Combination cancer therapy comprising anti-MSLN / CD137 antibody and PD-1 / PD-L1 inhibitor

Abstract

The present application relates to the use of antibody molecules that bind to MSLN and CD137 and a PD-1 / PD-L1 inhibitor 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 PD-1 / PD-L1 inhibitors for treating cancer in patients. Background Art

[0002] Antagonistic antibodies targeting immune checkpoint co-inhibitory receptors can reverse immune resistance in some tumors, but most patients do not respond to treatment or eventually experience resistance 1 Co-stimulatory pathways, such as the CD137 / 4-1BB pathway, are also important in driving effective anti-cancer immunity, and strong genetic evidence supports their role in mediating anti-cancer immune responses. 2-6 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. 7 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. 8 , 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 9-11 , providing strong protection against activation-induced T cell death and increasing cytotoxicity 12-14 .

[0004] The efficacy of anti-CD137 therapy has been demonstrated in multiple nonclinical tumor models 15-19 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 20 and increase the persistence of tumor-specific T cells 21 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, 22,23, 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. 24 .

[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. 25 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. 16,26,27 .

[0006] Mesothelin (MSLN) is a 40 kD membrane-bound protein that is overexpressed in various cancers, including mesothelioma, ovarian cancer, lung cancer, and pancreatic cancer. 28-39 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. 40 .

[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) form, in which the Fab part binds to the tumor antigen MSLN, and the modified CH3 domain binds to CD137. M9657 has a human IgG1-LALA main chain, which does not bind to Fcγ receptors, but retains FcRn binding and IgG-like pharmacokinetics (PK). The high expression of MSLN on tumor cells increases the binding of antibodies to tumor cells, the cross-linking of antibody molecules, and the interaction of antibody molecules with CD137 trimers, thereby increasing CD137 excitement. Therefore, aggregated M9657 can serve 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] Monoclonal antibodies that block the interaction between programmed cell death protein 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1), such as anti-PD-1 / PD-L1 antibodies, can enhance the immune response against cancer and are promising immune checkpoint antagonists. Currently, the FDA has approved eight checkpoint inhibitors, including six anti-PD-1 / PD-L1 monoclonal antibodies, one anti-CTLA-4 monoclonal antibody, and one LAG3 monoclonal antibody, for the treatment of more than a dozen major cancer types. 41,42 The successful application of anti-PD-1 / PD-L1 monoclonal antibodies in various clinical trials has demonstrated its great potential in cancer immunotherapy. However, clinical results are not always satisfactory, often showing large individual differences between patients, and usually only a few patients respond to treatment.

[0009] Therefore, combination therapy has become a new research focus for the development of therapies based on PD-1 / PD-L1 blockade. Anti-PD-1 / PD-L1 antibodies and CD137 agonists have different and complementary mechanisms of action in cancer immunotherapy. Specifically, anti-PD-1 / PD-L1 antibodies can eliminate negative signals that weaken T cell activation or inhibit effector T cell activity at the tumor site during the priming process, while CD137 agonists enhance T cell activation via antigen presentation and cytokine secretion. The combination of PD-1 / PD-L1 blockade and CD137 agonists has been shown to result in improved T cell activation. 43,44 . Summary of the invention

[0010] As described in the Background section, the combination of PD-1 / PD-L1 inhibitors and CD137 agonists has been shown to improve T cell activation. However, the effect of this combination in the treatment of cancer patients is not limited to the tumor microenvironment, which raises concerns about the efficacy and specificity of this treatment. The development of CD137 agonist molecules as anticancer therapies has also been hampered by concerns about liver inflammation and clinical efficacy, while anti-PD-1 / PD-L1 monoclonal antibody therapies have shown large differences in outcomes between patients.

[0011] The inventors recognized the need to enhance target-specific T cell activation and anti-tumor activity, and that this could be achieved by combining MSLN expression-dependent CD137 co-stimulation with PD-1 / PD-L1 inhibition. Unexpectedly, the inventors were able to demonstrate that the combination of an antibody molecule that binds MSLN and CD137 and a PD-1 / PD-L1 inhibitor produced an anti-tumor effect in a mouse tumor model that was greater than the sum of the anti-tumor effects observed when mice were treated with either an antibody molecule that binds MSLN and CD137 or a PD-1 / PD-L1 inhibitor alone. In other words, the anti-tumor effect of the combination therapy is not only additive, but also synergistic. This was unexpected. 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. 45 . Therefore, the present inventors have found that the combination of an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor synergistically enhances the anti-tumor effect in a mouse tumor model. When human patients are treated with a combination of an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor, a similar synergistic anti-tumor effect is expected.

[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 mouse MSLN and the modified CH3 domain 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 and mouse CD137 is similar to that of M9657 to human MSLN and human CD137.

[0013] As already outlined above, the inventors demonstrated that the combination of FS122m and anti-mPD-1 was able to slow tumor growth or reduce tumor volume in E0771, JC and Eph4-1424 mouse tumor models to a greater extent than the sum of the tumor growth slowdown or tumor volume reduction observed when mice were treated with FS122m or anti-mPD-1 alone. The inventors also demonstrated that combined treatment with FS122m and anti-mPD-1 increased the median survival in the same mouse tumor model and increased the percentage of mice with complete tumor regression compared to the sum of the increases in the median survival or percentage of mice with complete tumor regression observed when mice were treated with FS122m or anti-mPD-1 alone. Thus, the inventors demonstrated that the combination of FS122m and anti-mPD-1 synergistically enhanced anti-tumor activity in E0771, JC and Eph4-1424 mouse tumor models, as measured by tumor growth slowdown / 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 PD-1 / PD-L1 inhibitors enhances anti-tumor activity in a synergistic manner. These findings suggest that combination therapy with antibody molecules that bind to MSLN and CD137 with PD1 / PD-L1 inhibitors is a new therapeutic strategy to improve cancer treatment.

[0015] The inventors were also able to demonstrate that the combination of M9657 and the anti-PD-1 antibody pembrolizumab increased T cell activation, tumor cell killing, and cytokine release in vitro in a synergistic manner. This supports the expectation that the combination of a bispecific anti-MSLN / CD137 antibody molecule and a PD-1 / PD-L1 inhibitor will also show a synergistic anti-tumor effect in the treatment of cancer in human patients.

[0016] Therefore, the present invention provides an antibody molecule that binds to MSLN and CD137, which is used in a method of treating cancer in a patient, wherein the method comprises administering an antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor. The present invention also relates to a PD-1 / PD-L1 inhibitor, which is used in a method of treating cancer in a patient, wherein the method comprises administering a PD-1 / PD-L1 inhibitor in combination with an antibody molecule that binds to MSLN and CD137.

[0017] 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.

[0018] 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.

[0019] 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 CH3 domain of the antibody molecule has the sequence shown in SEQ ID NO:86.

[0020] 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.

[0021] A number of Fab regions that bind to MSLN are known from WO 2020 / 011976. An 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].

[0022] Many bispecific antibody molecules that bind to MSLN and CD137 are also 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).

[0023] PD-1 / PD-L1 inhibitors inhibit the PD-1 immune checkpoint. PD-1 / PD-L1 inhibitors can inhibit PD-1 activation directly or indirectly, but preferably directly inhibit PD-1 activation. PD-1 activation can be directly inhibited by inhibiting the binding of PD-L1 to PD-1 and / or by inhibiting PD-L1-mediated PD-1 activation. PD-1 activation can be indirectly inhibited, for example, by reducing PD-L1 expression.

[0024] PD-1 / PD-L1 inhibitors include small molecules, peptides and antibody molecules. Antibody molecules herein include immunoglobulins, such as IgG1, chimeric antibody molecules, antibody fusion proteins or antigen-binding antibody fragments, such as scFv, Fab, Fcab, VhH, monovalent IgG, double antibodies, three antibodies, immunoglobulin new antigen receptors (IGNAR), variable domains of single-domain shark new antigen receptors (V-NAR), human chimeric IgG (hcIgG), mini antibodies or nano antibodies. In a preferred embodiment, the PD-1 / PD-L1 inhibitor is an antibody molecule or an antigen-binding fragment thereof that binds to PD-1 or PD-L1.

[0025] The antibody molecule or antigen-binding fragment thereof that binds to PD-1 can be selected from: nivolumab, pembrolizumab, cemiplimab, dostarlimab, sintilimab, camrelizumab, toripalimab, tislelizumab, spartalizumab, zimberelimab, penpulimab and candonilimab. In a preferred embodiment, the antibody molecule or antigen-binding fragment thereof that binds to PD-1 can be selected from: nivolumab, pembrolizumab and cemiplimab. More preferably, the antibody molecule that binds to PD-1 is pembrolizumab. The heavy and light chain sequences of pembrolizumab are known in the art and are shown in SEQ ID NO: 93 and SEQ ID NO: 94, respectively.

[0026] The antibody molecule or antigen-binding fragment thereof that binds to PD-L1 can be selected from: avelumab, atezolizumab, durvalumab, sugemalimab and envafolimab. Preferably, the antibody molecule or antigen-binding fragment thereof that binds to PD-L1 can be selected from: avelumab, atezolizumab and durvalumab. More preferably, the antibody molecule that binds to PD-L1 is avelumab.

[0027] Small molecule inhibitors of PD-1 / PD-L1 can be selected from: BMS202, CA-170, fraxinellone, BMS-1166, N-deacetylated BMS-202, BMS-1001 hydrochloride, INCB086550, tomivosertib, PD-1 / PD-L1-IN-9, PROTAC PD-1 / PD-L1 degrader-1, PD-L1-IN-1, sulfamethoxazole, PD-1-IN-17, PD-1 / PD-L1-IN-10, PD-1-IN-24, BMS-8, evixapodlin, PD-1-IN-18, PD-1-IN-17TFA, BMS-1166 hydrochloride, ARB272572, PD-1, PD-L1-IN-13, sulindac sodium, PD-1 / PD-L1-IN 5. PD-1-IN-22, PD-1 / PD-L1-IN-NP19, PD-1 / PD-L1-IN-27, PD-1-IN-20, PD-1 / PD-L1-IN-23, PD-1 / PD-L1-IN 5TFA, PD-1 / PD-L1-IN 6. PD-1 / PD-L1-IN-14, PD-1 / PD-L1-IN-26, PD1-PDL1-IN 1. [D-Leu-4]-OB3、PD-1 / PD-L1-IN-16、PD-1 / PD-L1-IN-20、PD-1 / PD-L1-IN-19、PD-1 / PD-L1-IN-17、BMSpep-5 7. PD-1 / PD-L1-IN-24, PD-1 / PD-L1-IN-21, HE-S2, PD-1 / PD-L1-IN-22, PD-1 / PD-L1-IN-18 and PD-1 / PD-L1-IN-15.

[0028] The membrane-bound protein mesothelin (MSLN) has been shown to be expressed in several cancers. Specifically, ovarian cancer, pancreatic adenocarcinoma, mesothelioma, and non-small cell lung cancer 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 results 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.

[0029] Therefore, the cancer to be treated is preferably a cancer that expresses or has been determined to express MSLN. Preferably, the cancer is selected from ovarian cancer, pancreatic adenocarcinoma, mesothelioma, cervical cancer and non-small cell lung cancer.

[0030] As described above, the combination of FS122m and anti-mPD-1 produced greater anti-tumor activity in the E0771, JC, and Eph4-1424 mouse tumor models than the sum of the anti-tumor activity observed when mice were treated with FS122m or anti-PD-1 alone. In other words, the anti-tumor effect of the combination was synergistic.

[0031] Therefore, in one embodiment, treatment with an antibody molecule that binds MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor produces an anti-tumor effect greater than the anti-tumor effect observed when treating a patient with an antibody molecule that binds MSLN and CD137 or a PD-1 / PD-L1 inhibitor alone. Preferably, treatment with an antibody molecule that binds MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor produces an anti-tumor effect greater than the sum of the anti-tumor effects observed when treating a patient with an antibody molecule that binds MSLN and CD137 or a PD-1 / PD-L1 inhibitor alone. The anti-tumor effect may be tumor growth inhibition or slowing. Therefore, the anti-tumor effect may be a reduction in tumor volume. The anti-tumor effect may be an increase in the median survival of the patient. The anti-tumor effect may be an increase in the percentage of patients who experience complete tumor regression (such as a clinical complete response or a pathological complete response). The determination of these anti-tumor effects is within the capabilities of the technician.

[0032] The bispecific antibody molecules and PD-1 / PD-L1 inhibitors that bind to MSLN and CD137 can be administered to the subject in any suitable manner. Therefore, in one embodiment, the antibody molecules and / or PD-1 / PD-L1 inhibitors that bind to MSLN and CD137 are administered parenterally. The antibody molecules and / or PD-1 / PD-L1 inhibitors that bind to MSLN and CD137 can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally. Alternatively, the antibody molecules and / or PD-1 / PD-L1 inhibitors that bind to MSLN and CD137 can be administered by injection or infusion.

[0033] The antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 can be administered parenterally. The antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 can be administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0034] The antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor 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 PD-1 / PD-L1 inhibitor can be administered to the patient simultaneously or sequentially, but is preferably administered sequentially.

[0035] When the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor 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 administered sequentially to the patient on the same day.

[0036] The present invention also provides a method for treating cancer, comprising administering an antibody molecule that binds MSLN and CD137 and a PD-1 / PD-L1 inhibitor 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 PD-1 / PD-L1 inhibitor 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.

[0037] 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 PD-1 / PD-L1 inhibitor. The present invention also provides the use of a PD-1 / PD-L1 inhibitor in the manufacture of a medicament for treating cancer, wherein the PD-1 / PD-L1 inhibitor is administered in combination with an antibody molecule that binds to MSLN and CD137.

[0038] The present invention also provides a kit, which includes an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient, as well as a PD-1 / PD-L1 inhibitor and a pharmaceutically acceptable excipient.

[0039] The present invention therefore provides:

[0040] [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 PD-1 / PD-L1 inhibitor.

[0041] [2] A PD-1 / PD-L1 inhibitor for use in a method of treating cancer in a patient, wherein the method comprises administering the PD-1 / PD-L1 inhibitor in combination with an antibody molecule that binds to MSLN and CD137.

[0042] [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 PD-1 / PD-L1 inhibitor.

[0043] [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 PD-1 / PD-L1 inhibitor.

[0044] [5] Use of a PD-1 / PD-L1 inhibitor in the preparation of a medicament for treating cancer, wherein the PD-1 / PD-L1 inhibitor is administered in combination with an antibody molecule that binds to MSLN and CD137

[0045] [6] A kit comprising

[0046] (a) an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient; and

[0047] (b) a PD-1 / PD-L1 inhibitor and a pharmaceutically acceptable excipient.

[0048] [7] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit used according to [1] to [6],

[0049] The antibody molecules that bind to MSLN and CD137 include:

[0050] (a) MSLN antigen binding site based on complementarity determining regions (CDRs); and

[0051] (b) The CD137 antigen binding site located in the CH3 domain of the antibody molecule.

[0052] [8] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit according to [7], wherein the antibody molecule that binds to MSLN and CD137 comprises two or more of the following

[0053] (a) MSLN antigen binding site based on complementarity determining regions (CDRs); and

[0054] (b) The CD137 antigen binding site located in the CH3 domain of the antibody molecule.

[0055] [9] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit used 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.

[0056]

[10] The antibody molecule or PD-1 / PD-L1 inhibitor, 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.

[0057]

[11] The antibody molecule or PD-1 / PD-L1 inhibitor, 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.

[0058]

[12] The antibody molecule or PD-1 / PD-L1 inhibitor, 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.

[0059]

[13] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [7] to

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

[0060] (i) SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-271], respectively;

[0061] (ii) SEQ ID NOs 20, 22, 24, 12, 14 and 28, respectively [FS28-024-052];

[0062] (iii) SEQ ID NOs 4, 6, 8, 12, 14 and 34 [FS28-256-021], respectively;

[0063] (iv) SEQ ID NOs 4, 6, 8, 12, 14 and 39 [FS28-256-012], respectively;

[0064] (v) SEQ ID NOs 43, 6, 45, 12, 14 and 34 [FS28-256-023], respectively;

[0065] (vi) SEQ ID NOs 4, 6, 8, 12, 14 and 49 [FS28-256-024], respectively;

[0066] (vii) SEQ ID NOs 43, 6, 45, 12, 14 and 49 [FS28-256-026], respectively;

[0067] (viii) SEQ ID NOs 4, 6, 8, 12, 14 and 16 [FS28-256-027], respectively;

[0068] (ix) SEQ ID NOs 53, 6, 55, 12, 14 and 34 [FS28-256-001], respectively;

[0069] (x) SEQ ID NOs 53, 6, 55, 12, 14 and 49 [FS28-256-005], respectively;

[0070] (xi) SEQ ID NOs 60, 6, 62, 12, 14 and 39, respectively [FS28-256-014];

[0071] (xii) SEQ ID NOs 43, 6, 45, 12, 14 and 39, respectively [FS28-256-018];

[0072] (xiii) SEQ ID NOs 67, 6, 55, 12, 14 and 39 [FS28-256], respectively;

[0073] (xiv) SEQ ID NOs 21, 23, 72, 12, 14 and 28, respectively [FS28-024-051];

[0074] (xv) SEQ ID NOs 21, 23, 77, 12, 14 and 28, respectively [FS28-024-053]; or

[0075] (xvi) SEQ ID NOs 21, 23, 82, 12, 14 and 28, respectively [FS28-024]; and

[0076] 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.

[0077]

[14] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit used according to

[13] ,

[0078] in

[0079] (i) the first sequence is located between positions 14 and 17 of the CH3 domain of the antibody molecule; and / or

[0080] (ii) wherein the second sequence is located between 91 and 99 of the CH3 domain of the antibody molecule; and

[0081] The amino acid residues are numbered according to the IMGT numbering scheme.

[0082]

[15] The antibody molecule or PD-1 / PD-L1 inhibitor, 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.

[0083]

[16] The antibody molecule or PD-1 / PD-L1 inhibitor, 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.

[0084]

[17] The antibody molecule or PD-1 / PD-L1 inhibitor, 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:

[0085] (i) FS22-172-003-AA / FS28-256-271 shown in SEQ ID NOs 2 and 10, respectively;

[0086] (ii) FS22-172-003-AA / FS28-024-052 shown in SEQ ID NOs 18 and 26, respectively;

[0087] (iii) FS22-172-003-AA / FS28-256-021 shown in SEQ ID NOs 30 and 32, respectively;

[0088] (iv) FS22-172-003-AA / FS28-256-012 shown in SEQ ID NOs 36 and 37, respectively;

[0089] (v) FS22-172-003-AA / FS28-256-023 shown in SEQ ID NOs 41 and 32, respectively;

[0090] (vi) FS22-172-003-AA / FS28-256-024 shown in SEQ ID NOs 30 and 47, respectively;

[0091] (vii) FS22-172-003-AA / FS28-256-026 shown in SEQ ID NOs 41 and 47, respectively;

[0092] (viii) FS22-172-003-AA / FS28-256-027 shown in SEQ ID NOs 30 and 10, respectively;

[0093] (ix) FS22-172-003-AA / FS28-256-001 shown in SEQ ID NOs 51 and 32, respectively;

[0094] (x) FS22-172-003-AA / FS28-256-005 shown in SEQ ID NOs 51 and 47, respectively;

[0095] (xi) FS22-172-003-AA / FS28-256-014 shown in SEQ ID NOs 58 and 37, respectively;

[0096] (xii) FS22-172-003-AA / FS28-256-018 shown in SEQ ID NOs 41 and 37, respectively;

[0097] (xiii) FS22-172-003-AA / FS28-256 shown in SEQ ID NOs 65 and 37, respectively;

[0098] (xiv) FS22-172-003-AA / FS28-024-051 shown in SEQ ID NOs 70 and 26, respectively;

[0099] (xv) FS22-172-003-AA / FS28-024-053 shown in SEQ ID NOs 75 and 26, respectively; or

[0100] (xvi) FS22-172-003-AA / FS28-024 shown in SEQ ID NOs 80 and 26, respectively

[0101]

[18] The antibody molecule or PD-1 / PD-L1 inhibitor, 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].

[0102]

[19] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit according to [1] to

[18] , wherein the PD-1 / PD-L1 inhibitor is selected from small molecule inhibitors, peptides, antibodies, chimeric antibodies, antibody fusion proteins and antigen-binding fragments thereof.

[0103]

[20] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit used according to

[19] , wherein the antigen binding fragment is selected from scFv, Fab, Fcab, VhH, monovalent IgG, diabody or triabody, immunoglobulin new antigen receptor (IGNAR), single domain shark new antigen receptor variable domain (V-NAR), hIgG, minibody or nanobody.

[0104]

[21] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[20] , wherein the PD-1 / PD-L1 inhibitor is an antibody or an antigen-binding fragment thereof that binds to PD-1 or PD-L1.

[0105]

[22] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[21] , wherein the antibody or antigen-binding fragment thereof that binds to PD-1 or PD-L1 is an IgG, IgA, IgE, IgM molecule or a fragment thereof.

[0106]

[23] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[21] or

[22] , wherein the antibody or antigen-binding fragment thereof that binds to PD-1 is selected from nivolumab, pembrolizumab and cemiplizumab.

[0107]

[24] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[23] , wherein the antibody or antigen-binding fragment thereof that binds to PD-1 is pembrolizumab.

[0108]

[25] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[21] or

[22] , wherein the antibody or antigen-binding fragment thereof that binds to PD-L1 is selected from avelumab, atezolizumab and durvalumab.

[0109]

[26] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[25] , wherein the antibody or antigen-binding fragment thereof that binds to PD-L1 is avelumab.

[0110]

[27] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

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

[0111]

[28] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit according to [1] to

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

[0112]

[29] The antibody molecule or PD-1 / PD-L1 inhibitor, 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 PD-1 / PD-L1 inhibitor produces higher anti-tumor activity than monotherapy with the antibody molecule that binds to MSLN and CD137 or monotherapy with the PD-1 / PD-L1 inhibitor.

[0113]

[30] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[29] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor produces an anti-tumor activity that is higher than the sum of the anti-tumor activities of monotherapy with the antibody molecule that binds to MSLN and CD137 and monotherapy with the PD-1 / PD-L1 inhibitor.

[0114]

[31] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[30] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor produces a greater reduction in tumor growth, tumor volume, median survival and / or number of complete tumor regressions than monotherapy with the antibody molecule that binds to MSLN and CD137 or monotherapy with the PD-1 / PD-L1 inhibitor.

[0115]

[32] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[31] , wherein treatment with the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor produces a greater tumor growth reduction, tumor volume reduction, median survival increase and / or number of complete tumor regressions than the sum of tumor growth reduction, tumor volume reduction, median survival increase and / or number of complete tumor regressions treated with monotherapy of the antibody molecule that binds to MSLN and CD137 and monotherapy of the PD-1 / PD-L1 inhibitor.

[0116]

[33] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[32] , wherein the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered parenterally.

[0117]

[34] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit according to

[33] , wherein the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally

[0118]

[35] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[33] or

[34] , wherein the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered by injection or infusion.

[0119]

[36] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[32] , wherein the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered parenterally.

[0120]

[37] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit according to

[36] , wherein the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0121]

[38] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to [1] to

[37] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient simultaneously or sequentially.

[0122]

[39] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[38] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient sequentially.

[0123]

[40] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[39] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient within no more than 4 days of each other.

[0124]

[41] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[39] or

[40] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient within no more than 3 days of each other.

[0125]

[42] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[39] to

[41] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient within no more than 2 days of each other.

[0126]

[43] The antibody molecule or PD-1 / PD-L1 inhibitor, method, use or kit for use according to

[39] to

[42] , wherein the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient on the same day.

[0127]

[44] The antibody molecule or PD-1 / PD-L1 inhibitor or method for use according to [1] to [3] and [7] to

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

[0128] Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which:

[0129] Figure 1 Shows the results of a bioluminescent reporter assay, in which the bioluminescent signal was measured as a marker of PD-1 in co-culture with CHO tumor cells expressing PD-L1aAPC / CHO-K1 and MSLN + Function of NF-κB expression in CD137 effector cells. Cells were treated with different concentrations of M9657, M9657+pembrolizumab, M9657+anti-HEL-hIgG1-LALA antibody (isotype control), CD137L+pembrolizumab or CD137L+anti-HEL-hIgG1-LALA antibody. Treatment with M9657+pembrolizumab produced significantly higher bioluminescent signals than M9657, M9657+anti-HEL-hIgG1-LALA antibody, CD137L+pembrolizumab or CD137L+anti-HEL-hIgG1-LALA antibody. Mean ± standard error of the mean (SEM) is shown, and data represent 3 replicates.

[0130] Figure 2 Results are shown for an assay measuring CD8 +Cytotoxicity and cytokine release of T cells against target cells. Cytotoxicity was measured as % killing of target cells (A) and the normalized area under the curve (AUC) of the results in (A) (B). Cytokine release was measured as the INFγ level in the supernatant (C) and the normalized area under the curve (AUC) of the results in (C) (D). Cells were treated with different concentrations of M9657+pembrolizumab, M9657, anti-HEL-hIgG1-LALA antibody (isotype control) or BiTE (anti-CD3×anti-EGFR BiTE). Treatment with M9657+pembrolizumab increased CD8 + T cell cytotoxicity. Killing % (A) and AUC (B) were measured in cells from five different donors and are presented as mean ± SEM. IFNγ concentration (C) and AUC (D) were measured in cells from six different donors and are presented as mean ± SEM.

[0131] Figure 3 The therapeutic efficacy in the E0771 orthotopic mouse breast cancer model of C57BL / 6 mice is shown. The progression of the mouse cancer model was measured as the average tumor volume (A), median survival (B), % weight change (C), and changes in individual tumor volume over time (D). Mice were treated with anti-HEL-hIgG1-LALA antibody (isotype control), FS122m, anti-mPD-1, or FS122m+anti-mPD-1. Treatment with FS122m+anti-mPD-1 showed a decrease in average tumor volume over time, while FS122m and anti-mPD-1 monotherapy only slowed tumor growth (A) compared to anti-HEL-hIgG1-LALA isotype control during the study. FS122m and anti-mPD-1 combination therapy also prolonged median survival relative to FS122m and anti-mPD-1 monotherapy (B), and induced complete tumor regression in 7 of 9 mice, compared with no complete tumor regression in 9 mice treated with FS122m monotherapy and only 1 complete tumor regression in 9 mice treated with anti-mPD-1 monotherapy. 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.

[0132] Figure 4 The therapeutic efficacy in the JC subcutaneous mouse mammary tumor model of BALB / c mice is shown. The progression of the mouse cancer model was measured as the average tumor volume (A), median survival (B), weight change % (C), and individual tumor volume changes over time (D). Mice were treated with anti-HEL-hIgG1-LALA, FS122m, anti-mPD-1, or FS122m+anti-mPD-1. During the study, treatment with FS122m+anti-mPD-1 slowed tumor growth to a much greater extent than FS122m and anti-mPD-1 monotherapy compared to anti-HEL-hIgG1-LALA isotype control (A). FS122m and anti-mPD-1 combination therapy also prolonged median survival relative to FS122m and anti-mPD-1 monotherapy (B), and induced complete tumor regression in 3 of 10 mice, compared to no mice treated with FS122m monotherapy or anti-mPD-1 monotherapy. 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.

[0133] Figure 5The therapeutic efficacy in the Eph4-1424 subcutaneous mouse mammary tumor model of BALB / c mice is shown. The progression of the mouse cancer mouse model was measured as the average tumor volume over time (A), median survival (B), % weight change (C), and changes in individual tumor volume over time (D). Mice were treated with anti-HEL-hIgG1-LALA, FS122m, anti-mPD-1, or FS122m+anti-mPD-1. Treatment with FS122m+anti-mPD-1 showed a decrease in average tumor volume over time, while FS122m and anti-mPD-1 monotherapy only slowed tumor growth (A) compared to treatment with anti-HEL-hIgG1-LALA isotype controls during the study. Combination treatment with FS122m and anti-mPD-1 also prolonged median survival relative to FS122m and anti-mPD-1 monotherapy (B), and induced complete tumor regression in 10 of 10 mice, while only 1 of 10 mice treated with FS122m monotherapy was observed to have complete tumor regression, and 6 of 10 mice treated with anti-mPD-1 monotherapy were observed to have complete tumor regression. Body weight changes were comparable between all treatments (including anti-HEL-hIgG1LALA 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, where ** = 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

[0134] The present invention relates to an antibody molecule that binds to MSLN and CD137, which is used for treating cancer in combination with a PD-1 / PD-L1 inhibitor. The present invention also relates to a PD-1 / PD-L1 inhibitor, which is used for treating cancer in combination with an antibody molecule that binds to MSLN and CD137.

[0135] The term "PD-1 / PD-L1 inhibitor" describes a molecule that inhibits the PD-1 immune checkpoint. PD-1 / PD-L1 inhibitors can inhibit PD-1 activation directly or indirectly, but preferably directly inhibit PD-1 activation. PD-1 activation can be directly inhibited by inhibiting the binding of PD-L1 to PD-1 and / or by inhibiting PD-L1-mediated PD-1 activation. PD-1 activation can be indirectly inhibited by, for example, reducing PD-L1 expression. PD-1 / PD-L1 inhibitors can be small molecule inhibitors, peptides, antibodies, chimeric antibodies, antibody fusion proteins, or antibody fragments, such as scFv, Fab, Fcab, VhH, monovalent IgG, double antibodies or three antibodies, IGNAR, V-NAR, hcIgG, mini antibodies, or nano antibodies. For example, a PD-1 / PD-L1 inhibitor can be an antibody or an antigen-binding fragment thereof that binds to PD-1 or PD-L1.

[0136] 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, M, A, E and D, and their isotype subclasses, such as IgG1, IgG2, IgG3 and IgG4, and antigen-binding 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.

[0137] The PD-1 / PD-L1 inhibitor may be natural or partially or completely synthetically produced. For example, the PD-1 / PD-L1 inhibitor may be a recombinant antibody molecule.

[0138] The antibody or antigen-binding fragment thereof that binds to PD-1 or PD-L1 may be an anti-PD-1 antibody or an antigen-binding fragment thereof. Preferably, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab and cemiplizumab. More preferably, the anti-PD-1 antibody is pembrolizumab.

[0139] The antibody or antigen-binding fragment thereof that binds to PD-1 or PD-L1 may be an anti-PD-L1 antibody or an antigen-binding fragment thereof. Preferably, the anti-PD-1 antibody is selected from avelumab, atezolizumab and durvalumab. More preferably, the anti-PD-L1 antibody is avelumab.

[0140] 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.

[0141] Therefore, the PD-1 / PD-L1 inhibitor may be any anti-PD-1 / PD-L1 antibody not listed above, which is included in the prior art or not yet included in the prior art, and can be obtained using commonly available techniques known in the art.

[0142] The term "bispecific antibody molecule" is used below to refer to an antibody molecule that binds to MSLN and CD137.

[0143] The bispecific antibody molecule can be an immunoglobulin or an antigen-binding fragment thereof.

[0144] 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.

[0145] 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.

[0146] The bispecific antibody molecule may be natural or partially or completely synthetically produced. For example, the antibody molecule may be a recombinant antibody molecule.

[0147] 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.

[0148] 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 an antigen-binding fragment thereof. In a preferred embodiment, the bispecific antibody molecule is a complete immunoglobulin molecule.

[0149] In other embodiments, the bispecific antibody molecule can be an antigen binding fragment comprising a CDR-based MSLN antigen binding site and a CD137 antigen binding site located in a constant domain. 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).

[0150] 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.

[0151] 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.

[0152] 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).

[0153] Bispecific antibody molecules may or may not carry LALA mutations. In a preferred embodiment, bispecific antibody molecules 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 antibodies without LALA mutations. LALA mutations involve replacing leucine residues (L1.3A and L1.2A) at positions 1.3 and 1.2 (according to the IMGT numbering scheme) of the CH2 domain with alanine. According to the Kabat numbering system, the LALA mutation constitutes an L247A L248A substitution. Alternatively, complement activation (C1q binding) and ADCC are known to be 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. These two types of mutations can also be combined to produce antibody molecules with further reduced ADCC or CDC activity or without ADCC or CDC activity.

[0154] 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.

[0155] In another embodiment, the antibody molecule may comprise a CH2, wherein the CH2 domain comprises an alanine residue at position 114. For example, the CH2 domain may have the amino acid sequence shown in SEQ ID NO: 91. In another embodiment, 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] For example,

[0161] (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;

[0162] (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;

[0163] (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;

[0164] (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;

[0165] (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;

[0166] (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;

[0167] (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;

[0168] (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;

[0169] (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;

[0170] (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;

[0171] (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;

[0172] (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;

[0173] (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;

[0174] (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;

[0175] (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

[0176] (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;

[0177] The CDR sequences are defined according to the IMGT numbering scheme.

[0178] For example,

[0179] (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;

[0180] (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 28, respectively;

[0181] (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;

[0182] (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;

[0183] (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;

[0184] (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;

[0185] (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;

[0186] (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;

[0187] (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;

[0188] (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;

[0189] (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;

[0190] (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;

[0191] (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;

[0192] (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;

[0193] (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

[0194] (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;

[0195] The CDR sequences are defined according to the IMGT numbering scheme.

[0196] For example,

[0197] (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;

[0198] (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;

[0199] (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;

[0200] (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;

[0201] (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;

[0202] (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;

[0203] (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;

[0204] (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;

[0205] (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;

[0206] (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;

[0207] (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;

[0208] (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;

[0209] (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;

[0210] (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;

[0211] (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

[0212] (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;

[0213] The CDR sequences are defined according to the Kabat numbering scheme.

[0214] (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;

[0215] (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;

[0216] (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;

[0217] (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;

[0218] (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;

[0219] (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;

[0220] (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;

[0221] (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;

[0222] (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;

[0223] (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;

[0224] (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;

[0225] (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;

[0226] (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;

[0227] (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;

[0228] (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

[0229] (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;

[0230] The CDR sequences are defined according to the Kabat numbering scheme.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] The bispecific antibody molecule of the present invention comprises a CD137 antigen binding site. The CD137 antigen binding site is located in the constant domain of the antibody molecule, preferably in the CH3 domain. The CD137 antigen binding site is 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.

[0235] The CD137 antigen binding site of the bispecific antibody molecule includes 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 NO 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.

[0236] 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.

[0237] 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.

[0238] In a preferred embodiment, the bispecific antibody molecule comprises the heavy chain and / or light chain, preferably the heavy chain and light chain, of the following antibodies:

[0239] (i) FS22-172-003-AA / FS28-256-271 shown in SEQ ID NOs 2 and 10, respectively;

[0240] (ii) FS22-172-003-AA / FS28-024-052 shown in SEQ ID NOs 18 and 26, respectively;

[0241] (iii) FS22-172-003-AA / FS28-256-021 shown in SEQ ID NOs 30 and 32, respectively;

[0242] (iv) FS22-172-003-AA / FS28-256-012 shown in SEQ ID NOs 36 and 37, respectively;

[0243] (v) FS22-172-003-AA / FS28-256-023 shown in SEQ ID NOs 41 and 32, respectively;

[0244] (vi) FS22-172-003-AA / FS28-256-024 shown in SEQ ID NOs 30 and 47, respectively;

[0245] (vii) FS22-172-003-AA / FS28-256-026 shown in SEQ ID NOs 41 and 47, respectively;

[0246] (viii) FS22-172-003-AA / FS28-256-027 shown in SEQ ID NOs 30 and 10, respectively;

[0247] (ix) FS22-172-003-AA / FS28-256-001 shown in SEQ ID NOs 51 and 32, respectively;

[0248] (x) FS22-172-003-AA / FS28-256-005 shown in SEQ ID NOs 51 and 47, respectively;

[0249] (xi) FS22-172-003-AA / FS28-256-014 shown in SEQ ID NOs 58 and 37, respectively;

[0250] (xii) FS22-172-003-AA / FS28-256-018 shown in SEQ ID NOs 41 and 37, respectively;

[0251] (xiii) FS22-172-003-AA / FS28-256 shown in SEQ ID NOs 65 and 37, respectively;

[0252] (xiv) FS22-172-003-AA / FS28-024-051 shown in SEQ ID NOs 70 and 26, respectively;

[0253] (xv) FS22-172-003-AA / FS28-024-053 shown in SEQ ID NOs 75 and 26, respectively; or

[0254] (xvi) FS22-172-003-AA / FS28-024 shown in SEQ ID NOs 80 and 26, respectively.

[0255] 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.

[0256] 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 sequence changes or mutations and screening methods. 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).

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] In one 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.

[0265] In embodiments where one or more amino acids are substituted with another amino acid, the substitution may be 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.

[0266]

[0267] In some embodiments, the substitution may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may 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.

[0268] 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.

[0269] 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 or metastatic 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.

[0270] 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.

[0271] Therefore, the cancer to be treated using the bispecific antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor 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.

[0272] 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.

[0273] Cancer can be a primary or secondary cancer. Thus, antibody molecules that bind to MSLN and CD137 as described herein can be used in combination with a PD-1 / PD-L1 inhibitor for treating a method of treating cancer in an individual, wherein the cancer is a primary tumor and / or a secondary or metastatic tumor.

[0274] The cancer to be treated can be a solid cancer.

[0275] 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.

[0276] 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.

[0277] The inventors showed that combination treatment with M9657 (SEQ ID NO: 2 and SEQ ID NO: 10) and pembrolizumab (SEQ ID NO: 93 and SEQ ID NO: 94) resulted in significantly higher T cell activation, CD8 + The cells showed increased tumor target cell killing and cytokine release and greater overall T cell activation, CD8 + The inventors also showed that the combination therapy of FS122m and anti-mPD-1 resulted in 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 observed when mice were treated with FS122m or anti-mPD-1 alone. Thus, the inventors found that the combination treatment with M9657 and pembrolizumab increased T cell activation, CD8 + Similarly, combination treatment with F122m (SEQ ID NO: 84 and SEQ ID NO: 85) and anti-mPD-1 synergistically slowed tumor growth, reduced tumor volume, increased median survival, and increased the number of complete tumor regressions.

[0278] In one embodiment, the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor results in a 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. Preferably, the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor results in a statistically significant 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 treatment with an antibody molecule that binds to MSLN and CD137 or monotherapy treatment with a PD-1 / PD-L1 inhibitor. More preferably, the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor results in a greater tumor growth reduction, tumor volume reduction, median survival increase and / or increase in the number of complete tumor regressions than the sum of the tumor growth reduction, tumor volume reduction, median survival increase and / or increase in the number of complete tumor regressions treated with monotherapy with the antibody molecule that binds to MSLN and CD137 and monotherapy with the PD-1 / PD-L1 inhibitor.

[0279] In another embodiment, an antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor increases T cell activation, CD8 + Preferably, the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor results in statistically significantly greater T cell activation, CD8 T cells, and cytokine release than monotherapy with a bispecific antibody molecule that binds to MSLN and CD137 or a PD-1 / PD-L1 inhibitor. + More preferably, the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor results in T cell activation, CD8 T cells, and cytokine release compared to monotherapy treatment with an antibody molecule that binds to MSLN and CD137 and monotherapy treatment with a PD-1 / PD-L1 inhibitor. + The sum of tumor target cell killing and cytokine release by cells is greater than that of T cell activation, CD8 + Tumor target cell killing and cytokine release of cells.

[0280] Bio-Glo can be measured in the presence of antibody molecules that bind to MSLN and CD137 in combination with PD-1 / PD-L1 inhibitors. TM The ability of antibody molecules binding to MSLN and CD137 in combination with PD-1 / PD-L1 inhibitors to activate T cells was determined by the maximum bioluminescent signal emitted by T cells in a -NL luciferase assay.

[0281] The ability of the antibody molecule that binds MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor to activate T cells can also be determined by measuring IFNγ release in a cytokine release assay in the presence of the bispecific antibody molecule in combination with a PD-1 / PD-L1 inhibitor.

[0282] In one embodiment, the antibody molecule binding to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered as a first-line ("front-line") treatment (e.g., initial or first treatment). In another embodiment, the antibody molecule binding to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered as a second-line treatment (e.g., after initial treatment with the same or different therapy, including after relapse and / or when the first treatment fails).

[0283] 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 PD-1 / PD-L1 inhibitors 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 other than 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 administration routes, such as oral, intranasal, vaginal, rectal, sublingual or topical.

[0284] Therefore, in some embodiments, the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered parenterally. The antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or spinally. Alternatively, the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 can be administered by injection or infusion.

[0285] In other embodiments, the antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 is administered non-parenterally. The antibody molecule and / or PD-1 / PD-L1 inhibitor that binds to MSLN and CD137 can be administered orally, intranasally, vaginally, rectally, sublingually or topically.

[0286] "Simultaneous administration" describes the simultaneous administration of an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor in the same formulation or in separate formulations. "Sequential administration" refers to the administration of a bispecific antibody molecule and a PD-1 / PD-L1 inhibitor in separate formulations separated by time.

[0287] Therefore, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor can be part of the same formulation or part of separate formulations. Preferably, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are provided as separate formulations.

[0288] In one embodiment of the invention, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered simultaneously. For example, the bispecific antibody molecule that binds to MSLN and CD137 can be administered together with the PD-1 / PD-L1 inhibitor in the same formulation. Alternatively, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor can be administered in separate formulations immediately before or after each other.

[0289] Preferably, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor are administered to the patient sequentially, more preferably, the antibody molecule that binds to MSLN and CD137 and the PD-1 / PD-L1 inhibitor 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.

[0290] The present invention also relates to a method for treating cancer, comprising administering an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor to an individual in need thereof.

[0291] Administration can be in a "therapeutically effective amount", which is sufficient to show benefit to an individual. The actual amount applied, as well as the rate and time course of administration treatment 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.

[0292] 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 PD-1 / PD-L1 inhibitor.

[0293] A kit is also provided, comprising an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor. Preferably, the kit may include an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient, as well as a PD-1 / PD-L1 inhibitor and a pharmaceutically acceptable excipient.

[0294] 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 PD-1 / PD-L1 inhibitor. The package may include instructions for use of the antibody molecule that binds to MSLN and CD137 in combination with a PD-1 / PD-L1 inhibitor for treating cancer in an individual.

[0295] In one embodiment, the kit may 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 PD-1 / PD-L1 inhibitor. Preferably, the kit may comprise 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 PD-1 / PD-L1 inhibitor and a pharmaceutically acceptable excipient. More preferably, the kit may further comprise a package insert comprising instructions for using the drug to treat cancer in an individual.

[0296] In another embodiment, the kit can be a package comprising a first container and a second container, the first container comprising an antibody molecule that binds to MSLN and CD137, and the second container comprising a PD-1 / PD-L1 inhibitor. The first container may 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 may contain at least one dose of a drug comprising a PD-1 / PD-L1 inhibitor and a pharmaceutically acceptable excipient. The package may further contain an insert containing instructions for using the drug to treat cancer in an individual.

[0297] 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 the disclosed function, or a method or process for obtaining the disclosed results, may be used alone or in any combination of such features to implement the invention in its various forms.

[0298] 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.

[0299] 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.

[0300] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0301] 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.

[0302] 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%.

[0303] Example

[0304] Materials and methods

[0305] Compared with monotherapy with antibody molecules that bind to MSLN and CD137 or PD-1 / PD-L1 inhibitors, the effectiveness of the combination of antibody molecules that bind to MSLN and CD137 and PD-1 / PD-L1 inhibitors in enhancing anti-cancer immune responses is evaluated by studying different parameters. These parameters include in vitro T cell activation in human cells, cytotoxicity to target cells and cytokine release or in vivo average tumor volume and prolonged survival over time in mouse tumor models. These methods will be described in detail below.

[0306] Bio-Glo TM -NL luciferase assay

[0307] To investigate the combined effects of PD-1 blockade and CD137 stimulation, the Bio-Glo TM -NL luciferase assay to measure the effects of combination treatment with M9657 and pembrolizumab on PD-1 +The efficacy of NF-κB expression in CD137 effector cells. The assay was performed according to the manufacturer's instructions. The day before the assay, PD-L1 aAPC / CHO-K1 cells expressing PD-L1 and T cell activators were thawed and recovered in 14.5mL cell recovery medium (10% FBS / F-12). The cells were then plated in a white 96-well plate and incubated overnight at 37°C in a 5% CO2 incubator. On the day of the assay, M9657 (250nM 4-fold serial dilution to 0.003815nM), pembrolizumab (167.875nM), CD137L (250nM 4-fold serial dilution to 0.003815nM) and anti-HIS-tagged antibodies (125nM 4-fold serial dilution to 0.0019nM) (anti-HIS-tagged antibodies were added to CD137L for cross-linking) were prepared. CD137 ligand (CD137L) interacts with CD137 to induce T cell activation. Here CD137L is used as a positive control in the reporter assay. Antibodies against irrelevant targets (anti-HEL-hIgG1-LALA antibodies) are used as negative controls. Aspirate the culture medium from the PD-L1aAPC / CHO-K1 cells and add a total of 40 μL of the prepared antibody solution to each well. + CD137 effector cells were thawed and recovered, and 40 μL of cell suspension was added to each well. CHO cells expressing MSLN were added to the wells at a 1:1 ratio of PD-L1 aAPC / CHO-K1 (75,000 cells):CHO-MSLN cells (75,000 cells). The plates were incubated for 6 hours, and at the end of the incubation period, the Bio-Glo-NL luciferase reagent was prepared by mixing 1 volume of Bio-Glo-NL luciferase assay substrate with 50 volumes of Bio-Glo-NL luciferase assay buffer. The assay plate was removed from the incubator, 80 μL of Bio-Glo-NL reagent was added to each well, and the plate was incubated at room temperature for 5 minutes. After incubation, the plate was read using a Tecan plate reader with a luminescence setting. Statistical analysis and plotting were performed using GraphPad Prism V9 software. To calculate the efficacy of combined treatment with an antibody molecule that binds to MSLN and CD137 and a PD-1 / PD-L1 inhibitor in inducing luciferase activity compared with monotherapy with an antibody molecule that binds to MSLN and CD137 or a PD-1 / PD-L1 inhibitor, the half-maximal effective concentration (EC 50 ) at the time of antibody concentration.

[0308] Target-dependent cytotoxicity and cytokine release assays

[0309] The effects of combined PD-1 blockade and CD137 stimulation on CD8+ The efficacy of T cells in tumor target cell killing and cytokine release was monitored ex vivo. + T cell toxicity and IFNγ release against co-cultured NCI-H226 target cancer cells. NCI-H226 cells were cultured in RPMI-1640 medium containing 10% FBS. Human CD8 + T cells were purchased from Hemacare. All cells were cultured at 37°C, 5% CO2, and 95% relative humidity. NCI-H226 cells were treated with 10 ng / mL human IFNγ for 48 hours, washed and labeled with IncuCyte Cytolight Rapid Red dye the day before the experiment, and then seeded in the assay plate. + T cells were thawed and recovered in 250U / ml IL-2 for 24 hours and then added to the assay plate on the day of the experiment. (NCI-H226 cells expressed high levels of MSLN and EGFR in the presence or absence of 2pM anti-CD3×anti-EGFR BiTE. Subsequently, a fixed concentration of 2pM anti-CD3×anti-EGFR BiTE, 10μg / ml pembrolizumab and 1μM Cytolight Rapid Dye and serial dilutions of M9657 or control antibodies were added to the cells and incubated for 72 hours. In vivo imaging system detects cell growth. Cytotoxicity to target cells is detected by The killing percentage of the BiTE group alone was determined by calculating the live cell analysis system. Total cells and labeled cells were monitored using phase contrast and red fluorescence channels, respectively. IFNγ release was detected by AlphaLisa according to the manufacturer's instructions.

[0310] Mouse tumor models

[0311] Wild-type BALB / c and C57BL / 6 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 housed 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 performed in accordance with the guidelines of EMD Serono Research Institutional (Scientific Experimental Plan 17-008, 20-005) and Wuxi WuXi AppTec Animal Care and Use Committee (IACUC).

[0312] 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 on 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).

[0313] The E0771, JC, and Eph4-1424 tumor models were used to study the anti-tumor effects of the combination of antibody molecules binding to MSLN and CD137 and PD-1 / PD-L1 inhibitors compared with monotherapy with antibody molecules binding to MSLN and CD137 or PD-1 / PD-L1 inhibitors. Since M9657 does not show cross-reactivity to mouse MSLN and mouse CD137, anti-mMSLN-mCD137-huIgG1-LALA (FS122m) (SEQ ID NO: 84 and 85) was developed as an alternative antibody to M9657 for in vivo pharmacology studies. For the E0771 model, 0.5×10 6 E0771 tumor cells were orthotopically inoculated into the right mammary fat pad of female C57BL / 6 mice. When the average tumor volume reached approximately 50-100 mm 3 At 4 pm, mice were randomly assigned to treatment groups (n = 9 mice / group). For the JC model, JC tumor cells (5 × 10 6 The mice were inoculated subcutaneously (sc) in the right upper 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 Eph4-1424 model, Eph4-1424 tumor cells (1 × 10 6The mice were inoculated subcutaneously (sc) in the right upper flank of female BALB / c mice. When the average tumor volume reached approximately 50-100 mm 3 At 4 hr, mice were randomly assigned to treatment groups (n=10 mice / group).

[0314] Mortality checks were performed daily. Animals were examined daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (visual inspection only), eye / hair matting, and any other abnormal effects as specified in the scientific experimental plan. Body weights were recorded twice weekly, and any mouse that lost 20% of its initial body weight was humanely sacrificed. Mice with subcutaneous tumors reaching 2500 mm 3 If the volume reaches , the mice are humanely euthanized.

[0315] 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.

[0316] 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). Consult with veterinary staff to discuss any possible exceptions.

[0317] If the animal is found to be moribund, it is euthanized. Clinical examples of morbidity may include hunching of the animal, 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, coughing, rales), gastrointestinal signs (diarrhea lasting >2 days, jaundice). Any animal showing any of the above clinical problems is humanely killed by CO2.

[0318] 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.

[0319] 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.

[0320] Example 1: NF-κB expression

[0321] Activation of NF-κB expression is an indicator of T cell activation. When CD137 aggregates and interacts with its cognate ligand CD137L, it activates the NF-κB signaling pathway. Agonistic antibody molecules mimic the aggregation and activation of ligand-driven CD137, thereby activating the NF-κB signaling pathway. On the other hand, anti-PD-1 / PD-L1 antibodies limit the negative effects of PD-1 signaling on T cell activation by eliminating the interaction of PD-L1 with PD-1. NF-κB expression is measured as a function of luciferase activity that produces a bioluminescent signal in a luciferase reporter assay described in the Materials and Methods. The expression of luciferase in the assay is controlled by the NF-κB-sensitive promoter and is therefore directly correlated with NF-κB expression.

[0322] To investigate the combined effects of PD-1 blockade and CD137 stimulation, Bio-Glo TM -NL luciferase assay measures the effect of combination treatment with M9657 and pembrolizumab. PD-1 + CD137 effector cells were co-cultured with PD-L1aAPC / CHO K1 cells and treated with any of M9657+pembrolizumab, M9657, M9657+anti-HEL-hIgG1-LALA, CD137L+anti-HEL-hIgG1-LALA, CD137L+pembrolizumab, or CD137L+anti-HEL-hIgG1-LALA. Since the bispecific antibody molecule M9657 needs to bind to MSLN to exert its biological activity, and PD-L1 aAPC / CHO-K1 cells do not express MSLN, CHO cells expressing MSLN were added to PD-L1aAPC / CHO-K1 and CHO-MSLN cells. Luciferase activity was measured as a function of bioluminescence using a Tecan instrument and analyzed using GraphPad Prism V9 software.

[0323] Treatment with M9657 triggered a concentration-dependent bioluminescent signal, with an EC of 0.08024 nM. 50Enhanced T cell activation. Combination treatment with M9567 + pembrolizumab enhanced the bioluminescent signal compared to treatment with any of M9657, M9657 + anti-HEL-hIgG1-LALA, CD137L + anti-HEL-hIgG1-LALA, CD137L + pembrolizumab, and CD137L + anti-HEL-hIgG1-LALA. Pembrolizumab showed an EC of 0.06069 nM. 50 ( Figure 1 ).

[0324] Example 2: Cytotoxicity and cytokine release

[0325] Two other markers of T cell activation are human CD8 + T cell-mediated tumor target cell killing and cytokine release. To this end, NCI-H226 cancer cells were crossed with CD8 + T cells were co-cultured ex vivo to study the effects of combined CD137 stimulation and PD-1 blockade (Materials and Methods). Tumor target cell killing was measured as the percentage of killing of NCI-H226 cancer cells, while CD8 + Cytokine release by T cells was measured as the level of INFγ in the T cell culture supernatant.

[0326] To calculate the effect of treating co-cultured cells with M9657+pembrolizumab, M9657, isotype control (anti-HEL-hIgG1-LALA), or BiTE (anti-CD3×anti-EGFR BiTE), cytotoxicity and INFγ release were measured. Treatment with M9657 resulted in concentration-dependent tumor target cell killing and INFγ release, which was further increased in cells treated with the combination of M9657 and pembrolizumab ( Figure 2 ). CD8 + The killing of target cells by T cells increased, and the increase was particularly sharp between 10 and 100 pM ( Figure 2 A).

[0327] Example 3: In vivo proof of concept

[0328] CD137 / MSLN mAb has been shown 2 Combination with anti-PD-1 / PD-L1 antibodies resulted in improved T cell activation, target cell killing, and T cell cytokine release, and the effect of this combination was also tested in an in vivo mouse tumor mouse model.

[0329] Due to the lack of cross-reactivity between M9657 and murine CD137, an anti-mMSLN-mCD137-huIgG1-LALA (FS112m) bispecific antibody was developed as an alternative antibody for use in mouse tumor models, with a binding affinity for murine CD137 similar to that of M9657 for human CD137. FS112m was combined with anti-mPD-1 to test the anti-tumor efficacy of the two combined antibodies compared to FS112m and anti-mPD-1 monotherapy in three different mouse breast tumor models. To this end, female mice were inoculated with E0771, JC or Eph4-1424 breast cancer cells and inoculated with anti-mPD-1 monotherapy at an average tumor volume of approximately 50-100 mm. 3 Mice were inoculated, treated, and terminated as described in Materials and Methods.

[0330] In all three mouse breast tumor models, the combination of FS112m and anti-mPD-1 was compared to FS112m and anti-mPD-1 monotherapy as well as monotherapy with an anti-HEL-hlgG1-LALA isotype control antibody. The results are summarized below.

[0331] 3.1E0771 Mouse Breast Tumor Model

[0332] In the E0771 mouse mammary tumor model, FS122m and anti-mPD-1 monotherapy induced minimal or moderate tumor growth inhibition (TGI) (26.6% and 55.5%, respectively) relative to isotype control (P < 0.01, P < 0.0001, day 14, respectively) and prolonged median survival (18 and 24 days, respectively) relative to isotype control (16 days) ( Figure 3 When mice were treated with the combination of FS122m and anti-mPD1 (102%), TGI was further enhanced relative to FS122m (P < 0.0001, day 14) and anti-mPD1 (P < 0.0001, day 14) monotherapy ( Figure 3 A). Combination FS122m and anti-mPD-1 treatment also prolonged median survival ( Figure 3 B), and induced complete tumor regression in 7 out of 9 mice treated with FS122m monotherapy, compared with no complete tumor regression in 9 mice and only 1 out of 9 mice treated with anti-mPD-1 monotherapy ( Figure 3 D). Mice in the monotherapy and combination treatment groups showed no additional mortality or clinical signs, and body weight (BW) changes were comparable to those of the isotype control group, indicating that all treatments were well tolerated ( Figure 3 C).

[0333] 3.2 JC mouse breast tumor model

[0334] In the JC mouse mammary tumor model, anti-mPD1 monotherapy did not show any antitumor effect (TGI = 7%), while FS122m monotherapy showed a slight TGI (47.7%) relative to isotype control (P < 0.0001, day 22), as well as a slightly prolonged median survival (26 days vs. 33 days) relative to isotype control ( Figure 4 A, B, and D). Treatment with the combination of FS122m and anti-mPD1 increased TGI (85.1%) relative to FS122m (P < 0.0001, day 22) and anti-mPD1 (P < 0.0001, day 22) monotherapy ( Figure 4 A). Combined FS122m and anti-mPD-1 treatment also prolonged median survival (50 days) ( Figure 4 B), and induced complete tumor regression in 3 out of 10 mice, while neither FS112 nor anti-mPD-1 monotherapy achieved complete tumor regression in any of the 10 mice ( Figure 4 D). No relevant changes in body weight were observed between treatment groups compared to isotype controls, indicating that all treatments were well tolerated ( Figure 4 C).

[0335] 3.3 Eph-1424 mouse breast tumor model

[0336] The antitumor efficacy of FS122m in combination with anti-mPD1 was also evaluated in the Eph4-1424 subcutaneous mammary tumor model in BALB / c mice. Both anti-mPD1 and FS122m monotherapy showed significant antitumor effects (TGI 68.8% and 51.3%, respectively, P < 0.0001), and the median survival was significantly prolonged relative to isotype controls (43 days vs. 56 days vs. undefined) ( Figure 5 A, B, and D). Treatment with the combination of FS122m and anti-mPD1 increased TGI (105.6%) relative to FS122m (P < 0.0001, day 28) and anti-mPD1 (P < 0.0001, day 28) monotherapy ( Figure 5 A). Combined FS122m and anti-mPD-1 treatment also prolonged median survival ( Figure 5 B), and induced complete tumor regression in 10 of 10 mice, compared with only 1 of 10 mice treated with FS122m monotherapy and 6 of 10 mice treated with anti-mPD-1 monotherapy ( Figure 5 D). No relevant changes in body weight were observed between treatment groups compared to isotype controls, indicating that all treatments were well tolerated ( Figure 5C).

[0337] Overall, the combination treatment of FS122m and anti-mPD-1 produced statistically significantly higher TGI than monotherapy treatment with FS122m or anti-mPD-1. The combination of FS122m and anti-mPD-1 achieved complete tumor regression in the majority of mice in two of the three mouse breast tumor models ( Figure 3 The difference was particularly evident in the E0771 mouse tumor model, where the combination of FS122m and anti-mPD1 resulted in complete tumor regression in 7 of 9 mice compared to 1 of 9 mice treated with anti-mPD1 monotherapy and no mice in the FS122m monotherapy group (Figure 5D). Figure 3 D). In the JC mouse tumor model, some mice also achieved complete tumor regression as a result of the combined FS122m and anti-mPD-1 therapy, whereas neither FS112 nor anti-mPD-1 monotherapy resulted in complete tumor regression in any of the treated mice ( Figure 4 D). In all three mouse tumor models, the combination therapy resulted in prolonged median survival compared with monotherapy ( Figure 3 B, 4B and 5B).

[0338] Sequence Listing

[0339] Heavy chain annotation

[0340] 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).

[0341] 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).

[0342] iii. Provides the CDR amino acid sequences according to IMGT and Kabat.

[0343] Light chain annotation

[0344] 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).

[0345] 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).

[0346] iii. Provides the CDR amino acid sequences according to IMGT and Kabat.

[0347] FS22-172-003-AA / FS28-256-271 mAb 2 The amino acid sequence

[0348]

[0349] FS22-172-003-AA / FS28-024-052mAb 2 The amino acid sequence

[0350]

[0351] FS22-172-003-AA / FS28-256-021mAb 2 The amino acid sequence

[0352]

[0353] FS22-172-003-AA / FS28-256-012mAb 2 The amino acid sequence

[0354] FS22-172-003-AA / FS28-256-023mAb 2 The amino acid sequence

[0355]

[0356] FS22-172-003-AA / FS28-256-024mAb 2 The amino acid sequence

[0357]

[0358] FS22-172-003-AA / FS28-256-026mAb 2 The amino acid sequence

[0359]

[0360] FS22-172-003-AA / FS28-256-027mAb 2 The amino acid sequence

[0361]

[0362] FS22-172-003-AA / FS28-256-001mAb 2 The amino acid sequence

[0363] FS22-172-003-AA / FS28-256-005mAb 2 The amino acid sequence

[0364] FS22-172-003-AA / FS28-256-014mAb 2 The amino acid sequence

[0365]

[0366] FS22-172-003-AA / FS28-256-018mAb 2 The amino acid sequence

[0367]

[0368] FS22-172-003-AA / FS28-256 mAb 2 The amino acid sequence

[0369]

[0370] FS22-172-003-AA / FS28-024-051mAb 2 The amino acid sequence

[0371]

[0372] FS22-172-003-AA / FS28-024-053mAb 2 The amino acid sequence

[0373] FS22-172-003-AA / FS28-024 mAb 2 The amino acid sequence

[0374] Amino acid sequence of FS122m (replacing anti-mMSLNmCD137Fcab G1-AA):

[0375] 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

[0376]

[0377] Amino acid sequences of CH2 domains containing LALA mutation, PA mutation or LALA-PA mutation (mutations in bold and Underlined)

[0378]

[0379] Amino acid sequence of pembrolizumab

[0380]

[0381] References

[0382] 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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[0394] 12 Lee, H.W et al. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol 169, 4882-4888, doi:10.4049 / jimmunol.169.9.4882(2002).

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[0396] 14 Chacon, J.A. et al. Co-stimulation through 4-1BB / CD137 improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy. PLoS One 8, e60031, doi:10.1371 / journal.pone.0060031 (2013).

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[0401] 19 Narazaki, H., Zhu, Y., Luo, L., Zhu, G. and Chen, L. CD137 agonist antibody prevents cancer recurrence: contribution of CD137 on both hematopoietic and nonhematopoietic cells. Blood 115, 1941 - 1948, doi:10.1182 / blood-2008-12-192591 (2010).

[0402] 20 Wilcox, R.A et al. Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J Clin Invest 109, 651 - 659, doi:10.1172 / JCI14184 (2002).

[0403] 21 Weigelin, B et al. Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb. Proc Natl Acad Sci U S A 112, 7551-7556, doi:10.1073 / pnas.1506357112 (2015).

[0404] 22 Segal, N.H et al. Results from an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody. Clin Cancer Res 23, 1929-1936, doi:10.1158 / 1078-0432.CCR-16-1272 (2017).

[0405] 23 Timmerman, J et al. Urelumab alone or in combination with rituximab in patients with relapsed or refractory B-cell lymphoma. Am J Hematol 95, 510-520, doi:10.1002 / ajh.25757 (2020).

[0406] 24 Chin, S.M et al. Structure of the 4-1BB / 4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nat Commun 9, 4679, doi:10.1038 / s41467-018-07136-7 (2018).

[0407] 25 Eskiocak, U et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 5, doi:10.1172 / jci.insight.133647 (2020).

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[0409] 27 Mardiana, S et al. A Multifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting Antitumor Response by Chimeric Antigen Receptor T Cells. Cancer Res 77, 1296 - 1309, doi:10.1158 / 0008-5472.CAN-16-1831 (2017).

[0410] 28 N.G. Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol 27, 1418 - 1428, doi:10.1097 / 00000478-200311000-00003 (2003).

[0411] 29 Chang, K. and Pastan, I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 93, 136 - 140, doi:10.1073 / pnas.93.1.136 (1996).

[0412] 30 Miettinen, M. and Sarlomo-Rikala, M. Expression of calretinin, thrombomodulin, keratin 5, and mesothelin in lung carcinomas of different types: an immunohistochemical analysis of 596 tumors in comparison with epithelioid mesotheliomas of the pleura. Am J Surg Pathol 27, 150 - 158, doi:10.1097 / 00000478-200302000-00002(2003).

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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 molecule in combination with a PD-1 / PD-L1 inhibitor.

2. A method for 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 PD-1 / PD-L1 inhibitor.

3. A kit comprising (a) an antibody molecule that binds to MSLN and CD137 and a pharmaceutically acceptable excipient; and (b) a PD-1 / PD-L1 inhibitor 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, the method for treating cancer or the kit according to any one of the preceding claims, wherein the PD-1 / PD-L1 inhibitor is an antibody that binds to PD-1 or PD-L1.

11. The antibody molecule for use, the method for treating cancer or the kit according to claim 10, wherein the antibody molecule that binds to PD-1 is selected from nivolumab, pembrolizumab and cemiplizumab.

12. The antibody molecule for use, the method for treating cancer or the kit according to claim 10, wherein the antibody molecule that binds to PD-L1 is selected from avelumab, atezolizumab and durvalumab.

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 is selected from the group consisting of: 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 PD-1 / PD-L1 inhibitor produces an anti-tumor effect that is greater than the sum of the anti-tumor effects produced by treating the patient with the antibody molecule that binds to MSLN and CD137 or the PD-1 / PD-L1 inhibitor 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 PD-1 / PD-L1 inhibitor are administered to the patient simultaneously or sequentially.

17. The antibody molecule for use or method of 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.

Images