysine residue (K) immediately at the C-terminus of the CH3 domain sequence.

[0020] A number of Fabs that bind to MSLN are known from WO 2020 / 011976. A MSLN binding site based on the complementarity determining regions (CDRs) may comprise CDRs 1-6 of any of these Fabs. Thus, an antibody molecule that binds to MSLN and CD137 may comprise CDRs 1-6 as shown in: SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-271]; SEQ ID NOs 20, 22, 24, 12, 14 and 28 [FS28-024-052]; SEQ ID NOs 4, 6, 8, 12, 14 and 34 [FS28-256-021]; SEQ ID NOs 4, 6, 8, 12, 14 and 39 [FS28-256-012]; SEQ ID NOs 43, 6, 45, 12, 14 and 34 [FS28-256-023]; SEQ ID NOs 4, 6, 8, 12, 14 and 49 [FS28-256-024]; SEQ ID NOs 43, 6, 45, 12, 14 and 49 [FS28-256-026]; SEQ ID NO 4, 6, 8, 12, 14 and 16 [FS28-256-027]; SEQ ID NO 53, 6, 55, 12, 14 and 34 [FS28-256-001]; SEQ ID NO 53, 6, 55, 12, 14 and 49 [FS28-256-005]; SEQ ID NO 60, 6, 62, 12, 14 and 39 [FS28-256-014]; SEQ ID NO 43, 6, 45, 12, 14 and 39 [FS28-256-018]; SEQ ID NO 67, 6, 55, 12, 14 and 39 [FS28-256]; SEQ ID NO 21, 23, 72, 12, 14 and 28 [FS28-024-051]; SEQ ID NOs 21, 23, 77, 12, 14 and 28 [FS28-024-053]; or SEQ ID NOs 21, 23, 82, 12, 14 and 28 [FS28-024].

[0021] Many bispecific antibody molecules that bind to MSLN and CD137 are known from WO 2020 / 011976. The antibody M9657 of the present application is the same as the antibody FS22-172-003-AA / FS28-256-271 of WO 2020 / 011976. Any of these antibodies can be used and are hereby incorporated by reference. Thus, the antibody molecule that binds to MSLN and CD137 may comprise the heavy and light chains shown in the following, respectively: SEQ ID NOs 2 and 10 (FS22-172-003-AA / FS28-256-271), SEQ ID NOs 18 and 26 (FS22-172-003-AA / FS28-024-052), SEQ ID NOs 30 and 32 (FS22-172-003-AA / FS28-256-021), SEQ ID NOs 36 and 37 (FS22-172-003-AA / FS28-256-012), SEQ ID NOs 41 and 32 (FS22-172-003-AA / FS28-256-023), SEQ ID NOs 30 and 47 (FS22-172-003-AA / FS28-256-024), SEQ ID NO 41 and 47 (FS22-172-003-AA / FS28-256-026), SEQ ID NO 30 and 10 (FS22-172-003-AA / FS28-256-027), SEQ ID NO 51 and 32 (FS22-172-003-AA / FS28-256-001), SEQ ID NO 51 and 47 (FS22-172-003-AA / FS28-256-005), SEQ ID NO 58 and 37 (FS22-172-003-AA / FS28-256-014), SEQ ID NO 41 and 37 (FS22-172-003-AA / FS28-256-018), SEQ ID NO 65 and 37 (FS22-172-003-AA / FS28-256), SEQ ID NO 70 and 26 (FS22-172-003-AA / FS28-024-051), SEQ ID NO 75 and 26 (FS22-172-003-AA / FS28-024-053), or SEQ ID NO 80 and 26 (FS22-172-003-AA / FS28-024). Preferably, the antibody molecule that binds to MSLN and CD137 comprises the heavy chain sequence shown in SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 10 (FS22-172-003-AA / FS28-256-271).

[0022] The chemotherapeutic agent can be an alkylating agent or antimetabolite. Preferably, the antimetabolite is selected from azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine (cladribine), clofarabine (clofarabine), cytarabine (cytarabine) (Ara-C), decitabine (decitabine), floxuridine, fludarabine (fludarabine), gemcitabine (Gemzar), hydroxyurea, methotrexate (methotrexate), nelarabine (nelarabine), pemetrexed (pemetrexed) (Alimta), pentostatin (pentostatin), pralatrexate (pralatrexate), thioguanine and trifluridine / tipiracil (tipiracil) combination. More preferably, the antimetabolite can be gemcitabine.

[0023] The alkylating agent is selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (CPA), dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa and trabectedin. More preferably, the alkylating agent can be cisplatin.

[0024] The membrane-bound protein mesothelin (MSLN) has been shown to be expressed in several cancers. All ovarian cancers, pancreatic adenocarcinomas, mesotheliomas, and non-small cell lung cancers have been shown to express high levels of MSLN. The inventors have found that this is also true for cervical cancer. Without being bound by theory, it is believed that the binding of the antibody molecule to MSLN is expected to result in antibody cross-linking, binding to CD137 expressed on the surface of immune cells, followed by aggregation and activation of CD137, ultimately leading to activation of immune cells.

[0025] Therefore, the cancer to be treated is preferably a cancer that expresses or has been determined to express MSLN. More preferably, the cancer is selected from ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer and non-small cell lung cancer.

[0026] Combination therapy of FS122m with cisplatin or gemcitabine produced statistically significantly greater antitumor activity in the CT26 and JC mouse tumor models than the sum of the antitumor activity observed when mice were treated with FS122m or cisplatin or gemcitabine alone. An effect is synergistic if the effect achieved by the combination of two agents is greater than the sum of the individual effects of the two agents combined. 47 . Therefore, the combination therapy of FS122m with cisplatin or gemcitabine enhances anti-tumor activity in CT26 and JC mouse tumor models in a synergistic manner. Therefore, in one embodiment, treatment with an antibody molecule that binds MSLN and CD137 in combination with a chemotherapeutic agent produces an anti-tumor effect greater than the anti-tumor effect observed when the patient is treated with an antibody molecule or chemotherapeutic agent that binds MSLN and CD137 alone. Preferably, treatment with an antibody molecule that binds MSLN and CD137 in combination with a chemotherapeutic agent produces an anti-tumor effect greater than the sum of the anti-tumor effects observed when the patient is treated with an antibody molecule or chemotherapeutic agent that binds MSLN and CD137 alone. The anti-tumor effect may be an inhibition or slowing of tumor growth, a reduction in tumor volume, an increase in median survival, or an increase in the percentage of patients who experience complete tumor regression. Preferably, the anti-tumor effect is an inhibition or delay of tumor growth or a reduction in tumor volume. Therefore, the anti-tumor effect may be an inhibition or delay of tumor growth. The anti-tumor effect may be a reduction in tumor volume. The anti-tumor effect may be an increase in the median survival of patients. The anti-tumor effect can be an increase in the percentage of patients who experience complete regression of the tumor, such as a clinical complete response or a pathological complete response. The determination of these anti-tumor effects is within the capabilities of the skilled artisan.

[0027] The bispecific antibody molecules and chemotherapeutic agents that bind to MSLN and CD137 can be administered to a subject by any suitable means. Therefore, in one embodiment, the antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 are administered parenterally. The antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally. The antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 can be administered by injection or infusion.

[0028] The antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 can be administered parenterally. The antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 can be administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0029] The antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent can be part of the same preparation or part of a separate preparation, but is preferably provided as a separate preparation. Therefore, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent can be administered to the patient simultaneously or sequentially, but is preferably administered sequentially.

[0030] When the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient sequentially, they are preferably administered to the patient within 4 days of each other, more preferably within 3 days of each other, more preferably within 2 days of each other, or sequentially administered to the patient on the same day.

[0031] The present invention also provides a method for treating cancer, comprising administering an antibody molecule that binds MSLN and CD137 and a chemotherapeutic agent to an individual in need. Preferably, the method for treating cancer comprises administering a therapeutically effective amount of an antibody molecule that binds MSLN and CD137 and a therapeutically effective amount of a chemotherapeutic agent to an individual in need. In one embodiment, the method may include determining whether the patient's cancer expresses MSLN, and if it is determined that the cancer expresses MSLN, then treating the patient. Alternatively, the method may include sorting the test results that determine whether the patient's cancer expresses MSLN, and if the test results show that the cancer expresses MSLN, then treating the patient.

[0032] The present invention also provides the use of an antibody molecule that binds to MSLN and CD137 in the manufacture of a medicament for treating cancer, wherein the antibody molecule that binds to MSLN and CD137 is administered in combination with a chemotherapeutic agent. The present invention also provides the use of a chemotherapeutic agent in the manufacture of a medicament for treating cancer, wherein the chemotherapeutic agent is administered in combination with an antibody molecule that binds to MSLN and CD137.

[0033] The present invention also provides a kit, which comprises an antibody molecule binding to MSLN and CD137 and a pharmaceutically acceptable excipient, as well as a chemotherapeutic agent and a pharmaceutically acceptable excipient.

[0034] The present invention therefore provides:

[0035] [1] An antibody molecule that binds to MSLN and CD137, for use in a method of treating cancer in a patient, wherein the method comprises administering the antibody in combination with a chemotherapeutic agent.

[0036] [2] A chemotherapeutic agent for use in a method of treating cancer in a patient, wherein the method comprises administering the chemotherapeutic agent in combination with an antibody molecule that binds to MSLN and CD137.

[0037] [3] A method for treating cancer in an individual, the method comprising administering to the individual an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent.

[0038] [4] Use of an antibody molecule that binds to MSLN and CD137 in the manufacture of a medicament for treating cancer, wherein the antibody molecule that binds to MSLN and CD137 is administered in combination with a chemotherapeutic agent.

[0039] [5] Use of a chemotherapeutic agent in the preparation of a medicament for treating cancer, wherein the chemotherapeutic agent is administered in combination with an antibody molecule that binds to MSLN and CD137

[0040] [6] A kit comprising

[0041] (a) an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient; and

[0042] (b) a chemotherapeutic agent and a pharmaceutically acceptable excipient.

[0043] [7] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to [6], wherein the antibody molecule that binds to MSLN and CD137 comprises:

[0044] (a) MSLN antigen binding site based on complementarity determining regions (CDRs); and

[0045] (b) The CD137 antigen binding site located in the CH3 domain of the antibody molecule.

[0046] [8] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [7], wherein the antibody molecule that binds to MSLN and CD137 comprises two or more of the following

[0047] (a) MSLN antigen binding site based on complementarity determining regions (CDRs); and

[0048] (b) The CD137 antigen binding site located in the CH3 domain of the antibody molecule.

[0049] [9] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to [8], wherein the antibody molecule that binds to MSLN and CD137 is an IgG, IgA, IgE, IgM molecule or an antigen-binding fragment thereof.

[0050]

[10] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [9], wherein the antibody molecule that binds to MSLN and CD137 is an IgG molecule or an antigen-binding fragment thereof.

[0051]

[11] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[10] , wherein the antibody molecule that binds to MSLN and CD137 is an IgG1 or IgG2 molecule, or an antigen-binding fragment thereof.

[0052]

[12] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[11] , wherein the antibody molecule that binds to MSLN and CD137 is an IgG1 molecule or an antigen-binding fragment thereof.

[0053]

[13] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [8] to

[12] , wherein the CDR-based MSLN antigen binding site comprises CDRs 1-6 shown below:

[0054] (i) SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-271], respectively;

[0055] (ii) SEQ ID NOs 20, 22, 24, 12, 14 and 28, respectively [FS28-024-052];

[0056] (iii) SEQ ID NOs 4, 6, 8, 12, 14 and 34 [FS28-256-021], respectively;

[0057] (iv) SEQ ID NOs 4, 6, 8, 12, 14 and 39 [FS28-256-012], respectively;

[0058] (v) SEQ ID NOs 43, 6, 45, 12, 14 and 34 [FS28-256-023], respectively;

[0059] (vi) SEQ ID NOs 4, 6, 8, 12, 14 and 49 [FS28-256-024], respectively;

[0060] (vii) SEQ ID NOs 43, 6, 45, 12, 14 and 49, respectively [FS28-256-026]; (viii) SEQ ID NOs 4, 6, 8, 12, 14 and 16, respectively [FS28-256-027];

[0061] (ix) SEQ ID NOs 53, 6, 55, 12, 14 and 34, respectively [FS28-256-001]; (x) SEQ ID NOs 53, 6, 55, 12, 14 and 49, respectively [FS28-256-005];

[0062] (xi) SEQ ID NOs 60, 6, 62, 12, 14 and 39, respectively [FS28-256-014]; (xii) SEQ ID NOs 43, 6, 45, 12, 14 and 39, respectively [FS28-256-018]; (xiii) SEQ ID NOs 67, 6, 55, 12, 14 and 39, respectively [FS28-256];

[0063] (xiv) SEQ ID NOs 21, 23, 72, 12, 14 and 28, respectively [FS28-024-051]; (xv) SEQ ID NOs 21, 23, 77, 12, 14 and 28, respectively [FS28-024-053]; or

[0064] (xvi) SEQ ID NOs 21, 23, 82, 12, 14 and 28, respectively [FS28-024]; and

[0065] The CD137 antigen binding site includes a first sequence and a second sequence located in the AB and EF structural loops of the CH3 domain, respectively, wherein the first sequence and the second sequence have the sequences shown in SEQ ID NOs 87 and 88, respectively.

[0066]

[14] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[13] , wherein

[0067] (i) the first sequence is located between positions 14 and 17 of the CH3 domain of the antibody molecule; and / or

[0068] (ii) wherein the second sequence is located between 91 and 99 of the CH3 domain of the antibody molecule; and

[0069] The amino acid residues are numbered according to the IMGT numbering scheme.

[0070]

[15] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[14] , wherein the antibody molecule that binds to MSLN and CD137 comprises the CH3 domain sequence shown in SEQ ID NO: 86.

[0071]

[16] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[15] , wherein the CH3 domain comprises an additional lysine residue (K) immediately at the C-terminus of the CH3 domain sequence.

[0072]

[17] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[16] , wherein the antibody molecule that binds to MSLN and CD137 comprises the heavy chain and light chain of the following antibody:

[0073] (i) FS22-172-003-AA / FS28-256-271 shown in SEQ ID NOs 2 and 10, respectively;

[0074] (ii) FS22-172-003-AA / FS28-024-052 shown in SEQ ID NOs 18 and 26, respectively;

[0075] (iii) FS22-172-003-AA / FS28-256-021 shown in SEQ ID NOs 30 and 32, respectively;

[0076] (iv) FS22-172-003-AA / FS28-256-012 shown in SEQ ID NOs 36 and 37, respectively;

[0077] (v) FS22-172-003-AA / FS28-256-023 shown in SEQ ID NOs 41 and 32, respectively;

[0078] (vi) FS22-172-003-AA / FS28-256-024 shown in SEQ ID NOs 30 and 47, respectively;

[0079] (vii) FS22-172-003-AA / FS28-256-026 shown in SEQ ID NOs 41 and 47, respectively;

[0080] (viii) FS22-172-003-AA / FS28-256-027 shown in SEQ ID NOs 30 and 10, respectively;

[0081] (ix) FS22-172-003-AA / FS28-256-001 shown in SEQ ID NOs 51 and 32, respectively;

[0082] (x) FS22-172-003-AA / FS28-256-005 shown in SEQ ID NOs 51 and 47, respectively;

[0083] (xi) FS22-172-003-AA / FS28-256-014 shown in SEQ ID NOs 58 and 37, respectively;

[0084] (xii) FS22-172-003-AA / FS28-256-018 shown in SEQ ID NOs 41 and 37, respectively;

[0085] (xiii) FS22-172-003-AA / FS28-256 shown in SEQ ID NOs 65 and 37, respectively;

[0086] (xiv) FS22-172-003-AA / FS28-024-051 shown in SEQ ID NOs 70 and 26, respectively;

[0087] (xv) FS22-172-003-AA / FS28-024-053 shown in SEQ ID NOs 75 and 26, respectively; or

[0088] (xvi) FS22-172-003-AA / FS28-024 shown in SEQ ID NOs 80 and 26, respectively

[0089]

[18] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[17] , wherein the antibody molecule that binds to MSLN and CD137 comprises the heavy chain sequence shown in SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 10 [FS22-172-003-AA / FS28-256-271].

[0090]

[19] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[18] , wherein the chemotherapeutic agent is an alkylating agent or an antimetabolite.

[0091]

[20] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[19] , wherein the alkylating agent is selected from: altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (CPA), dacarbazine, ifosfamide, lomustine, nitrogen mustard, melphalan, oxaliplatin, temozolomide, thiotepa and trabectedin.

[0092]

[21] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[19] or

[20] , wherein the alkylating agent is cisplatin.

[0093]

[22] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[18] , wherein the antimetabolite is selected from azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine and trifluridine / tipiracil combination.

[0094]

[23] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[20] or

[22] , wherein the antimetabolite is gemcitabine.

[0095]

[24] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[23] , wherein the cancer expresses MSLN, or has been determined to express MSLN.

[0096]

[25] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[24] , wherein the cancer is selected from ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer and non-small cell lung cancer.

[0097]

[26] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[25] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent produces higher anti-tumor activity than monotherapy treatment with the antibody molecule that binds to MSLN and CD137 or monotherapy treatment with the chemotherapeutic agent.

[0098]

[27] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[26] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent produces an anti-tumor activity that is higher than the sum of the anti-tumor activities of monotherapy treatment with the antibody molecule that binds to MSLN and CD137 and monotherapy treatment with the chemotherapeutic agent.

[0099]

[28] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[27] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent produces a greater reduction in tumor growth, a reduction in tumor volume, an increase in median survival and / or an increase in the number of complete tumor regressions than monotherapy with the antibody molecule that binds to MSLN and CD137 or monotherapy with the chemotherapeutic agent.

[0100]

[29] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[28] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent produces a greater reduction in tumor growth, reduction in tumor volume, increase in median survival and / or increase in the number of complete tumor regressions than the sum of the reduction in tumor growth, reduction in tumor volume, increase in median survival and / or increase in the number of complete tumor regressions obtained by monotherapy with the antibody molecule that binds to MSLN and CD137 and monotherapy with the chemotherapeutic agent.

[0101]

[30] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[29] , wherein the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered parenterally.

[0102]

[31] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[30] , wherein the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally.

[0103]

[32] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[30] or

[31] , wherein the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered by injection or infusion.

[0104]

[33] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[29] , wherein the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered parenterally.

[0105]

[34] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[33] , wherein the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0106]

[35] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to [1] to

[34] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient simultaneously or sequentially.

[0107]

[36] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[35] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient sequentially.

[0108]

[37] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[36] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient within no more than 4 days of each other.

[0109]

[38] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[36] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient within no more than 3 days of each other.

[0110]

[39] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[36] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient within no more than 2 days of each other.

[0111]

[40] The antibody molecule or chemotherapeutic agent, method, use or kit for use according to

[36] , wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient on the same day.

[0112]

[41] The antibody molecule or chemotherapeutic agent or method for use according to [1] to [3] and [7] to

[40] , wherein the method comprises determining whether the cancer expresses MSLN, and if the cancer expresses MSLN, treating the individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0114] Figure 1 The therapeutic efficacy in the CT26 colon tumor mouse model of BALB / c is shown. The progress of the tumor mouse model is measured as the average tumor volume (A), median survival (B), weight change % (C) and the change (D) of individual tumor volume over time. Mice are treated with anti-HEL-hIgG1-LALA, FS122m, cisplatin or FS122m+cisplatin. Compared with the anti-HEL-hIgG1-LALA isotype control, the degree of tumor volume growth slowed down by the treatment of FS122m+cisplatin is much larger than FS122m and cisplatin monotherapy (A). FS122m+cisplatin also prolongs the median survival (B) relative to FS122m or cisplatin monotherapy, and compared with 2 tumors completely regressed in 10 mice treated with FS122m monotherapy, and there was no complete regression of tumors in 10 mice treated with cisplatin monotherapy, 7 of 10 mice induced complete regression of tumors (D). The weight changes between all treatments, including the anti-HEL-hIgG1-LALA isotype control, were comparable, indicating that all treatments were well tolerated (C). Tumor volume data were log transformed and subjected to two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, where ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Survival rates are expressed as median survival percentages, and mean tumor volume and weight changes are expressed as mean ± SEM.

[0115] Figure 2The therapeutic efficacy in the JC tumor mouse model of BALB / c mice is shown. The progress of the tumor mouse model is measured as the average tumor volume (A), median survival (B), weight change % (C) and the change (D) of individual tumor volume over time. Mice are treated with anti-HEL-hIgG1-LALA, FS122m, cisplatin or FS122m+cisplatin. Compared with the anti-HEL-hIgG1-LALA isotype control, the degree of tumor volume growth slowed down by the treatment of FS122m+cisplatin is much larger than FS122m and cisplatin monotherapy (A). FS122m+cisplatin also prolongs the median survival (B) relative to FS122m or cisplatin monotherapy, and compared with 2 tumors completely regressed in 10 mice treated with FS122m monotherapy, and there was no complete tumor regression in 10 mice treated with cisplatin monotherapy, and 2 induced complete tumor regression (D) in 10 mice. The weight changes between all treatments, including the anti-HEL-hIgG1-LALA isotype control, were comparable, indicating that all treatments were well tolerated (C). Tumor volume data were log transformed and subjected to two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test, where ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Survival rates are expressed as median survival percentages, and mean tumor volume and weight changes are expressed as mean ± SEM.

[0116] Figure 3The therapeutic efficacy in the JC tumor mouse model of BALB / c mice is shown. The progress of the tumor mouse model is measured as the average tumor volume (A), median survival (B), weight change % (C) and the change (D) of individual tumor volume over time. Mice are treated with anti-HEL-hIgG1-LALA, FS122m, gemcitabine or FS122m+gemcitabine. Compared with the anti-HEL-hIgG1-LALA isotype control, the degree of tumor volume growth slowed down by the treatment of FS122m+gemcitabine is much greater than FS122m and gemcitabine monotherapy (A). FS122m + gemcitabine also prolonged median survival relative to FS122m or gemcitabine monotherapy (B), and induced complete tumor regression in 5 of 10 mice compared to 1 in 10 mice treated with FS122m monotherapy and no complete tumor regression in 10 mice treated with gemcitabine monotherapy (D). Body weight changes were comparable between all treatments, including the anti-HEL-hIgG1-LALA isotype control, indicating that all treatments were well tolerated (C). Tumor volume data were log transformed and subjected to two-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test, with ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Survival rates are expressed as median survival percentages, and mean tumor volume and body weight changes are expressed as mean ± SEM.

[0117] Figure 4The therapeutic efficacy in the CT26 colon tumor mouse model of BALB / c mice is shown. The progress of the tumor mouse model is measured as the average tumor volume (A), median survival (B), weight change % (C) and the change (D) of individual tumor volume over time. Mice are treated with anti-HEL-hIgG1-LALA, FS122m, gemcitabine or FS122m+gemcitabine. Compared with the anti-HEL-hIgG1-LALA isotype control, the degree of tumor volume growth slowed down by the treatment of FS122m+gemcitabine is much greater than FS122m and gemcitabine monotherapy (A). FS122m + gemcitabine also prolonged median survival relative to FS122m or gemcitabine monotherapy (B), and induced complete tumor regression in 1 of 9 mice compared to 3 of 9 mice with FS122m monotherapy and none of 9 mice with gemcitabine monotherapy (D). Body weight changes were comparable between all treatments, including the anti-HEL-hIgG1-LALA isotype control, indicating that all treatments were well tolerated (C). Tumor volume data were log transformed and subjected to two-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test, with ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. Survival rates are expressed as median survival percentages, and mean tumor volume and body weight changes are expressed as mean ± SEM. DETAILED DESCRIPTION

[0118] The present invention relates to an antibody molecule that binds to MSLN and CD137, which is used in combination with a chemotherapeutic agent to treat cancer in a patient. The present invention also relates to a chemotherapeutic agent, which is used in combination with an antibody molecule that binds to MSLN and CD137 to treat cancer.

[0119] The term "bispecific" refers to a molecule that will not show any significant binding to other molecules except its two specific binding partners. The term may also refer to specific epitopes of the two binding partners, which may be carried by other antigens, in which case the antibody may also bind to the antigen carrying the specific epitope. In a preferred embodiment, the bispecific antibody molecule does not show any significant binding activity to OX40, GITR, CD40, CEACAM-5, E-cadherin, thrombomodulin or EpCAM.

[0120] 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 may preferably be a monoclonal antibody. Examples of antibody molecules are immunoglobulin isotypes, such as immunoglobulin G, and their isotype subclasses, such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof. The antibody molecule may be isolated, i.e. free of contaminants, such as antibody molecules capable of binding to other polypeptides and / or serum components.

[0121] The term "bispecific antibody molecule" is used below to refer to an antibody molecule that binds to MSLN and CD137.

[0122] In one embodiment, the bispecific antibody molecule independently binds to MSLN and CD137. In one embodiment, the bispecific antibody binds to MSLN and CD137 simultaneously.

[0123] The bispecific antibody molecule may be natural or partially or completely synthetically produced. For example, the antibody molecule may be a recombinant antibody molecule.

[0124] The bispecific antibody molecule may comprise at least one, preferably more than one, MSLN binding site based on a complementarity determining region (CDR), and at least one, preferably more than one, CD137 binding site in a constant domain, preferably at least one CH3 domain, of the bispecific antibody molecule.

[0125] The bispecific antibody molecule can be an immunoglobulin or an antigen-binding fragment thereof. For example, the bispecific antibody molecule can be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an IgG1, IgG2, IgG3 or IgG4 molecule, more preferably an IgG1 or IgG2 molecule, most preferably an IgG1 molecule, or a fragment thereof. In a preferred embodiment, the bispecific antibody molecule is a complete immunoglobulin molecule.

[0126] In other embodiments, the bispecific antibody molecule can be an antigen binding fragment comprising a MSLN antigen binding site based on CDR and a CD137 antigen binding site located in a constant domain. The antigen binding fragment can be a scFv, Fab, Fcab, VhH, monovalent IgG, double antibody or three antibodies, IGNAR, V-NAR, hclgG, mini antibody or nano antibody. For example, the antigen binding fragment can be a scFv-Fc fusion, wherein the scFv binds to MSLN and the Fc binds to CD137, or a mini antibody comprising a scFv connected to a CH3 domain (Hu et al. (1996), Cancer Res., 56 (13): 3055-61).

[0127] In a preferred embodiment, the bispecific antibody molecule is a mAb 2 (TM) bispecific antibody. mAb referred to herein 2 Bispecific antibodies are IgG immunoglobulins that include a CDR-based antigen binding site in each of their variable regions and at least one antigen binding site in the constant domains of the antibody molecule.

[0128] Antibodies and their construction and use methods are well known in the art and are described in, for example, Holliger and Hudson, 2005. Monoclonal antibodies and other antibodies can be used, and the technology of recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such technology can involve introducing the CDR or variable region of an antibody molecule into different antibody molecules (EP-A-184187, GB 2188638A and EP-A-239400). New antibodies for known targets can be routinely produced, and those skilled in the art can obtain without excessive burden.

[0129] Many antibody molecules that bind to MSLN and CD137 are known from WO 2020 / 011976. The antibody M9657 of the present application is the same as the antibody FS22-172-003-AA / FS28-256-271 of WO 2020 / 011976. Any of these antibodies can be used. Therefore, the CDR-based antigen binding site of the bispecific antibody molecule may comprise three VH CDRs or three VLCDRs, preferably three VH CDRs and three VL CDR: 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 / FS2 8-256-023, FS22-172-003-AA / FS28-256-024, FS22-172-003-AA / FS28-256-026, F S22-172-003-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 / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-00 3-AA / FS28-024-053 or FS22-172-003-AA / FS28-024, preferably the antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably the antibody FS22-172-003-AA / FS28-256-271.

[0130] The sequences of the CDRs can be readily determined from the VH and VL domain sequences of the antibody molecule using conventional techniques. Described herein are 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 / FS28-256-028. The VH and VL domain sequences of S28-256-001, 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, and the three VH domain CDRs and three VL domain CDRs of the antibodies can therefore be determined from the sequences. The CDR sequences can be determined, for example, according to Kabat et al., 1991 or the international ImMunoGeneTics information system (IMGT) (Lefranc et al., 2015).

[0131] Bispecific antibody molecules may or may not carry LALA mutations. LALA mutations describe a type of mutation that destroys the antibody effector function of an antibody molecule or its fragment. LALA mutations are associated with several favorable antibody properties (such as reduced toxicity) (Lo et al. (2017), The Journal of Biological Chemistry, 292 (9): 3900-3908). The mutation eliminates the binding of an antibody molecule or its fragment to an Fcγ receptor and is located in the CH2 domain. The sequences of the VH domain and VL domain of an antibody containing a LALA mutation and therefore the sequences of VH domain CDR1, CDR2 and CDR3 and VL domain CDR1, CDR2 and CDR3 are the same as those of an antibody without a LALA mutation. LALA mutations involve substitution of leucine residues (L1.3A and L1.2A) at positions 1.3 and 1.2 of the CH2 domain (according to the IMGT numbering scheme) with alanine. According to the Kabat numbering system, LALA mutations constitute L247A L248A substitutions. It is also known that complement activation (C1q binding) and ADCC are attenuated by mutating the proline at position 114 (according to the IMGT numbering system) of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). According to the Kabat numbering system, this mutation constitutes a P348A or P348G substitution. This mutation and the LALA mutation can also be combined to produce antibody molecules with further reduced ADCC or CDC activity or no ADCC or CDC activity.

[0132] Thus, the bispecific antibody molecule may comprise a CH2 domain, wherein the CH2 domain comprises an alanine residue at position 1.3 and an alanine residue at position 1.2, wherein the amino acid numbering is according to the IMGT numbering system. The bispecific antibody molecule may comprise a CH2 domain, wherein the CH2 domain comprises an alanine residue at position 247 and an alanine residue at position 248, wherein the amino acid numbering is according to the Kabat numbering system. For example, the CH2 domain may have the amino acid sequence shown in SEQ ID NO:90. In an alternative embodiment, the antibody molecule may comprise a CH2, wherein the CH2 domain comprises an alanine residue at position 91. The antibody molecule may comprise a CH2, wherein the CH2 domain comprises an alanine residue at position 1.3, an alanine residue at position 1.2 and an alanine residue at position 114. For example, the CH2 domain may have the amino acid sequence shown in SEQ ID NO:92.

[0133] The VH domain CDR1, CDR2 and CDR3 sequences of the bispecific antibody molecule according to IMGT numbering may be sequences located at positions 27-38, 56-65 and 105-117 of the VH domain of the antibody molecule, respectively.

[0134] The VH domain CDR1, CDR2, and CDR3 sequences of the bispecific antibody molecule according to Kabat numbering may be sequences located at positions 31-35, 50-65, and 95-102 of the VH domain, respectively.

[0135] The VL domain CDR1, CDR2 and CDR3 sequences of the bispecific antibody molecule according to IMGT numbering may be sequences located at positions 27-38, 56-65 and 105-117 of the VL domain, respectively.

[0136] The VL domain CDR1, CDR2, and CDR3 sequences of the bispecific antibody molecule according to Kabat numbering may be sequences located at positions 24-34, 50-56, and 89-97 of the VL domain, respectively.

[0137] For example,

[0138] (i) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-271 can be shown in SEQ ID NOs 4, 6 and 8, respectively;

[0139] (ii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-052 can be shown in SEQ ID NOs 20, 22 and 24, respectively;

[0140] (iii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-021 can be shown in SEQ ID NOs 4, 6 and 8, respectively;

[0141] (iv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-012 can be shown in SEQ ID NOs 4, 6 and 8, respectively;

[0142] (v) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-023 can be shown in SEQ ID NOs 42, 6 and 44, respectively;

[0143] (vi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-024 can be shown in SEQ ID NOs 4, 6 and 8, respectively;

[0144] (vii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-026 can be shown in SEQ ID NOs 43, 6 and 45, respectively;

[0145] (viii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-027 can be shown in SEQ ID NOs 4, 6 and 8, respectively;

[0146] (ix) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-001 can be shown in SEQ ID NOs 53, 6 and 55, respectively;

[0147] (x) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-005 can be shown in SEQ ID NOs 53, 6 and 55, respectively;

[0148] (xi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-014 can be shown in SEQ ID NOs 60, 6 and 62, respectively;

[0149] (xii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-018 can be shown in SEQ ID NOs 43, 6 and 45, respectively;

[0150] (xiii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256 can be shown in SEQ ID NOs 67, 6 and 55, respectively;

[0151] (xiv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-051 can be shown in SEQ ID NOs 21, 23 and 72, respectively;

[0152] (xv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-053 can be shown in SEQ ID NOs 21, 23 and 77, respectively; and

[0153] (xvi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024 can be shown in SEQ ID NOs 21, 23 and 82, respectively;

[0154] The CDR sequences are defined according to the IMGT numbering scheme.

[0155] For example,

[0156] (i) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-256-271 can be shown in SEQ ID NOs 12, 14 and 16, respectively;

[0157] (ii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-024-052 can be shown in SEQ ID NOs 12, 14 and 18, respectively;

[0158] (iii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-021 can be shown in SEQ ID NOs 12, 14 and 34, respectively;

[0159] (iv) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-012 can be shown in SEQ ID NOs 12, 14 and 39, respectively;

[0160] (v) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-023 can be shown in SEQ ID NOs 12, 14 and 34, respectively;

[0161] (vi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-024 can be shown in SEQ ID NOs 12, 14 and 49, respectively;

[0162] (vii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-256-026 can be shown in SEQ ID NOs 12, 14 and 49, respectively;

[0163] (viii) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-256-027 are shown in SEQ ID NOs 12, 14 and 16, respectively;

[0164] (ix) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-256-001 can be shown in SEQ ID NOs 12, 14 and 34, respectively;

[0165] (x) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-005 can be shown in SEQ ID NOs 12, 14 and 49, respectively;

[0166] (xi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-014 can be shown in SEQ ID NOs 12, 14 and 39, respectively;

[0167] (xii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-018 can be shown in SEQ ID NOs 12, 14 and 39, respectively;

[0168] (xiii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256 can be shown in SEQ ID NOs 12, 14 and 39, respectively;

[0169] (xiv) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-024-051 can be shown in SEQ ID NOs 12, 14 and 28, respectively;

[0170] (xv) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-024-053 can be shown in SEQ ID NOs 12, 14 and 28, respectively; and

[0171] (xvi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-024 can be shown in SEQ ID NOs 12, 14 and 28, respectively;

[0172] The CDR sequences are defined according to the IMGT numbering scheme.

[0173] For example,

[0174] (i) The sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-271 can be shown in SEQ ID NOs 5, 7 and 9, respectively;

[0175] (ii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-052 can be shown in SEQ ID NOs 21, 23 and 25, respectively;

[0176] (iii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-021 can be shown in SEQ ID NOs 5, 31 and 9, respectively;

[0177] (iv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-012 can be shown in SEQ ID NOs 5, 31 and 9, respectively;

[0178] (v) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-023 can be shown in SEQ ID NOs 44, 31 and 46, respectively;

[0179] (vi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-024 can be shown in SEQ ID NOs 5, 31 and 9, respectively;

[0180] (vii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-026 can be shown in SEQ ID NOs 44, 31 and 46, respectively;

[0181] (viii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-027 can be shown in SEQ ID NOs 5, 31 and 9, respectively;

[0182] (ix) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-001 can be shown in SEQ ID NOs 54, 31 and 56, respectively;

[0183] (x) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-005 can be shown in SEQ ID NOs 54, 31 and 56, respectively;

[0184] (xi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-014 can be shown in SEQ ID NOs 61, 31 and 63, respectively;

[0185] (xii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256-018 can be shown in SEQ ID NOs 44, 31 and 46, respectively;

[0186] (xiii) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-256 can be shown in SEQ ID NOs 68, 31 and 56, respectively;

[0187] (xiv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-051 can be shown in SEQ ID NOs 22, 24 and 73, respectively;

[0188] (xv) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024-053 can be shown in SEQ ID NOs 22, 24 and 78, respectively; and

[0189] (xvi) the sequences of CDR1, CDR2 and CDR3 of the VH domain of FS22-172-003-AA / FS28-024 can be shown in SEQ ID NOs 22, 24 and 83, respectively;

[0190] The CDR sequences are defined according to the Kabat numbering scheme.

[0191] (i) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-271 can be shown in SEQ ID NOs 13, 15 and 16, respectively;

[0192] (ii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-024-052 can be shown in SEQ ID NOs 13, 15 and 28, respectively;

[0193] (iii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-021 can be shown in SEQ ID NOs 13, 15 and 34, respectively;

[0194] (iv) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-012 can be shown in SEQ ID NOs 13, 15 and 39, respectively;

[0195] (v) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-023 can be shown in SEQ ID NOs 13, 15 and 34, respectively;

[0196] (vi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-024 can be shown in SEQ ID NOs 13, 15 and 49, respectively;

[0197] (vii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-026 can be shown in SEQ ID NOs 13, 15 and 49, respectively;

[0198] (viii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-027 can be shown in SEQ ID NOs 13, 15 and 16, respectively;

[0199] (ix) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-001 can be shown in SEQ ID NOs 13, 15 and 34, respectively;

[0200] (x) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-005 can be shown in SEQ ID NOs 13, 15 and 49, respectively;

[0201] (xi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-014 can be shown in SEQ ID NOs 13, 15 and 39, respectively;

[0202] (xii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256-018 can be shown in SEQ ID NOs 13, 15 and 39, respectively;

[0203] (xiii) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-256 can be shown in SEQ ID NOs 13, 15 and 39, respectively;

[0204] (xiv) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-024-051 can be shown in SEQ ID NOs 13, 15 and 28, respectively;

[0205] (xv) the sequences of the VL domain CDR1, CDR2 and CDR3 of FS22-172-003-AA / FS28-024-053 can be shown in SEQ ID NOs 13, 15 and 28, respectively; and

[0206] (xvi) the sequences of CDR1, CDR2 and CDR3 of the VL domain of FS22-172-003-AA / FS28-024 can be shown in SEQ ID NOs 13, 15 and 28, respectively;

[0207] The CDR sequences are defined according to the Kabat numbering scheme.

[0208] The CDR-based antigen binding site may include the VH or VL domains of the following antibodies, preferably the VH and VL domains: 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 / FS2 8-256-012, FS22-172-003-AA / FS28-256-023, FS22-172-003-AA / FS28-256-024, FS22-1 72-003-AA / FS28-256-026, FS22-172-003-AA / FS28-256-027, FS22-172-003-AA / FS28-25 6-001, FS22-172-003-AA / FS28-256-005, FS22-172-003-AA / FS28-256-014, FS22-172-0 03-AA / FS28-256-018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, F S22-172-003-AA / FS28-024-053 or FS22-172-003-AA / FS28-024, preferably the antibody FS22-172-003-AA / FS28-256-271 or FS22-172-003-AA / FS28-024-052, most preferably the antibody FS22-172-003-AA / FS28-256-271.

[0209] 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 The VH domains of S22-172-003-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 / FS28-024-054 may have SEQ The sequences shown in ID NOs 3, 19, 3, 3, 42, 3, 42, 3, 52, 52, 59, 42, 66, 71, 76 and 81.

[0210] 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 The VL domains of S22-172-003-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 / FS28-024-054 may have SEQ The sequences shown in ID NOs 11, 27, 33, 38, 33, 48, 48, 11, 33, 48, 38, 38, 38, 27, 27 and 27.

[0211] In one embodiment, the bispecific antibody molecule of the present invention comprises a CD137 antigen binding site. The CD137 antigen binding site may be located in the constant domain of the antibody molecule, preferably in the CH3 domain. The CD137 antigen binding may be included in one or more modified structural loops in the constant domain of the antibody molecule. It is known in the art that the antibody constant domain structural loop is engineered to produce an antigen binding site for a target antigen, and is described, for example, in Wozniak-Knopp G et al. (2010) Protein Eng Des. 23 (4): 289–297; WO2006 / 072620 and WO2009 / 132876. The CD137 constant domain antigen binding site included in the antibody molecule of the present invention is determined after extensive selection and affinity maturation procedures, and preferentially binds to dimers rather than monomeric human CD137.

[0212] The CD137 antigen binding site of the bispecific antibody molecule may include a first sequence and a second sequence, wherein the first sequence and the second sequence are located in the AB and EF structural loops of the constant domain (preferably CH3 domain) of the bispecific antibody molecule. The first sequence and the second sequence are preferably the first sequence and the second sequence of FS22-172-003 shown in SEQ ID NOs 87 and 88, respectively. The first sequence and the second sequence are preferably located at the 14th and 17th, and between the 91st and 99th of the CH3 domain of the bispecific antibody molecule, respectively, wherein the residue numbering is according to IMGT numbering. The CD loop sequence of the bispecific antibody molecule is preferably unmodified, i.e., wild type.

[0213] Thus, the CD loop sequence preferably has the sequence shown in SEQ ID NO: 89. The CD loop sequence is preferably located at positions 43 to 78 of the CH3 domain of the bispecific antibody molecule, wherein the residue numbering follows the IMGT numbering.

[0214] In a preferred embodiment, the bispecific antibody molecule comprises a CH3 domain comprising, having or consisting of the CH3 domain sequence of FS22-172-003 shown in SEQ ID NO: 86. The CH3 domain of the bispecific antibody molecule may optionally comprise an additional lysine residue (K) immediately at the C-terminus of the CH3 domain sequence.

[0215] In a preferred embodiment, the antibody molecule comprises the heavy chain and / or light chain, preferably the heavy chain and light chain, of the following antibodies:

[0216] (i) FS22-172-003-AA / FS28-256-271 shown in SEQ ID NOs 2 and 10, respectively;

[0217] (ii) FS22-172-003-AA / FS28-024-052 shown in SEQ ID NOs 18 and 26, respectively;

[0218] (iii) FS22-172-003-AA / FS28-256-021 shown in SEQ ID NOs 30 and 32, respectively;

[0219] (iv) FS22-172-003-AA / FS28-256-012 shown in SEQ ID NOs 36 and 37, respectively;

[0220] (v) FS22-172-003-AA / FS28-256-023 shown in SEQ ID NOs 41 and 32, respectively;

[0221] (vi) FS22-172-003-AA / FS28-256-024 shown in SEQ ID NOs 30 and 47, respectively;

[0222] (vii) FS22-172-003-AA / FS28-256-026 shown in SEQ ID NOs 41 and 47, respectively;

[0223] (viii) FS22-172-003-AA / FS28-256-027 shown in SEQ ID NOs 30 and 10, respectively;

[0224] (ix) FS22-172-003-AA / FS28-256-001 shown in SEQ ID NOs 51 and 32, respectively;

[0225] (x) FS22-172-003-AA / FS28-256-005 shown in SEQ ID NOs 51 and 47, respectively;

[0226] (xi) FS22-172-003-AA / FS28-256-014 shown in SEQ ID NOs 58 and 37, respectively;

[0227] (xii) FS22-172-003-AA / FS28-256-018 shown in SEQ ID NOs 41 and 37, respectively;

[0228] (xiii) FS22-172-003-AA / FS28-256 shown in SEQ ID NOs 65 and 37, respectively;

[0229] (xiv) FS22-172-003-AA / FS28-024-051 shown in SEQ ID NOs 70 and 26, respectively;

[0230] (xv) FS22-172-003-AA / FS28-024-053 shown in SEQ ID NOs 75 and 26, respectively; or

[0231] (xvi) FS22-172-003-AA / FS28-024 shown in SEQ ID NOs 80 and 26, respectively.

[0232] In a more preferred embodiment, the bispecific antibody molecule comprises the following heavy chain and / or light chain, preferably the heavy chain and light chain: 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, wherein the heavy chain and light chain sequences of these antibodies are as shown above.

[0233] The bispecific antibody molecules of the present invention may also comprise a first sequence, a second sequence or a 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 disclosed herein and / or a variant of a heavy chain sequence. Suitable variants can be obtained by a method of sequence change or mutation and screening. In a preferred embodiment, an antibody molecule comprising one or more variant sequences retains one or more functional features of a parent antibody molecule, such as binding specificity and / or binding affinity to MSLN and CD137. For example, an antibody molecule comprising one or more variant sequences preferably binds to MSLN and / or CD137 with the same affinity or with an affinity higher than that of the (parent) antibody molecule. A parent antibody molecule is an antibody molecule that does not include amino acid substitutions, deletions and / or insertions (which are incorporated into the variant antibody molecule).

[0234] Contains antibodies FS22-172-003-AA / FS28-256-271, FS22-172-003-AA / FS28-024-052FS22-172-003-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 CDR of -018, FS22-172-003-AA / FS28-256, FS22-172-003-AA / FS28-024-051, FS22-172-003-AA / FS28-024-053 or FS22-172-003-AA / FS28-024 1-6. The antibody molecule of the VH domain and / or heavy chain may comprise an amino acid substitution at position 55 or 57 of the VH domain, wherein the amino acid residue numbering is according to the IMGT numbering scheme.

[0235] For example, the antibody molecule may comprise CDRs 1-6, VH domain and / or 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 amino acid residue numbering is according to the IMGT numbering scheme. For example, an antibody molecule of the invention may comprise a first, second or third sequence, AB, CD or EF structural loop sequence, CH3 domain, CH2 domain, CDR, VH domain, VL domain, light chain and / or heavy chain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a structural loop, CH3 domain, CH2 domain, CDR, VH domain, VL domain, light chain or heavy chain sequence disclosed herein.

[0236] In preferred embodiments, the bispecific antibody molecules of the invention comprise a CH3 domain 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 as disclosed herein.

[0237] In a further preferred embodiment, the bispecific antibody molecule has or comprises a CH2 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 a CH2 domain as disclosed herein.

[0238] Sequence identity is usually defined with reference to algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Default parameters are usually used, and gap generation penalty is equal to 12, and gap extension penalty is equal to 4. GAP can be preferably used but other algorithms can also be used, such as BLAST (it uses Altschul et al., 1990 method), FASTA (it uses Pearson and Lipman, 1988 method) or Smith-Waterman algorithm (Smith and Waterman, 1981) or Altschul et al., 1990 TBLASTN programs on the same, usually using default parameters. In particular, psi-Blast algorithm (Altschul et al., 1997) can be used.

[0239] The bispecific antibody molecules of the present invention may also comprise a first sequence, a second sequence or a third sequence, an AB, CD or EF structural loop sequence, a CH3 domain, a CH2 domain, a Fcab, a CDR, a VH domain, a VL domain, a light chain or a heavy chain sequence disclosed herein, which has one or more amino acid sequence changes (addition, deletion, substitution and / or insertion of amino acid residues), preferably 20 changes or less, 15 changes or less, 10 changes or less, 5 changes or less, 4 changes or less, 3 changes or less, 2 changes or less or 1 change. The first sequence, the second sequence or the third sequence, the AB, CD or EF structural loop sequence, the CH3 domain, the CH2 domain, the VH domain, the VL domain, the light chain and / or the heavy chain. Specifically, changes can be made in one or more framework regions outside the VH and VL domain sequences of the antibody molecule and / or in the framework region of one or more CH3 domains. For example, changes may be made outside of the sequences described herein as the first sequence, the second sequence, and the third sequence, or the sequences described as the AB, CD, or EF structural loop sequences of the CH3 domain.

[0240] The bispecific antibody molecule may comprise a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 having one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2 and / or VL CDR3 as disclosed herein, preferably 3 alterations or less, 2 alterations or less or 1 alteration.

[0241] In a preferred embodiment, the bispecific antibody molecule of the invention comprises a CH3 domain sequence having one or more amino acid sequence alterations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to a CH3 domain as disclosed herein, preferably 20 alterations or less, 15 alterations or less, 10 alterations or less, 5 alterations or less, 4 alterations or less, 3 alterations or less, 2 alterations or less or 1 alteration.

[0242] In preferred embodiments where one or more amino acids are substituted with another amino acid, the substitution is a conservative substitution, e.g., according to the following table. In some embodiments, amino acids in the same category in the middle column are substituted with each other, i.e., a non-polar amino acid is substituted with, e.g., another non-polar amino acid. In some embodiments, amino acids in the same row in the rightmost column are substituted with each other.

[0243]

[0244]

[0245] In some embodiments, the substitution is functionally conservative. That is, in some embodiments, the substitution does not affect (or does not significantly affect) one or more functional properties (e.g., binding affinity) of the antibody molecule comprising the substitution compared to an equivalent unsubstituted antibody molecule.

[0246] The term "chemotherapeutic agent" describes a variety of agents used to treat cancer. Chemotherapeutic agents may be naturally occurring compounds, or may be partially or completely synthetically produced.

[0247] For example, the chemotherapeutic agent can be any one of a group of agents including an alkylating agent, a nitrosourea, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, or trans-retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, or vorinostat. The topoisomerase inhibitor can be a type I topoisomerase inhibitor or a type II topoisomerase inhibitor. The mitotic inhibitor can be a taxane or a vinca alkaloid.

[0248] Preferably, the chemotherapeutic agent is an alkylating agent or antimetabolite. Preferably, the antimetabolite is selected from azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine and trifluridine / tipiracil combination. More preferably, the antimetabolite is gemcitabine. Preferably, the alkylating agent is selected from hexamethylmelamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (CPA), dacarbazine, ifosfamide, lomustine, nitrogen mustard, melphalan, oxaliplatin, temozolomide, thiotepa and trabectedin. More preferably, the alkylating agent is cisplatin.

[0249] Preferably, nitrosourea is selected from camustine, lomustine and streptozocin. Preferably, cytotoxic antibiotics are selected from daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin and mitomycin C. Preferably, type I topoisomerase inhibitors are selected from irinotecan, liposomal irinotecan and topotecan. Preferably, type II topoisomerase inhibitors are selected from etoposide, mitoxantrone and teniposide. Preferably, the taxane is selected from cabazitaxel, docetaxel, nab-paclitaxel and paclitaxel. Preferably, the vinca alkaloid is selected from vinblastine, vincristine, liposomal vincristine and vinorelbine. Preferably, the corticosteroid is selected from prednisone, methylprednisolone and dexamethasone.

[0250] The term "tumor" refers to a mass of cells that is abnormal in size and / or composition due to increased proliferation and / or prolonged survival of the cells. Tumors can be benign or malignant. In the latter case they are referred to as "cancers". Thus, a "tumor" cell is a cell that has an abnormally increased ability to divide and / or resist cell death compared to other cells of the same cell type.

[0251] Cancer is characterized by the abnormal proliferation of malignant tumor cells. In the case of certain types of cancer, such as ovarian cancer, it refers to the abnormal proliferation of malignant cells of related tissues (such as breast tissue). Secondary cancers that are located in the breast but caused by the abnormal proliferation of malignant cells of another tissue (such as ovarian tissue) are not breast cancer as referred to herein but ovarian cancer.

[0252] MSLN is expressed on the surface of some tumor cells, and high expression levels of soluble MSLN are associated with poor prognosis in several cancers. Anti-MSLN antibodies have been studied as anticancer therapeutics. These anti-MSLN antibodies directly induce cell killing through their ADCC activity, or are used in the form of ADC.

[0253] Therefore, the cancer to be treated using the bispecific antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent preferably expresses or has been determined to express MSLN. More preferably, the cells of the cancer to be treated contain or have been determined to contain MSLN at their cell surface, i.e. contain cell surface-bound MSLN.

[0254] The cancer preferably comprises or has been determined to comprise tumor infiltrating lymphocytes (TILs) expressing CD 137. In particular, the TILs preferably comprise or have been determined to comprise CD137 on their cell surface.

[0255] The cancer may be a primary or secondary cancer. Thus, the antibody molecules that bind to MSLN and CD137 as described herein can be used in combination with a chemotherapeutic agent in a method for treating cancer in an individual, wherein the cancer is a primary tumor and / or tumor metastasis.

[0256] The cancer to be treated can be a solid cancer.

[0257] As mentioned above, the cancer to be treated may be a cancer expressing MSLN or a cancer determined to express MSLN. Preferably, the cancer is selected from the group consisting of: ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer and non-small cell lung cancer.

[0258] If the cancer expresses MSLN, the patient to be treated can be selected for treatment. If the cancer has been determined to express MSLN, the patient can be selected. Preferably, if the cancer is any of ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer or non-small cell lung cancer and expresses MSLN, the patient is selected for treatment.

[0259] The inventors show that combined treatment with FS122m (SEQ ID NO: 84 and SEQ ID NO: 85) and cisplatin or gemcitabine results in greater tumor growth slowing, tumor volume reduction, median survival increase, and percentage increase in mice with complete tumor regression than the sum of the tumor growth slowing, tumor volume reduction, median survival increase, and percentage increase in mice with complete tumor regression observed when mice were treated with FS122m alone or with cisplatin or gemcitabine. Thus, the inventors show that combined treatment with FS122m and cisplatin or gemcitabine slows tumor growth, reduces tumor volume, increases median survival, and increases percentage of mice with complete tumor regression in a synergistic manner in ST26 and JC mouse tumor models.

[0260] In one embodiment, the antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent thus reduces tumor volume, delays tumor growth, improves survival rate, and / or increases the percentage of patients with complete tumor regression. Preferably, the antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent reduces tumor volume, delays tumor growth, improves survival rate, and / or increases the percentage of patients with complete tumor regression to a degree that is statistically significantly greater than a monotherapy with a bispecific antibody molecule that binds to MSLN and CD137 or a monotherapy with a chemotherapeutic agent. More preferably, the bispecific antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent reduces tumor volume, delays tumor growth, increases survival rate, and / or increases the percentage of patients with complete tumor regression in a synergistic manner, i.e., compared to the sum of the slowing of tumor growth, reduction of tumor volume, increase in median survival time, and / or increase in the percentage of patients with complete tumor regression when the patient is treated with an antibody molecule that binds to MSLN and CD137 or a chemotherapeutic agent alone.

[0261] In one embodiment, the antibody molecule and chemotherapeutic agent that binds to MSLN and CD137 are used as first-line ("front-line") treatment (e.g., initial or first treatment). In another embodiment, the antibody molecule and chemotherapeutic agent that binds to MSLN and CD137 are used as second-line treatment (e.g., after initial treatment with the same or different therapy, including after relapse and / or when the first treatment fails).

[0262] As used herein, "administering" refers to the physical introduction of therapeutic agents into subjects using various methods and delivery systems known to any person skilled in the art. Exemplary routes of administration of antibody molecules and / or chemotherapeutics in conjunction with MSLN and CD137 include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Therapeutic agents can be administered via non-parenteral routes. Non-parenteral routes include topical, epidermal or mucosal routes of administration, such as oral, intranasal, vaginal, rectal, sublingual or topical.

[0263] Therefore, in some embodiments, the antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 are administered parenterally. The antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally. Alternatively, the antibody molecules and / or chemotherapeutic agents that bind to MSLN and CD137 can be administered by injection or infusion.

[0264] In other embodiments, the antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 is administered non-parenterally. The antibody molecule and / or chemotherapeutic agent that binds to MSLN and CD137 can be administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0265] "Simultaneous administration" describes that the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered simultaneously in the same formulation or in separate formulations. "Sequential administration" refers to the administration of the bispecific antibody molecule and the chemotherapeutic agent in separate formulations separated by time.

[0266] Thus, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent may be part of the same formulation or part of separate formulations.Preferably, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are provided as separate formulations.

[0267] In one embodiment of the invention, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered simultaneously. For example, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent can be administered together in the same formulation. Alternatively, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent can be administered in separate formulations immediately before or after each other.

[0268] Preferably, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient sequentially, more preferably, the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient within 4 days of each other, preferably within 3 days of each other, more preferably within 2 days of each other, or sequentially administered to the patient on the same day.

[0269] The present invention also relates to a method of treating cancer, comprising administering an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent to an individual in need thereof.

[0270] Administration can be in a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount applied and the rate and time course of administration will depend on the nature and severity of the treatment, the specific individual treated, the individual's clinical condition, the cause of the disease, the delivery site of the composition, the type of antibody molecule, the method of administration, the time schedule of administration, and other factors known to medical practitioners. Prescribing treatment, such as determining the dosage, etc., is within the responsibility of general practitioners and other doctors, and may depend on the severity and / or progression of the symptoms of the disease being treated. The appropriate dosage of antibody molecules is well known in the art (Ledermann et al., 1991; Bagshawe et al., 1991). The therapeutically effective amount or suitable dosage of antibody molecules can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses for humans in mice and other experimental animals are known. The exact dosage will depend on many factors, including the size and location of the area to be treated, and the exact nature of the antibody molecule.

[0271] Therefore, the present invention may relate to a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody molecule that binds to MSLN and CD137 and a therapeutically effective amount of a chemotherapeutic agent.

[0272] Also provided is a kit comprising an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent. Preferably, the kit comprises an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient as well as a chemotherapeutic agent and a pharmaceutically acceptable excipient.

[0273] The kit may be a package comprising a first container comprising an antibody molecule that binds to MSLN and CD137 and a second container comprising a chemotherapeutic agent. The package may include instructions for use of the antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent for treating cancer in an individual.

[0274] In one embodiment, the kit can be a package comprising at least one dose of a drug comprising an antibody molecule that binds to MSLN and CD137 and one dose of a drug comprising a chemotherapeutic agent. Preferably, the kit comprises at least one dose of a drug comprising an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient and one dose of a drug comprising a chemotherapeutic agent and a pharmaceutically acceptable excipient. More preferably, the kit comprises a package insert comprising instructions for using the drug to treat cancer in an individual.

[0275] In another embodiment, the kit can be a package comprising a first container comprising an antibody molecule that binds to MSLN and CD137 and a second container comprising a chemotherapeutic agent. The first container can contain at least one dose of a drug comprising an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient, and the second container can contain at least one dose of a drug comprising a chemotherapeutic agent. The package can further contain an insert containing instructions for using the drug to treat cancer in an individual.

[0276] The features disclosed in the foregoing description, in the appended claims or in the drawings, expressed where appropriate in their specific form or in the form of a means for performing a disclosed function or a method or process for obtaining a disclosed result, may be used alone or in any combination of such features to implement the invention in its various forms.

[0277] The features disclosed in the foregoing description, in the appended claims or in the drawings, expressed where appropriate in their specific form or in the form of a means for performing a disclosed function or a method or process for obtaining a disclosed result, may be used alone or in any combination of such features to implement the invention in its various forms.

[0278] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art after obtaining the present disclosure. Therefore, the exemplary embodiments of the present invention described above should be considered illustrative rather than restrictive. Various changes may be made to the embodiments without departing from the spirit and scope of the present invention.

[0279] To avoid any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0280] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0281] Throughout this specification, including the appended claims, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprises", "comprising" and "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0282] It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the preposition "about", it is understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and, for example, means + / - 10%.

[0283] Example

[0284] Materials and methods

[0285] The efficacy of the combination of an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent in enhancing anti-cancer immune responses was evaluated by studying the average tumor volume over time and prolonged survival in mouse tumor models compared to monotherapy with an antibody molecule that binds to MSLN and CD137 or a chemotherapeutic agent. These mouse tumor models will be described in detail below.

[0286] CT26 cells were obtained from ATCC and cultured in RPMI medium containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, 1500 mg / L sodium bicarbonate, and 10% fetal bovine serum (FBS). JC cells were obtained from ATCC and maintained in vitro as monolayer cultures in RPMI 1640 supplemented with 10% heat-inactivated FBS at WuXi. These tumor cell lines were maintained individually as monolayer cultures in vitro and incubated at 37°C in 5% CO 2 atmosphere. The cells were passaged twice a week by TrypLE treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation. Wild-type BALB / c female mice were purchased from Charles River Laboratories or Lingchang Biotechnology Co., Ltd. All mice were 8-12 weeks old at the beginning of the study and were raised and maintained under conditions consistent with the 8th edition of the Guide for the Care and Use of Laboratory Animals, 8th Edition as described below. All animal experiments were conducted in accordance with the EMD Serono Research Institutional (Scientific Experimental Program 17-008, 20-005) and Wuxi WuXi AppTec Animal Care and Use Committee (IACUC) guidelines.

[0287] Upon arrival at the institute's animal housing unit, all animals underwent a detailed physical examination performed by the research staff, including body weight measurement. All animals were found to be in satisfactory health. Animals were housed in a specific pathogen-free barrier animal facility at EMD Serono. Mice were kept in individually ventilated cages at constant temperature and humidity, with 5 animals per cage. The identification tag of each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and date of treatment start. Animals were marked by ear notch or ear tag. The animal housing unit was maintained at 20-26°C and 40-70% humidity. The lighting was a 12-h light / dark cycle. Animals had free access to a standard certified commercial laboratory diet. The maximum permissible concentration of contaminants in the diet was controlled by the manufacturer and analyzed regularly. Animals had free access to autoclaved municipal tap water suitable for human consumption. The dietary materials were considered free of known contaminants that could affect the growth of tumors. Approximately one week was allowed between the arrival of the animals and tumor inoculation to allow the animals to acclimate to the laboratory environment. All procedures related to animal handling, care, and treatment in the study were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of EMD Serono and followed the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0288] The anti-tumor effect of the combination of an antibody molecule binding to MSLN and CD137 and a chemotherapeutic agent compared to monotherapy with an antibody molecule binding to MSLN and CD137 or a chemotherapeutic agent was investigated using the CT26 and JC tumor models. Mice were treated with the anti-mMSLN-mCD137-huIgG1-LALA antibody molecule FS122m in combination with either of the two chemotherapeutic agents, cisplatin or gemcitabine. For the CT26 model, 0.3 × 10 6 CT26 cells were inoculated subcutaneously (sc) into the right flank of female BALB / c mice. When the average tumor volume reached approximately 50-100 mm 3 At 4 pm, mice were randomly assigned to treatment groups (n = 10 mice / group). For the JC model, JC tumor cells (5 × 10 6 ) were inoculated into the right upper flank of female BALB / c mice via sc injection. On day 14 after tumor inoculation, when the average tumor size reached approximately 80 mm 3 Treatment was started at 1:00 pm. FS122m and anti-HEL hIgG1LALA isotype control were given at 5 mg / kg via intraperitoneal (ip) injection on days 1, 3, and 5 of treatment (days 0, 2, and 4). Cisplatin was given at 10 mg / kg via ip injection on day 0, and gemcitabine was given at 120 mg / kg via ip injection on day 0.

[0289] Mortality checks were performed daily. Animals were checked daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (visually only), eye / hair matting, and any other abnormal effects as specified in the scientific experimental plan. Body weights were recorded twice a week, and any mouse with a 20% loss of initial body weight was humanely sacrificed. Mice with subcutaneous tumors reaching 2500 mm 3 If the volume reaches , the mice are humanely euthanized.

[0290] If tumor ulceration occurs, monitor animals with ulcerated tumors at least 3 times per week, increasing frequency to once daily based on clinical signs. Uncrusted ulcerated tumors should be cleaned with an appropriate wound cleansing solution (e.g., Novalsan). Antibiotic ointment should be applied to ulcers / lesions only if directed by veterinary staff.

[0291] Criteria for euthanasia include lesions that have not healed or crusted over within 1 week, lesions that are larger than 5 mm in diameter, lesions that have cavitated or developed signs of infection (such as the presence of pus) or bleeding, or the animal showing signs of discomfort (such as excessive licking and biting of the area) or systemic illness (lethargy, decreased activity, decreased food consumption, decreased body condition, or weight loss). Contact veterinary staff to discuss any possible exceptions.

[0292] If an animal is found to be moribund, it is euthanized. Clinical examples of morbidity may include hunching, persistent prostration, and lack of response to touch or other stimulation, signs of severe organ or system failure, emaciation, hypothermia, CNS deficits (convulsions), respiratory signs (rapid respiratory rate, labored breathing, cough, rales), and gastrointestinal signs (diarrhea persisting for >2 days, jaundice). Any animal displaying the above clinical problems is treated with CO 2 Humane execution.

[0293] Body weight was measured and recorded twice weekly until endpoint was reached. Tumor dimensions were measured twice weekly using calipers in three dimensions, and volume was reported in mm 3 Indicated using the following formula: width × length × height × 0.5236.

[0294] Two-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used to determine the difference in tumor growth between the treatment groups. The significance of survival rate was determined using the Log-rank (Mantel Cox) test. All analyses were performed using the GraphPad Prism software package (Prism 5 for Windows, 8.0 version, GraphPad Software Inc., San Diego, CA), and statistical significance was accepted at p≤0.05 level.

[0295] Example 1: In vivo proof of concept

[0296] To test whether the combination of an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent shows improved anti-tumor activity relative to monotherapy with a bispecific antibody molecule and monotherapy with a chemotherapeutic agent, an in vivo mouse tumor model was treated with an antibody molecule that binds to MSLN and CD137 in combination with a chemotherapeutic agent. The results of tumor growth slowing, tumor volume reduction, and survival rate were then compared with monotherapy with an antibody molecule that binds to MSLN and CD137 and monotherapy with a chemotherapeutic agent.

[0297] Due to the lack of cross-reactivity between the bispecific anti-MSLN / CD137 antibody molecule M9657 and mouse MSLN and mouse CD137, an anti-mMSLN-mCD137-huIgG1-LALA (FS112m) bispecific antibody was developed as an alternative antibody (SEQ ID NO: 84 and 85), the binding affinity of the antibody to mouse CD137 is similar to that of M9657 to human CD137. Cisplatin and gemcitabine are two chemotherapeutic agents belonging to the class of alkylating agents and antimetabolites, respectively. FS112m is combined with cisplatin or gemcitabine and the combined tumor efficacy is compared with monotherapy with FS112m and monotherapy with cisplatin or gemcitabine in two different mouse tumor models. To this end, female mice are inoculated with CT26 colon cancer or JC breast cancer cells and are inoculated with CT26 colon cancer or JC breast cancer cells when the average tumor volume reaches about 50-100 mm 3 Mice were inoculated, treated, and terminated as described in Materials and Methods.

[0298] 1.1 Combination of FS221m and cisplatin

[0299] The antitumor efficacy of FS122m in combination with cisplatin was first evaluated in the CT26 colon tumor model of BALB / c mice. Monotherapy with FS122m and cisplatin induced significant tumor growth inhibition (TGI) (61.3% and 77.8%, respectively) relative to isotype control (both P≤0.0001, day 17) and prolonged median survival (24 days and 36.5 days, respectively) relative to isotype control (17 days) ( Figure 1 A, B, and D). The combination of FS122m and cisplatin resulted in a higher TGI (96.3%) than that observed with FS122m monotherapy (P≤0.0001, day 17) and cisplatin monotherapy (P≤0.0001, day 17) ( Figure 1 A and D). The combination of FS122m and cisplatin also prolonged the median survival ( Figure 1B), and induced complete tumor regression in 7 out of 10 mice, compared with 2 out of 10 mice treated with FS122m monotherapy and no complete tumor regression in 10 mice treated with cisplatin monotherapy ( Figure 1 D).

[0300] In JC tumor-bearing mice, treatment with cisplatin induced a small but statistically significant TGI at day 22 after the start of treatment (44.9%, P≤0.0001), whereas FS122m monotherapy induced a moderate TGI at day 22 (60.1% P≤0.0001) compared with isotype controls ( Figure 2 A and D). Cisplatin and FS122m monotherapy prolonged survival relative to isotype control (22 days vs. 29 and 33 days, respectively) ( Figure 2 B). The combination of FS122m and cisplatin resulted in a higher TGI (83.3%) than that observed with cisplatin monotherapy (P≤0.0001) and FS122m monotherapy (P≤0.001) ( Figure 2 A and D). The combination of FS122m and cisplatin also prolonged the median survival (36.5 days) ( Figure 2 B), and induced complete tumor regression in 2 of 10 mice compared with 2 of 10 mice treated with FS122m monotherapy and no complete tumor regression in 10 mice treated with cisplatin monotherapy ( Figure 2 D).

[0301] In both CT26 colon tumor-bearing mice and JC tumor-bearing mice, the combination of FS122m and cisplatin resulted in a higher TGI than that observed when mice were treated with either FS122m monotherapy or cisplatin monotherapy. The difference compared to FS122m and cisplatin monotherapy was particularly evident in the CT26 mouse tumor model, where the combination of FS122m and cisplatin resulted in complete tumor regression in 7 out of 10 mice ( 10 mice with FS122m monotherapy had complete tumor regression, compared to 2 out of 10 mice with complete tumor regression in the FS122m monotherapy group and no mice with complete tumor regression in the cisplatin monotherapy group. Figure 1 D). No significant weight loss was observed in either mouse tumor model following treatment with the combination of FS122m and cisplatin, indicating that the combination treatment was well tolerated in the animals ( Figure 1 C and 2C).

[0302] 1.2 Combination of FS221m and gemcitabine

[0303] The antitumor efficacy of FS122m in combination with gemcitabine was first evaluated in JC tumor-bearing mice. Treatment with gemcitabine or FS122m monotherapy induced a small but statistically significant TGI (47.8% and 45.8%, respectively) relative to isotype control (P≤0.0001 for both, day 25) ( Figure 3 A and D). Monotherapy with gemcitabine and FS122m prolonged survival relative to isotype control (27 days vs. 36 and 34 days, respectively) ( Figure 3 B). The combination of FS122m and gemcitabine resulted in a higher TGI (80.2%) than gemcitabine monotherapy (P≤0.0001) and FS122m monotherapy (P≤0.001) ( Figure 3 A). The combination of FS122m and gemcitabine also prolonged median survival (74.5 days) ( Figure 3 B), and induced complete tumor regression in 5 out of 10 mice, compared with 1 out of 10 mice treated with FS122m monotherapy and no complete tumor regression in 10 mice treated with cisplatin monotherapy ( Figure 3 D).

[0304] In the CT26 colon tumor model in BALB / c mice, monotherapy with FS122m and gemcitabine induced a moderate TGI (61.4% and 58.2%, respectively) relative to isotype control (P≤0.0001, day 14) and prolonged median survival (35 days and 22 days, respectively) relative to isotype control (17 days) ( Figure 4 A, B, and D). The combination of FS122m and gemcitabine resulted in a higher TGI (86.1%) than gemcitabine monotherapy (P≤0.0001, day 18) and FS122m monotherapy (P=0.0113, day 18) ( Figure 4 A). The combination therapy also prolonged median survival (35 days) ( Figure 4 B), and induced complete tumor regression in 1 of 9 mice, compared with 3 of 9 mice treated with FS122m monotherapy and none of 9 mice treated with cisplatin monotherapy ( Figure 4 D).

[0305] In both JC tumor-bearing mice and CT26 colon tumor-bearing mice, treatment with the combination of FS122m and gemcitabine resulted in higher TGIs than those observed when mice were treated with either FS122m monotherapy or gemcitabine monotherapy. The difference compared to monotherapy with FS122m and gemcitabine was particularly evident in the JC mouse tumor model, where the combination of FS122m and cisplatin resulted in complete tumor regression in 5 out of 10 mice ( 1 out of 10 mice with FS122m monotherapy had complete tumor regression, and no mice in the gemcitabine monotherapy group had complete tumor regression. Figure 3 D). The combination of FS122m and gemcitabine did not cause significant weight loss in both mouse tumor models, indicating that the combination treatment was well tolerated in the animals ( Figure 3 C and 4C).

[0306] Sequence Listing

[0307] Heavy chain annotation

[0308] i. In mAb 2 In the heavy chain amino acid sequence of , the variable domain is shown in italics, the CDRs according to IMGT are shown in bold italics, the CDRs according to Kabat are shown in italics and underlined (thus any overlapping IMGT and Kabat CDR sequences are shown in bold, italics and underlined), the CH1 domain is underlined, the hinge region is double underlined, the CH2 domain is shown in bold (and, where applicable, the position of the LALA mutation is shown in bold and underlined), the CH3 domain is shown in normal font, and modified regions of the CH3 structural loops are underlined (no underlining if the loop is unchanged).

[0309] ii. In the amino acid sequences of the variable domains, the CDRs according to IMGT are shown in bold and italics and the CDRs according to Kabat are shown in italics and underlined (thus any overlapping IMGT and Kabat CDR sequences are shown in bold, italics and underlined).

[0310] iii. Provides the CDR amino acid sequences according to IMGT and Kabat.

[0311] Light chain annotation

[0312] i. In mAb 2 In the light chain amino acid sequences of , the variable domains are shown in italics, the CDRs according to IMGT are shown in bold and italics, and the CDRs according to Kabat are shown in italics and underlined (thus any overlapping IMGT and Kabat CDR sequences are shown in bold, italics and underlined).

[0313] ii. In the amino acid sequences of the variable domains, the CDRs according to IMGT are shown in bold and italics, and the CDRs according to Kabat are shown in italics and underlined (thus any overlapping IMGT and Kabat CDR sequences are shown in bold, italics and underlined).

[0314] iii. Provides the CDR amino acid sequences according to IMGT and Kabat.

[0315] Amino acid sequence of FS22-172-003-AA / FS28-256-271 mAb2

[0316] FS22-172-003-AA / FS28-024-052mAb 2 The amino acid sequence

[0317] FS22-172-003-AA / FS28-256-021mAb 2 The amino acid sequence

[0318] FS22-172-003-AA / FS28-256-012mAb 2 The amino acid sequence

[0319] FS22-172-003-AA / FS28-256-023mAb 2 The amino acid sequence

[0320]

[0321] FS22-172-003-AA / FS28-256-024mAb 2 The amino acid sequence

[0322]

[0323] FS22-172-003-AA / FS28-256-026mAb 2 The amino acid sequence

[0324]

[0325] FS22-172-003-AA / FS28-256-027mAb 2 The amino acid sequence

[0326]

[0327] FS22-172-003-AA / FS28-256-001mAb 2 The amino acid sequence

[0328] FS22-172-003-AA / FS28-256-005mAb 2 The amino acid sequence

[0329] FS22-172-003-AA / FS28-256-014mAb 2 The amino acid sequence

[0330]

[0331] FS22-172-003-AA / FS28-256-018mAb 2 The amino acid sequence

[0332]

[0333] FS22-172-003-AA / FS28-256 mAbs 2 The amino acid sequence

[0334]

[0335] FS22-172-003-AA / FS28-024-051mAb 2 The amino acid sequence

[0336]

[0337] FS22-172-003-AA / FS28-024-053mAb 2 The amino acid sequence

[0338] FS22-172-003-AA / FS28-024 mAbs 2 The amino acid sequence

[0339] Amino acid sequence of FS122m (replacing anti-mMSLNmCD137Fcab G1-AA):

[0340] All mAbs containing FS22-172-003Fcab 2 Amino acids of the CH3 domain of cloned and FS22-172-003 Fcab Sequences and amino acid sequences of the modified regions of the CH3 AB and EF structural loops

[0341]

[0342] Amino acid sequences of CH2 domains containing LALA mutation, PA mutation or LALA-PA mutation (mutations in bold and Underlined)

[0343]

[0344] References

[0345] Many publications are cited above to more fully describe and disclose the present invention and the prior art in the field to which the present invention belongs. The complete citations of these references are provided below. The full text of each of these references is incorporated herein.

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Claims

1. An antibody molecule that binds to MSLN and CD137 for use in a method of treating cancer in a patient, wherein the method comprises administering the antibody in combination with a chemotherapeutic agent.

2. A method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody molecule that binds to MSLN and CD137 and a chemotherapeutic agent.

3. A kit comprising (a) an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient; and (b) a chemotherapeutic agent and a pharmaceutically acceptable excipient.

4. An antibody molecule for use, a method for treating cancer or a kit according to any one of the preceding claims, wherein the antibody molecule that binds to MSLN and CD137 comprises (a) MSLN antigen binding site based on complementarity determining regions (CDRs); and (b) The CD137 antigen binding site located in the CH3 domain of the antibody molecule.

5. The antibody molecule for use, method for treating cancer or kit according to any one of the preceding claims, wherein the CDR-based MSLN antigen binding site comprises CDRs 1-6 as shown below: (i) SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-271], respectively; (ii) SEQ ID NOs 20, 22, 24, 12, 14 and 28, respectively [FS28-024-052]; (iii) SEQ ID NOs 4, 6, 8, 12, 14 and 34 [FS28-256-021], respectively; (iv) SEQ ID NOs 4, 6, 8, 12, 14 and 39 [FS28-256-012], respectively; (v) SEQ ID NOs 43, 6, 45, 12, 14 and 34 [FS28-256-023], respectively; (vi) SEQ ID NOs 4, 6, 8, 12, 14 and 49 [FS28-256-024], respectively; (vii) SEQ ID NOs 43, 6, 45, 12, 14 and 49 [FS28-256-026], respectively; (viii) SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-027], respectively; (ix) SEQ ID NOs 53, 6, 55, 12, 14 and 34 [FS28-256-001], respectively; (x) SEQ ID NOs 53, 6, 55, 12, 14 and 49 [FS28-256-005], respectively; (xi) SEQ ID NOs 60, 6, 62, 12, 14 and 39, respectively [FS28-256-014]; (xii) SEQ ID NOs 43, 6, 45, 12, 14 and 39, respectively [FS28-256-018]; (xiii) SEQ ID NOs 67, 6, 55, 12, 14 and 39 [FS28-256], respectively; (xiv) SEQ ID NOs 21, 23, 72, 12, 14 and 28, respectively [FS28-024-051]; (xv) SEQ ID NOs 21, 23, 77, 12, 14 and 28, respectively [FS28-024-053]; or (xvi) SEQ ID NOs 21, 23, 82, 12, 14 and 28, respectively [FS28-024]; and The CD137 antigen binding site comprises a first sequence and a second sequence respectively located in the AB and EF structural loops of the CH3 domain, wherein the first sequence and the second sequence have the sequences shown in SEQ ID NOs 87 and 88 respectively [FS22-172-003].

6. An antibody molecule for use, a method for treating cancer or a kit according to any one of the preceding claims, wherein (i) the first sequence is located between positions 14 and 17 of the CH3 domain of the antibody molecule; and / or (ii) wherein the second sequence is located between 91 and 99 of the CH3 domain of the antibody molecule; and The amino acid residues are numbered according to the IMGT numbering scheme.

7. The antibody molecule for use, the method for treating cancer or the kit according to any one of the preceding claims, wherein the antibody molecule comprises the CH3 domain sequence shown in SEQ ID NO: 86 [FS22-172-003].

8. The antibody molecule for use, the method for treating cancer or the kit according to any one of the preceding claims, wherein the antibody molecule comprises the heavy chain and the light chain of the following antibody: (i) FS22-172-003-AA / FS28-256-271 shown in SEQ ID NOs 2 and 10, respectively; (ii) FS22-172-003-AA / FS28-024-052 shown in SEQ ID NOs 18 and 26, respectively; (iii) FS22-172-003-AA / FS28-256-021 shown in SEQ ID NOs 30 and 32, respectively; (iv) FS22-172-003-AA / FS28-256-012 shown in SEQ ID NOs 36 and 37, respectively; (v) FS22-172-003-AA / FS28-256-023 shown in SEQ ID NOs 41 and 32, respectively; (vi) FS22-172-003-AA / FS28-256-024 shown in SEQ ID NOs 30 and 47, respectively; (vii) FS22-172-003-AA / FS28-256-026 shown in SEQ ID NOs 41 and 47, respectively; (viii) FS22-172-003-AA / FS28-256-027 shown in SEQ ID NOs 30 and 10, respectively; (ix) FS22-172-003-AA / FS28-256-001 shown in SEQ ID NOs 51 and 32, respectively; (x) FS22-172-003-AA / FS28-256-005 shown in SEQ ID NOs 51 and 47, respectively; (xi) FS22-172-003-AA / FS28-256-014 shown in SEQ ID NOs 58 and 37, respectively; (xii) FS22-172-003-AA / FS28-256-018 shown in SEQ ID NOs 41 and 37, respectively; (xiii) FS22-172-003-AA / FS28-256 shown in SEQ ID NOs 65 and 37, respectively; (xiv) FS22-172-003-AA / FS28-024-051 shown in SEQ ID NOs 70 and 26, respectively; (xv) FS22-172-003-AA / FS28-024-053 shown in SEQ ID NOs 75 and 26, respectively; or (xvi) FS22-172-003-AA / FS28-024 shown in SEQ ID NOs 80 and 26, respectively.

9. The antibody molecule for use, the method for treating cancer or the kit according to claim 8, wherein the antibody molecule comprises the heavy chain sequence shown in SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 10 [FS22-172-003-AA / FS28-256-271].

10. The antibody molecule for use, method for treating cancer or kit according to any one of the preceding claims, wherein the chemotherapeutic agent is an alkylating agent or an antimetabolite.

11. The antibody molecule, method or kit for treating cancer according to claim 10, wherein the alkylating agent is selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (CPA), dacarbazine, ifosfamide, lomustine, nitrogen mustard, melphalan, oxaliplatin, temozolomide, thiotepa and trabectedin.

12. The antibody molecule, method or kit for treating cancer according to claim 10, wherein the antimetabolite is selected from azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine and trifluridine / tipiracil combination.

13. The antibody molecule for use or the method for treating cancer according to any one of claims 1, 2 and 4 to 12, wherein the cancer expresses MSLN or has been determined to express MSLN and is selected from ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer and non-small cell lung cancer.

14. The antibody molecule for use or the method for treating cancer according to any one of claims 1, 2 and 4 to 13, wherein treatment with the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent produces an anti-tumor effect greater than the sum of the anti-tumor effects when the patient is treated with the antibody molecule that binds to MSLN and CD137 or the chemotherapeutic agent alone.

15. The antibody molecule for use or the method for treating cancer according to claim 14, wherein the anti-tumor effect is tumor growth inhibition, tumor volume reduction, median survival increase and / or an increase in the percentage of patients experiencing complete tumor regression.

16. The antibody molecule for use or the method for treating cancer according to any one of claims 1, 2 and 4 to 15, wherein the antibody molecule that binds to MSLN and CD137 and the chemotherapeutic agent are administered to the patient simultaneously or sequentially.

17. An antibody molecule for use, method or kit for treating cancer according to any one of claims 1, 2 and 4 to 16, wherein the method comprises determining whether the cancer expresses MSLN, and treating the individual if the cancer expresses MSLN.

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