Multispecific antibodies for the treatment of cancer

Abstract

The problem to be solved by the present invention is to provide more and better options for treating any solid tumor with high frequency microsatellite instability (high frequency MSI or MSI-H) alterations, cervical cancer, endometrial cancer, lung cancer, brain cancer and breast cancer.SOLUTION: The present invention relates to multispecific antibodies comprising an antigen binding site that binds an extracellular part of CD137 and an antigen binding site that binds an extracellular part of a second membrane protem for use in a method of treatment of cancers in a subject in need thereof. The invention further relates to such multispecific antibodies, as well as methods and other aspects related thereto.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of binding molecules. Specifically, the present invention relates to the field of therapeutic binding molecules for the treatment of diseases involving abnormal cells, particularly cancer cells. In particular, the present invention relates to multispecific antibodies that bind to the extracellular portions of two or more different membrane-associated proteins, thereby modulating biological activities expressed by the cells. [Background technology]

[0002] Cancer remains a leading cause of death in the world, despite many advances made in the treatment of the disease and increasing knowledge about the molecular events that lead to cancer.

[0003] Traditionally, most cancer drug discovery has focused on agents that block essential cellular functions and kill dividing cells. However, in cases of advanced cancer, no matter how aggressively applied, chemotherapy rarely results in a complete cure, even at the point where patients suffer life-threatening side effects from the treatment. In most cases, the patient's tumor only stops growing or shrinks temporarily (called remission), only to begin growing again, sometimes more rapidly (called relapse), and become increasingly difficult to treat. More recently, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to targeted, less toxic cell-suppressing therapies. Treatments for advanced cancers have been clinically tested in leukemia and several other cancers. However, in most carcinomas, targeted approaches have still not proven effective enough to completely eradicate cancer in most patients.

[0004] Cancer targeting has been achieved using a variety of different methods, including, for example, small molecules directed at signaling proteins that cancers depend on for survival and / or growth, vaccines that use tumor-specific proteins, cell therapies that use immune cells to actively kill tumor cells, and antibodies that target cytotoxic molecules to tumors, disrupt signal transduction, and / or (re)direct the host's immune system to tumor cells.

[0005] (Re)direction of the immune system can be achieved in several ways. One way is by activating T cell costimulatory molecules such as the tumor necrosis factor receptor superfamily, including CD137 (4-1BB, TNFRSF9). Activation of CD137 leads to increased T cell proliferation, cytokine production, and CD8 + This results in prolonged T cell survival. Another method is to block negative signals induced by molecules involved in immune checkpoints, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death (PD-1) expressed on T cells, or its cognate ligand, programmed cell death 1 ligand 1 (PD-L1), which may be expressed on tumor cells. Upon binding of PD-L1 to PD-1, signaling leads to attenuation of T cell receptor (TCR) signaling and T cell exhaustion. This is a mechanism used by tumors to evade and / or suppress the immune system.

[0006] This immune suppression can be blocked by immune checkpoint inhibitor therapy (ICI), such as antagonistic antibodies against PD-1 or PD-L1. ICI treatment inhibits activated CD8 + A subset of cancer patients has shown remarkable and durable responses that correlate with T cell infiltration and proliferation. Combinations of ICIs (e.g., anti-PD-1 and anti-CTLA-4) have been shown to further enhance efficacy, but at the cost of toxicity, with the majority of patients experiencing grade 3 or 4 treatment-related adverse events.

[0007] Dual targeting of the PD-1 / PD-L1 axis and CD137 may be beneficial for optimally engaging specific antitumor immunity. Currently, the two most advanced therapeutic CD137 agonist antibodies in clinical trials are urelumab (IgG4) and utomilumab (IgG2). Development of urelumab has been halted as a result of dose-dependent hepatitis caused by systemic activation of the CD137 pathway in patients. Safe administration of urelumab requires dose reduction, and 0.1 mg / kg was selected for combination studies with PD-1 inhibitors. Utomilumab is well tolerated by patients, but has modest antitumor activity as monotherapy and no clear synergistic effects with PD-1 blockade in combination therapy.

[0008] Thus, there remains a need to provide more and better options for healthcare professionals to treat cancer, particularly any solid tumor with high microsatellite instability (high MSI or MSI-H) alterations, cervical cancer, endometrial cancer, lung cancer, brain cancer, and breast cancer. Summary of the Invention

[0009] The present disclosure provides means and methods for the (re)direction of immune system components in the treatment of cancer, particularly any solid tumor with high-frequency MSI alterations, cervical cancer, endometrial cancer, lung cancer, brain cancer, and breast cancer.

[0010] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the multispecific antibody is administered at a dose of 25 to 300 mg, preferably 25 to 150 or 25 to 100 mg, more preferably 50 to 100 mg.

[0011] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the multispecific antibody is administered at a dose of 25-300 mg, or 25-150 mg, or 25-100 mg, or 25-75 mg, or 25-50 mg.

[0012] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is characterized by: Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and The cancer is selected from breast cancer, in particular metastatic breast cancer, and triple-negative breast cancer (TNBC).

[0013] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is characterized by: Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, e.g., non-small cell lung cancer (NSCLC), PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; Brain cancer, e.g., glioblastoma; and The cancer is selected from breast cancer, for example, metastatic breast cancer or triple-negative breast cancer (TNBC).

[0014] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is characterized by: Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and selected from breast cancer, particularly metastatic breast cancer and triple-negative breast cancer (TNBC); The multispecific antibody is administered at a dose of 10 to 1200 mg, preferably 25 to 600 or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 50 to 100 mg.

[0015] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is characterized by: Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, e.g., non-small cell lung cancer (NSCLC), PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; Brain cancer, e.g., glioblastoma; and selected from breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC); The multispecific antibody is administered at a dose of 10 to 1200 mg, or 25 to 600 mg, or 25 to 300 mg, or 25 to 150 mg, or 25 to 100 mg, or 25 to 75 mg, or 25 to 50 mg.

[0016] In certain embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to the subject 25 to 300 mg, preferably 25 to 150 or 25 to 100 mg, and more preferably 50 to 100 mg, of a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein.

[0017] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject 25-300 mg, or 25-150 mg, or 25-100 mg, or 25-75 mg, or 25-50 mg of a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein.

[0018] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to a patient with cancer a multispecific antibody comprising an antigen-binding site capable of binding to an extracellular portion of CD137 and an antigen-binding site capable of binding to an extracellular portion of a second membrane protein, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and The method is provided wherein the cancer is selected from breast cancer, particularly metastatic breast cancer or triple-negative breast cancer (TNBC).

[0019] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to a patient with cancer a multispecific antibody comprising an antigen-binding site capable of binding to an extracellular portion of CD137 and an antigen-binding site capable of binding to an extracellular portion of a second membrane protein, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, such as non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high expression of PD-L1; Brain cancer, e.g., glioblastoma; and The cancer is selected from breast cancer, for example, metastatic breast cancer or triple-negative breast cancer (TNBC).

[0020] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering 10 to 1200 mg, preferably 25 to 600 or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 50 to 100 mg of a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, to a subject with cancer, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and The method is provided wherein the cancer is selected from breast cancer, particularly metastatic breast cancer or triple-negative breast cancer (TNBC).

[0021] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering 10-1200 mg, or 25-600 mg, or 25-300 mg, or 25-150 mg, or 25-100 mg, or 25-75 mg, or 25-50 mg of a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, to a subject with cancer, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, such as non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high expression of PD-L1; Brain cancer, e.g., glioblastoma; and The cancer is selected from breast cancer, for example, metastatic breast cancer or triple-negative breast cancer (TNBC). [Brief explanation of the drawings]

[0022] [Figure 1] Ex vivo activity of exemplary multispecific antibodies that bind to CD137 and PD-L1. The top panel shows the relative proportions of T cell subsets in different segments and the percentage of CD3+ cells in single-cell suspensions derived from five human endometrial tumors, as determined by flow cytometry, in the center of the donut. The bottom panel shows IFNγ levels in the supernatants of tumor xenograft cultures after 6 days of incubation with 1) a negative control antibody, 2) a multispecific antibody that binds to CD137 and PD-L1, 3) a urelumab analog, 4) an atezolizumab analog, 5) a combination of urelumab and an atezolizumab analog, and soluble anti-CD3. ND = not performed; *p<0.01, **p<0.0001 as determined by one-way ANOVA and Tukey's test. [Figure 2]In vivo activity of exemplary multispecific antibodies that bind CD137 and PD-L1. A) Ly95 T cells expressing NY-ESO-specific TCR were transferred into NSG mice bearing A549 tumors expressing NY-ESO antigen and human PD-L1 and treated with 1) Ly95 cells alone, 2) Ly95 cells plus a multispecific antibody that binds CD137 and PD-L1, 3) Ly95 cells plus a urelumab analog, 4) Ly95 cells plus a urelumab analog, or 5) Ly95 cells plus a combination of urelumab and atezolizumab analog. Endpoint data are shown (n=7); B) Stacked histograms showing the percentage of human CD3+ lymphocytes among the total viable cell population in blood (open bars) and tumors (filled bars) after red blood cell (RBC) lysis from each treatment group in A); C) Proportion of NY-ESO-1-specific tumors among tumors from each treatment group in A), expressed as a percentage of CD3+ tumor-infiltrating lymphocytes (TILs) (n=7, error bars are SEM); and *p<0.05 (multispecific antibody compared to Ly95) as determined by one-way ANOVA and Tukey's test. [Figure 3] In vivo activity of exemplary multispecific antibodies that bind CD137 and PD-L1. A) Tumor volume in individual human CD34+ engrafted NSG mice (n=9 mice per group) after subcutaneous inoculation with MDA-MB-231 cells and treatment with 5 mg / kg or 0.5 mg / kg of multispecific antibody, or 5 mg / kg of reference antibody, once every 5 days. Treatment groups: a) negative control IgG, b) multispecific antibody, c) urelumab analog, d) atezolizumab, e) atezolizumab + urelumab analog, and f) pembrolizumab. Tumor growth and time of death are shown for individual mice per group. B) The proportion of CD8+ (top panel) and CD4+ (middle panel) tumor-infiltrating lymphocytes (TILs) in tumors from each treatment group is expressed as a percentage of the total population of tumor cells, and the proportion of PD-L1+ monocytes (bottom panel) in tumors from each treatment group is expressed as a percentage of all monocytes (CD11b+ cells); n=9, error bars are SEM; *p<0.05 as determined by one-way ANOVA and Tukey's test. [Figure 4] Percentage weight change in individual human CD34+ engrafted NSG mice after subcutaneous inoculation with MDA-MB-231 cells and the following treatments (n=9 mice per group): a) negative control, b) multispecific antibody, c) urelumab analog, d) atezolizumab, e) urelumab analog and atezolizumab, f) pembrolizumab. DETAILED DESCRIPTION OF THE INVENTION

[0023] In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein for use in a method for treating cancer in some or all patients in need of cancer treatment, wherein the multispecific antibody is administered at a dose of 25 to 300 mg, preferably 25 to 150 mg or 25 to 100 mg, more preferably 25 to 75 mg. In certain embodiments, the multispecific antibody is administered at a dose of 50 to 100 mg or 75 to 125 mg. In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein for use in a method for treating cancer in some or all patients in need of cancer treatment, wherein the multispecific antibody is administered at a dose of 25 to 300 mg, or 25 to 150 mg, or 25 to 100 mg, or 25 to 75 mg, or 25 to 50 mg. In certain embodiments, the multispecific antibody is administered in a dose of 50-100 mg or 75-125 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25-50 mg. In certain embodiments, the multispecific antibody is administered in a dose of 25, 30, 35, 40, 45, or 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25, 30, 35, 40, 45, or 50 mg.

[0024] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject 25-300 mg, preferably 25-150 mg or 25-100 mg, and more preferably 25-75 mg, of a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein. In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject 25-300 mg, 25-150 mg, 25-100 mg, 25-75 mg, or 25-50 mg of a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein. In certain embodiments, the multispecific antibody is administered at a dose of 50-100 mg or 75-125 mg. In certain embodiments, the multispecific antibody is administered as a flat dose of 25-50 mg. In certain embodiments, the multispecific antibody is administered in a dose of 25, 30, 35, 40, 45, or 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25, 30, 35, 40, 45, or 50 mg.

[0025] In certain embodiments, the cancer is Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and In certain embodiments, the cancer is selected from breast cancer, particularly metastatic breast cancer, and triple-negative breast cancer (TNBC). Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, such as non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high expression of PD-L1; Brain cancer, e.g., glioblastoma; and In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is selected from: Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and selected from breast cancer, particularly metastatic breast cancer and triple-negative breast cancer (TNBC); The multispecific antibody is administered at a dose of 10 to 1200 mg, preferably 25 to 600 or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 50 to 100 mg. In certain embodiments, the present disclosure provides a multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, such as non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high expression of PD-L1; Brain cancer, e.g., glioblastoma; and selected from breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC); The multispecific antibody is administered in a dose of 10 to 1200 mg, or 25 to 600 mg, or 25 to 300 mg, or 25 to 150 mg, or 25 to 100 mg, or 25 to 75 mg, or 25 to 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25 to 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25 to 50 mg. In certain embodiments, the multispecific antibody is administered in a dose of 25, 30, 35, 40, 45, or 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25, 30, 35, 40, 45, or 50 mg.

[0026] In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering 10 to 1200 mg, preferably 25 to 600 or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 50 to 100 mg of a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, to a subject with cancer, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Endometrial cancer, especially MSI-rich endometrial cancer; Lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and In certain embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to a subject having cancer 10-1200 mg, 25-600 mg, 25-300 mg, 25-150 mg, 25-100 mg, 25-75 mg, or 25-50 mg of a multispecific antibody comprising an antigen-binding site that binds to an extracellular portion of CD137 and an antigen-binding site that binds to an extracellular portion of a second membrane protein, wherein the cancer is Any solid tumor with high-frequency MSI alterations; Cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; Endometrial cancer, e.g., MSI-high endometrial cancer; Lung cancer, for example, non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high expression of PD-L1; Brain cancer, e.g., glioblastoma; and The multispecific antibody is administered in a flat dose of 25 to 50 mg. In certain embodiments, the multispecific antibody is administered in a dose of 25, 30, 35, 40, 45, or 50 mg. In certain embodiments, the multispecific antibody is administered in a flat dose of 25, 30, 35, 40, 45, or 50 mg.

[0027] CD137 can be expressed by activated T cells. It is also found on other cells, such as dendritic cells, natural killer cells, granulocytes, and cells in the vascular wall at sites of inflammation. This protein is known for its costimulatory activity for T cell activation. CD137 has been known by many different names, including: TNFRSF9; TNF receptor superfamily member 9; tumor necrosis factor receptor superfamily member 9; T cell antigen 4-1BB homolog; 4-1BB ligand receptor; T cell antigen ILA; CD137 antigen; CDw137; ILA; interleukin-activated receptor, homolog of mouse Ly63; induced by lymphocyte activation (ILA); homolog of mouse 4-1BB; receptor protein 4-1BB; cell antigen ILA; and 4-1BB. The external IDs for CD137 are HGNC:11924, Entrez Gene:3604, Ensembl:ENSG00000049249, OMIM:602250, and UniProtKB:Q07011. CD137 is an inducible receptor most commonly upregulated on activated CD8+ T cells. CD137 signaling enhances T cell function by activating NF-κB [Arch et al., 1998]. Other immune cell types, including CD4+ T cells, monocytes, B cells, dendritic cell (DC) subpopulations, and granulocytes and NK cells, can express CD137 at various levels [Shao et al., 2011]. On monocytes, CD137 is inducible upon activation by lipopolysaccharide (LPS) and IL-1b. On B lymphocytes, CD137 expression is induced by antibodies against cell surface immunoglobulins and transformation by EBV. In DCs, CD137 ligation induces maturation through upregulation of B7 costimulatory molecules (CD80 and CD86), in addition to increasing the production of proinflammatory cytokines (IL-6 and IL-12) and their survival [Makkouk et al., 2015]. The natural function of CD137 ligation on neutrophils is increased phagocytosis of bacterial and parasitic infections.Additionally, CD137 ligation blocks anti-apoptotic signals mediated by the IL-3 / IL-5 / GM-CSF receptor in neutrophils and eosinophils in vitro, thereby preventing granulocyte accumulation [Simon, 2001; Vinay et al., 2011]. On non-lymphoid cells, such as chondrocytes, endothelial cells, and tumor cells, CD137 expression is driven by cytokine stimulation, such as IL-1b in chondrocytes, the proinflammatory cytokines TNF-alpha / IFN-γ / IL-1b in endothelial cells, and IFN-γ in tumor cells. The CD137 stimulatory ligand (CD137L) is expressed on activated antigen-presenting cells. CD137 exists in the membrane as both a monomer and a dimer [Pollok et al., 1993].

[0028] The B7 family includes a number of structurally related cell surface proteins that bind to receptors on lymphocytes to regulate immune responses. Lymphocyte activation is initiated by engagement of the cell surface, antigen-specific T cell receptor, or B cell receptor. Additional signals simultaneously delivered by B7 ligands further determine the immune response of these cells. These so-called "costimulatory" or "coinhibitory" signals are delivered by B7 family members via the CD28 family of receptors on lymphocytes. Binding of B7 family members to costimulatory receptors enhances immune responses, while binding to costimulatory receptors attenuates immune responses. The following members are currently considered to be part of this family: B7.1 (CD80), B7.2 (CD86), inducible costimulatory ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3 (CD276), B7-H4, B7-H5, B7-H6, and B7-H7. B7 family members are expressed in lymphoid and non-lymphoid tissues. Their effects on regulating immune responses are demonstrated in mice with mutations in B7 family genes, which develop immunodeficiency and autoimmune diseases. Manipulation of the signals delivered by B7 ligands has shown promise in the treatment of autoimmune, inflammatory, and cancer diseases.

[0029] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis. PD-L1 is expressed in various types of cancer, particularly NSCLC (Boland et al., 2013; Velcheti et al., 2014), melanoma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, and various leukemias and multiple myeloma (Bernstein et al., 2014; Thompson et al., 2005). PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, although not all cancers or cells within tumors express PD-L1 (Dong et al., 2002). Multiple tumor microenvironment cells contribute to immune suppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because tumors protect themselves by inducing PD-L1 in response to IFN-γ produced by activated T cells (Sharma et al., 2017). PD-L1 can also be regulated by oncogenes, a mechanism known as intrinsic immune resistance (Akbay et al., 2013). Within the tumor microenvironment, PD-L1 is also expressed on myeloid cells and activated T cells (Tumeh et al., 2014). PD-L1 expression is induced by multiple proinflammatory molecules, including type I and type II IFN-γ, TNF-α, LPS, GM-CSF, and VEGF, as well as the cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer (Sznol and Chen, 2013).

[0030] The programmed cell death 1 protein (PD-1) is a cell surface receptor that belongs to the CD28 family of receptors and is expressed on T cells and pro-B cells. PD-1 is currently known to bind two ligands, PD-L1 and PD-L2. PD-1, which functions as an immune checkpoint, plays a key role in downregulating the immune system by inhibiting T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 is thought to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is also known by a number of different names, including PDCD1, programmed cell death 1, systemic lupus erythematosus susceptibility 2, protein PD-1, HPD-1, PD1, programmed cell death 1 protein, CD279 antigen, CD279, HPD-L, HSLE1, SLEB2, and PD-1. The external Ids for PD-1 are HGNC:8760, Entrez Gene:5133, Ensembl:ENSG00000188389, OMIM:600244, and UniProtKB:Q15116. PD-1 inhibitors, a new class of drugs that block the activity of PD-1, activate the immune system to attack tumors and thus have been used successfully to treat several types of cancer.

[0031] Binding of PD-L1 to PD-1 or B7.1 (CD80) transmits an inhibitory signal that reduces the proliferation of PD-1-expressing T cells. PD-1 is thought to be able to control the accumulation of foreign antigen-specific T cells through apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in the attenuation of immune responses to cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names, including CD274 molecule, CD274 antigen, B7 homolog 1, PDCD1 ligand 1, PDCD1LG1, PDCD1L1, B7H1, PDL1, programmed cell death 1 ligand 1, programmed death ligand 1, B7-H1, and B7-H. The external Ids for CD274 are HGNC:17635; Entrez Genes:29126; Ensembl:ENSG00000120217; OMIM:605402; UniProtKB:Q9NZQ7.

[0032] PD-L2 is the second ligand for PD-1. Engagement of PD-1 by PD-L2 inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2 / PD-1 binding inhibits B7-CD28 signaling. At high antigen concentrations, PD-L2 / PD-1 binding reduces cytokine production. PD-L expression is upregulated on antigen-presenting cells by interferon-gamma treatment. It is expressed in several normal tissues and various tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. The protein is known by several other names, including programmed cell death 1 ligand 2, B7 dendritic cell molecule, programmed death ligand 2, butyrophilin B7-DC, PDCD1 ligand 2, PD-1 ligand 2, PDCD1L2, B7-DC, CD273, B7DC, PDL2, PD-1-ligand 2, CD273 antigen, BA574F11.2, and Btdc. The external identifiers for PD-L2 are HGNC:18731, Entrez gene:80380, Ensembl:ENSG00000197646, OMIM:605723, and UniProtKB:Q9BQ51.

[0033] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure binds to a second membrane protein that is not a member of the TNF receptor superfamily. In certain embodiments, the second membrane protein is a member of the B7 family. In certain embodiments, the second membrane protein is PD-L1 or PD-L2, preferably PD-L1.

[0034] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure comprises one antigen-binding site that binds to the PD-1 binding domain of PD-L1.

[0035] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure binds to a second membrane protein that is not expressed to a significant extent by T cells.

[0036] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure binds to a second membrane protein expressed on an antigen-presenting cell, a tumor cell, a virus-infected cell, or a parasite-infected cell.

[0037] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure binds to a second membrane protein that is a membrane protein present in one or more zones on the cell membrane, preferably a cluster, domain, microdomain, or compartment on the cell membrane, preferably an immunological synapse.

[0038] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure binds to a second membrane protein that is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the aforementioned second membrane proteins, hi certain embodiments, the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

[0039] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure comprises one antigen-binding site that binds to the CD137L-binding domain of CD137.

[0040] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure comprises one antigen-binding site that blocks binding of a ligand to CD137 or that binds to an extracellular ligand-blocking binding site of CD137, preferably the CD137L-blocking binding site.

[0041] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure comprises a variable domain that binds to the extracellular portion of CD137, defined as a variable domain that, in a bivalent monospecific antibody format comprising two of the aforementioned variable domains that bind to CD137, does not stimulate the activity of CD137 on cells, or stimulates it at a reduced level compared to one of the aforementioned variable domains as part of a multispecific antibody with a second variable domain that binds to a tumor-associated antigen, preferably a member of the B7 family, more preferably PD-L1.

[0042] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure comprises a variable domain that binds to the extracellular portion of CD137, which, when combined in a multispecific antibody with a second variable domain that binds PD-L1, is capable of stimulating the activity of CD137 on a cell when in the presence of a first cell that expresses CD137 and a second cell that expresses PD-L1.

[0043] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is capable of simultaneously binding CD137 and PD-L1.

[0044] Multispecific antibodies according to the present disclosure that bind to CD137 and the extracellular portion of a second membrane protein, particularly a membrane protein that is a member of the B7 family, offer the advantage that they are particularly well able to promote a desired immune response because B7 family members deliver "costimulatory" or "coinhibitory" signals to lymphocytes, thereby enhancing or attenuating the immune response. Thus, by targeting a second transmembrane protein, particularly a transmembrane protein that is a member of the B7 family, it is possible to enhance stimulatory signals and / or inhibit inhibitory signals, thereby inducing or enhancing a desired immune response against abnormal cells, such as cancer cells. Thus, according to the present disclosure, multispecific antibodies are used to treat cancer in a subject in need thereof, when a desired immune response is elicited against abnormal cells found in cancers, particularly MSI-high cancers, cervical cancer, e.g., MSI-high endometrial cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), brain cancers such as glioblastoma, and breast cancer, e.g., mammary adenocarcinoma and triple-negative breast cancer (TNBC).

[0045] In certain embodiments, a multispecific antibody according to the present disclosure has one antigen-binding site capable of binding to the extracellular portion of CD137 and a second antigen-binding site capable of binding to the extracellular portion of a second membrane protein that is not a member of the TNF receptor superfamily, but is preferably a member of the B7 family, more preferably PD-L1. This offers the advantage that cis activation of (immune) cells, such as T cells, that express several different members of the TNF receptor superfamily is at least partially avoided, thereby reducing potential adverse side effects and toxicity due to nonspecific T cell activation. Prior art approaches may result in T cell activation in cis, meaning the absence of a secondary target, and may entail the risk of excessive T cell responses resulting, for example, in a cytokine storm. Consequently, such prior art approaches increase the likelihood of adverse side effects compared to a binding molecule according to the present invention that has an antigen-binding site capable of binding to CD137 and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein.

[0046] In certain embodiments, the present disclosure relates to multispecific antibodies that target both PD-L1 and CD137 to partially avoid cis-activation of (immune) cells such as T cells. In certain embodiments, the variable domain that binds to the extracellular portion of CD137 is such that, in a bivalent monospecific antibody format comprising two of such CD137-binding domains, it does not stimulate the activity of CD137 on cells or stimulates it at a reduced level compared to one of the aforementioned variable domains as part of a multispecific antibody with a second variable domain that binds to a tumor-associated antigen, preferably a member of the B7 family, more preferably PD-L1. Suitable CD137-binding arms are disclosed in WO2018 / 056821.

[0047] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an agonistic CD137 antibody, e.g., an antibody that can stimulate the activity of CD137. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an antagonistic CD137 antibody, e.g., an antibody that can reduce the activity of CD137.

[0048] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an agonistic B7 antibody, e.g., an antibody that can stimulate the activity of B7. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an antagonistic B7 antibody, e.g., an antibody that can reduce the activity of a B7 family member.

[0049] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an agonistic PD-L1 antibody, e.g., an antibody that can agonize the activity of PD-L1. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may be an antagonistic PD-L1 antibody, e.g., an antibody that can reduce the activity of PD-L1.

[0050] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure may stimulate CD137 activity upon binding to a B7 family member. In certain embodiments, the multispecific antibody stimulates CD137 activity upon binding to CD137 and PD-L1. In certain embodiments, the multispecific antibody induces or activates CD137 signaling only in the presence of PD-L1-expressing cells.

[0051] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure comprises an antigen-binding site consisting of one immunoglobulin variable domain that binds to CD137 and one immunoglobulin variable domain that binds to the extracellular portion of a second membrane protein.

[0052] In certain embodiments, the variable domain comprised by a multispecific antibody according to a use or method of the disclosure that binds to the extracellular portion of CD137 and at least partially blocks the binding of a CD137 ligand to CD137 is a variable domain comprising the amino acid sequence of the VH of MF6783 (SEQ ID NO: 1), MF6861 (SEQ ID NO: 5), MF6795 (SEQ ID NO: 9), MF6808 (SEQ ID NO: 13), MF6798 (SEQ ID NO: 17), MF6754 (SEQ ID NO: 20), MF6763 (SEQ ID NO: 24), MF6744 (SEQ ID NO: 28), MF6785 (SEQ ID NO: 31), MF6825 (SEQ ID NO: 35), MF6737 (SEQ ID NO: 39), MF6749 (SEQ ID NO: 43), MF6788 (SEQ ID NO: 46), or MF6797 (SEQ ID NO: 49).

[0053] In certain embodiments, a multispecific antibody according to a use or method of the disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of CD137, comprises a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF6754 (SEQ ID NO: 23), MF6763 (SEQ ID NO: 27), MF6785 (SEQ ID NO: 34), or MF6797 (SEQ ID NO: 52).

[0054] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of CD137, comprises a heavy chain variable region having a CDR2 region comprising the amino acid sequence of the CDR2 region of the variable heavy chain region of MF6754 (SEQ ID NO: 22), MF6763 (SEQ ID NO: 26), MF6785 (SEQ ID NO: 33), or MF6797 (SEQ ID NO: 51).

[0055] In certain embodiments, a multispecific antibody according to a use or method of the disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of CD137, comprises a heavy chain variable region having a CDR1 region comprising the amino acid sequence of the CDR1 region of the variable heavy chain region of MF6754 (SEQ ID NO: 21), MF6763 (SEQ ID NO: 25), MF6785 (SEQ ID NO: 32), or MF6797 (SEQ ID NO: 50).

[0056] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of CD137, comprises a heavy chain variable region having CDR1, CDR2, and CDR3 regions that comprise the CDR1, CDR2, and CDR3 amino acid sequences of the variable heavy chain region of one of the VHs listed for MF6754, MF6763, MF6785, or MF6797. The CDR1, CDR2, and CDR3 sequences are preferably selected from the same VH region.

[0057] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analog thereof, according to a use or method of the present disclosure, comprising a variable domain that binds to the extracellular portion of CD137, comprises the amino acid sequence of the variable heavy chain region of MF6754, MF6763, MF6785, or MF6797, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF. In certain embodiments, amino acid insertions, deletions, substitutions, or combinations thereof, if present, are absent from the amino acid sequences of the CDR regions.

[0058] In certain embodiments, a variable domain that binds to the extracellular portion of CD137, or a functional portion, derivative, and / or analog thereof, according to a use or method of the present disclosure, comprises a VH region having the amino acid sequence of CDR3, or a VH region having the amino acid sequence of CDR1, CDR2, and CDR3 of one of the VHs of MF6754, MF6763, MF6785, or MF6797. In certain embodiments, the variable domain that binds to the extracellular portion of CD137 comprises a VH region having the amino acid sequence of the VH of MF6754 (SEQ ID NO: 20), MF6763 (SEQ ID NO: 24), MF6785 (SEQ ID NO: 31), or MF6797 (SEQ ID NO: 49), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or combinations thereof relative to the amino acid sequence of the VH of the designated MF. In certain embodiments, amino acid insertions, deletions, substitutions, or combinations thereof, if present, are not included in the amino acid sequences of the CDR regions.

[0059] In certain embodiments, the variable domain comprised by a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the disclosure, that binds to the extracellular portion of PD-L1 and blocks PD-1 binding to PD-L1 is selected from the group consisting of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5359 (SEQ ID NO: 69), MF5560 (SEQ ID NO: 69), MF5579 (SEQ ID NO: 70), MF5570 (SEQ ID NO: 71), MF5571 (SEQ ID NO: 72), MF5572 (SEQ ID NO: 73), MF5573 (SEQ ID NO: 74), MF5574 (SEQ ID NO: 75), MF5575 (SEQ ID NO: 76), MF5576 (SEQ ID NO: 77), MF5578 (SEQ ID NO: 78), MF5579 (SEQ ID NO: 79), MF5579 (SEQ ID NO: 80), MF5579 (SEQ ID NO: 81), MF5579 (SEQ ID NO: 82), MF5579 (SEQ ID NO: 83), MF5579 (SEQ ID NO: 84), MF5579 (SEQ ID NO: 85), MF5579 (SEQ ID NO: 86), MF5579 (SEQ ID NO: 87), MF5579 (SEQ ID NO: 88), MF5579 ( ), MF5377 (SEQ ID NO: 73), MF5382 (SEQ ID NO: 77), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5439 (SEQ ID NO: 89), MF5442 (SEQ ID NO: 92), MF5553 (SEQ ID NO: 96), MF5557 (SEQ ID NO: 97), MF5561 (SEQ ID NO: 100), MF5576 (SEQ ID NO: 103), MF5594 (SEQ ID NO: 104), or MF5708 (SEQ ID NO: 107).

[0060] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of PD-L1, comprises a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF5554 (SEQ ID NO:56), MF5576 (SEQ ID NO:58), MF5578 (SEQ ID NO:61), MF9375 (SEQ ID NO:56), MF9376 (SEQ ID NO:56), MF7702 (SEQ ID NO:56), MF5424 (SEQ ID NO:84), MF5561 (SEQ ID NO:102), MF5439 (SEQ ID NO:91), MF5553 (SEQ ID NO:56), MF5594 (SEQ ID NO:106), MF5426 (SEQ ID NO:88), or MF5442 (SEQ ID NO:95).

[0061] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure comprises a heavy chain variable region that binds to the extracellular portion of PD-L1 comprising a CDR3 region having the amino acid sequence set forth in SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 102, or SEQ ID NO: 106, preferably SEQ ID NO: 56, SEQ ID NO: 91, SEQ ID NO: 95, or SEQ ID NO: 102, or a variant thereof.

[0062] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of PD-L1, comprises a heavy chain variable region having a CDR2 region comprising the amino acid sequence of the CDR2 region of the variable heavy chain region of MF5554 (SEQ ID NO: 55), MF5576 (SEQ ID NO: 55), MF5578 (SEQ ID NO: 3), MF9375 (SEQ ID NO: 63), MF9376 (SEQ ID NO: 66), MF7702 (SEQ ID NO: 55), MF5424 (SEQ ID NO: 83), MF5561 (SEQ ID NO: 101), MF5439 (SEQ ID NO: 79), MF5553 (SEQ ID NO: 55), MF5594 (SEQ ID NO: 105), MF5426 (SEQ ID NO: 87), or MF5442 (SEQ ID NO: 94).

[0063] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the disclosure comprises a variable domain that binds to the extracellular portion of PD-L1, wherein the antibody comprises a heavy chain variable region that binds to the extracellular portion of PD-L1 comprising a CDR2 region having the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:87, SEQ ID NO:94, SEQ ID NO:101, or SEQ ID NO:105, or a variant thereof.

[0064] In certain embodiments, a multispecific antibody, or functional part, derivative and / or analogue thereof, according to the uses or methods of the disclosure, comprising a variable domain that binds to the extracellular portion of PD-L1, comprises a heavy chain variable region having a CDR1 region comprising the amino acid sequence of the CDR1 region of the variable heavy chain region of MF5554 (SEQ ID NO: 54), MF5576 (SEQ ID NO: 54), MF5578 (SEQ ID NO: 60), MF9375 (SEQ ID NO: 60), MF9376 (SEQ ID NO: 65), MF7702 (SEQ ID NO: 68), MF5424 (SEQ ID NO: 82), MF5561 (SEQ ID NO: 93), MF5439 (SEQ ID NO: 90), MF5553 (SEQ ID NO: 68), MF5594 (SEQ ID NO: 74), MF5426 (SEQ ID NO: 86), or MF5442 (SEQ ID NO: 93).

[0065] In certain embodiments, a multispecific antibody or method of the disclosure comprising a variable domain that binds to the extracellular portion of PD-L1 comprises a heavy chain variable region that binds to the extracellular portion of PD-L1 comprising a CDR1 region having the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, or SEQ ID NO:93, or a variant thereof.

[0066] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the disclosure, comprising a variable domain that binds to the extracellular portion of PD-L1, comprises a heavy chain variable region having CDR1, CDR2, and CDR3 regions that comprise the CDR1, CDR2, and CDR3 amino acid sequences of the variable heavy chain region of one of the VHs listed for MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, or MF5442. The CDR1, CDR2, and CDR3 sequences are preferably selected from the same VH region.

[0067] In certain embodiments, the multispecific antibody, or functional part, derivative and / or analogue thereof, according to the uses or methods of the disclosure, comprising a variable domain that binds to the extracellular portion of PD-L1 is selected from the group consisting of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5424 (SEQ ID NO: 81), MF5561 (SEQ ID NO: 100), The amino acid sequence of the variable heavy chain region of MF5439 (SEQ ID NO: 89), MF5553 (SEQ ID NO: 96), MF5594 (SEQ ID NO: 104), MF5426 (SEQ ID NO: 85), or MF5442 (SEQ ID NO: 92) has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF. In certain embodiments, the amino acid insertions, deletions, substitutions, or a combination thereof, if present, are not contained in the amino acid sequences of the CDR regions.

[0068] In certain embodiments, particularly preferred combinations in a multispecific antibody, or functional part, derivative, and / or analogue according to the uses or methods of the disclosure are MF6797 (SEQ ID NO:49) and MF7702 (SEQ ID NO:67), MF6763 (SEQ ID NO:24) and MF7702 (SEQ ID NO:67), MF6785 (SEQ ID NO:31) and MF7702 (SEQ ID NO:67), MF6797 (SEQ ID NO:49) and MF5553 (SEQ ID NO:96), MF6763 (SEQ ID NO:24) and MF5553 (SEQ ID NO:96), MF6785 (SEQ ID NO:31) and combinations of variable domains comprising the shown sequences of MF5553 (SEQ ID NO: 96), MF6754 (SEQ ID NO: 20) and MF5424 (SEQ ID NO: 81), MF6763 (SEQ ID NO: 24) and MF5561 (SEQ ID NO: 100), MF6785 (SEQ ID NO: 31) and MF5439 (SEQ ID NO: 89), MF6754 (SEQ ID NO: 20) and MF5553 (SEQ ID NO: 96), MF6744 (SEQ ID NO: 28) and MF5594 (SEQ ID NO: 104), or MF6783 (SEQ ID NO: 1) and MF5594 (SEQ ID NO: 104) or variants thereof.

[0069] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analog thereof according to the uses or methods of the present disclosure comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3 or the amino acid sequences of CDR1, CDR2, and CDR3 of the VH of MF6797 (SEQ ID NO: 49); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of CDR1, CDR2, and CDR3, or the amino acid sequences of the CDR1, CDR2, and CDR3 of the VH of MF5554 (SEQ ID NO: 53), MF5576 (SEQ ID NO: 57), MF5578 (SEQ ID NO: 59), MF9375 (SEQ ID NO: 62), MF9376 (SEQ ID NO: 64), MF7702 (SEQ ID NO: 67), MF5594 (SEQ ID NO: 104), MF5424 (SEQ ID NO: 81), MF5426 (SEQ ID NO: 85), MF5553 (SEQ ID NO: 96), MF5442 (SEQ ID NO: 92), MF5561 (SEQ ID NO: 100), or MF5439 (SEQ ID NO: 89).

[0070] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analog thereof according to the uses or methods of the present disclosure comprises: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6797 (SEQ ID NO: 49), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6797 (SEQ ID NO: 49); a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5554 (SEQ ID NO:53), MF5576 (SEQ ID NO:57), MF5578 (SEQ ID NO:59), MF9375 (SEQ ID NO:62), MF9376 (SEQ ID NO:64), MF7702 (SEQ ID NO:67), MF5594 (SEQ ID NO:104), MF5424 (SEQ ID NO:81), MF5426 (SEQ ID NO:85), MF5553 (SEQ ID NO:96), MF5442 (SEQ ID NO:92), MF5561 (SEQ ID NO:100), or MF5439 (SEQ ID NO:89), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF.

[0071] Certain embodiments further provide a multispecific antibody, or a functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, comprising: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6763 (SEQ ID NO: 27); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5442 (SEQ ID NO: 95).

[0072] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analog thereof, which comprises: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6763 (SEQ ID NO: 24); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5442 (SEQ ID NO: 92).

[0073] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6763 (SEQ ID NO: 24), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6763; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5442 (SEQ ID NO: 92), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5442. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF.

[0074] Certain embodiments further provide a multispecific antibody, or a functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, comprising: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6797 (SEQ ID NO: 52); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF7702 (SEQ ID NO: 56).

[0075] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analog thereof, which comprises: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6797 (SEQ ID NO: 49); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF7702 (SEQ ID NO: 67).

[0076] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6797 (SEQ ID NO: 49), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6797; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF7702 (SEQ ID NO: 67), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF7702. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF shown.

[0077] Certain embodiments further provide a multispecific antibody, or a functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, comprising: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6754 (SEQ ID NO: 23), - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH CDR3 of MF5561 (SEQ ID NO: 102).

[0078] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analog thereof, which comprises: - a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6754 (SEQ ID NO: 20); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5561 (SEQ ID NO: 100).

[0079] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6754 (SEQ ID NO: 20), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6754; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5561 (SEQ ID NO: 100), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5561. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF.

[0080] Certain embodiments further provide a multispecific antibody, or a functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6785 (SEQ ID NO: 34); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5439 (SEQ ID NO: 91).

[0081] Also provided is a bispecific antibody, or a functional part, derivative, and / or analogue thereof, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6785 (SEQ ID NO: 31); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5439 (SEQ ID NO: 89).

[0082] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785 (SEQ ID NO: 31), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6785; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5439 (SEQ ID NO: 89), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5439. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF shown.

[0083] Certain embodiments further provide a multispecific antibody, or a functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6785 (SEQ ID NO: 34); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH CDR3 of MF5542 (SEQ ID NO: 95).

[0084] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analog thereof, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6785 (SEQ ID NO: 31); - a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5442 (SEQ ID NO: 92).

[0085] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785 (SEQ ID NO: 31), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6785; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5442 (SEQ ID NO: 92), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5542. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF.

[0086] Certain embodiments provide a multispecific antibody, or functional part, derivative, and / or analogue thereof, according to the uses or methods of the present disclosure, which comprises: - a variable domain comprising a CDR1 set forth in SEQ ID NO: 50, a CDR2 set forth in SEQ ID NO: 51, and a CDR3 set forth in SEQ ID NO: 52, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or - a variable domain comprising a CDR1 set forth in SEQ ID NO: 40, a CDR2 set forth in SEQ ID NO: 41, and a CDR3 set forth in SEQ ID NO: 42, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or - a variable domain comprising a CDR1 set forth in SEQ ID NO: 21, a CDR2 set forth in SEQ ID NO: 22, and a CDR3 set forth in SEQ ID NO: 23, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or a binding domain that binds to CD137, comprising a variable domain comprising CDR1 set forth in SEQ ID NO: 32, CDR2 set forth in SEQ ID NO: 33, and CDR3 set forth in SEQ ID NO: 34, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; and / or - a variable domain comprising a CDR1 set forth in SEQ ID NO: 68, a CDR2 set forth in SEQ ID NO: 55, and a CDR3 set forth in SEQ ID NO: 56, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or - a variable domain comprising a CDR1 set forth in SEQ ID NO: 93, a CDR2 set forth in SEQ ID NO: 94, and a CDR3 set forth in SEQ ID NO: 95, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or - a variable domain comprising a CDR1 set forth in SEQ ID NO: 93, a CDR2 set forth in SEQ ID NO: 101, and a CDR3 set forth in SEQ ID NO: 102, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof; or a binding domain that binds to PD-L1, comprising a variable domain comprising a CDR1 set forth in SEQ ID NO: 90, a CDR2 set forth in SEQ ID NO: 79, and a CDR3 set forth in SEQ ID NO: 91, wherein each CDR1, CDR2, and / or CDR3 has 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof. In certain embodiments, the amino acid sequences of the CDRs have up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof.

[0087] In certain embodiments, a multispecific antibody, or functional part, derivative, and / or analog thereof, according to the uses or methods of the present disclosure comprises a variable domain that binds to the extracellular portion of CD137 and blocks the binding of CD137 to a CD137 ligand, and a variable domain that binds to the extracellular portion of PD-L1 and blocks the binding of PD-L to PD-L1. In certain embodiments, the variable domain that binds to the extracellular portion of PD-L1 in the antibody, or functional part, derivative, and / or analog thereof, comprises a VH region having the amino acid sequence of CDR3, or the amino acid sequences of CDR1, CDR2, and CDR3 of the VH of one of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442. In certain embodiments, the variable domain that binds to the extracellular portion of PD-L1 comprises a VH region having the amino acid sequence of the VH of MF5554 (SEQ ID NO:53), MF5576 (SEQ ID NO:57), MF5578 (SEQ ID NO:59), MF9375 (SEQ ID NO:62), MF9376 (SEQ ID NO:64), MF7702 (SEQ ID NO:67), MF5424 (SEQ ID NO:81), MF5561 (SEQ ID NO:100), MF5439 (SEQ ID NO:89), MF5553 (SEQ ID NO:96), MF5594 (SEQ ID NO:104), MF5426 (SEQ ID NO:85), MF5442 (SEQ ID NO:92), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF. In certain embodiments, the amino acid sequence of the variable heavy chain region has up to 5, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the designated MF.

[0088] This is shown in the examples of WO2018 / 056821A1, where the binding of the CD137-specific VH of MF6797 (sequence number 49) is associated with the presence of amino acids including Arg66, Gly70, and Phe72 in the CD137 amino acid sequence.

[0089] Thus, in certain embodiments, the present disclosure provides uses or therapeutic methods of isolated, synthetic, or recombinant antibodies, or functional portions, derivatives, and / or analogs thereof, capable of binding to CD137, wherein the binding of said antibodies, or functional portions, derivatives, or analogs to CD137 is associated with the presence of amino acids including Arg66, Gly70, and Phe72 of the CD137 amino acid sequence (SEQ ID NO: 117). In certain embodiments, the binding of said antibodies, or functional portions, derivatives, or analogs to CD137 is also associated with amino acids including Val71 of the CD137 amino acid sequence.

[0090] The term "Arg66" refers to the arginine residue at position 66 of the CD137 sequence. The term "Gly70" refers to the glycine residue at position 70 of the CD137 sequence according to SEQ ID NO: 117. The term "Val71" refers to the valine residue at position 71 of the CD137 sequence. The term "Phe72" refers to the phenylalanine residue at position 72 of the CD137 sequence.

[0091] In certain embodiments, multispecific antibodies of the present disclosure comprise binding sites comprising a common light chain set forth in SEQ ID NO: 109 with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, each of the binding sites comprises a common light chain set forth in SEQ ID NO: 109 with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0092] In certain embodiments, the multispecific antibodies described herein comprise a common light chain variable domain, preferably a common light chain variable region according to SEQ ID NO: 110, with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof, preferably with 0, 1, 2, 3, or 4, more preferably with 0, 1, 2, or 3, more preferably with 0, 1, or 2, and most preferably with 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof. In certain embodiments, the multispecific antibodies described herein comprise a common light chain constant domain, preferably a common light chain constant region according to SEQ ID NO: 111, with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. The term "common light chain" according to the present invention refers to light chains that may be identical or may differ in some amino acid sequence, but in which the binding specificity of the full-length antibody is not affected. For example, preparing or finding a non-identical but still functionally equivalent light chain, such as by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a heavy chain, may fall within the scope of the definition of a common light chain used herein. The terms "common light chain," "common LC," "cLC," and "single light chain," whether with or without the addition of the term "rearranged," are all used interchangeably herein. The terms "common light chain variable region," "common VL," "common LCv," "cLCv," and "single VL," whether with or without the addition of the term "rearranged," are all used interchangeably herein. In certain embodiments of the present disclosure, a multispecific antibody has a common light chain (variable region) that can be combined with at least two, preferably multiple, heavy chains (variable regions) of different binding specificities to form an antibody with functional antigen-binding domains (WO2004 / 009618, WO2009 / 157771). In certain embodiments, the common light chain (variable region) is a human light chain (variable region). In certain embodiments, the common light chain (variable region) has a germline sequence. In certain embodiments, the germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield, and solubility.In certain embodiments, the common light chain is a rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01 (SEQ ID NO: 109). In certain embodiments, the common light chain variable region is the variable region of the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01. In certain embodiments, the common light chain comprises a light chain variable region set forth in SEQ ID NO: 110 with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof, preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1 amino acid insertions, deletions, substitutions, or a combination thereof. In certain embodiments, the common light chain further comprises a codon constant region, preferably a kappa light chain constant region. The nucleic acid encoding the common light chain can be codon-optimized for the cell system used to express the common light chain protein. The encoding nucleic acid can deviate from the germline nucleic acid sequence.

[0093] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is a full-length antibody or a fragment of an antibody, such as a Fab fragment or a single-chain variable fragment (scFv). In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is a full-length antibody.

[0094] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure is an IgG. In certain embodiments, the multispecific antibody is an IgG1 molecule without Fc effector functions.

[0095] The Fc region mediates antibody effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the application of a therapeutic antibody or Fc fusion protein, it may be desirable to reduce or increase effector function. Reduced effector function is preferred in the present disclosure. Reduced effector function may be desired when an immune response is activated, enhanced, or stimulated, as in some embodiments of the present disclosure. Antibodies with reduced effector function can be used to target, among other things, cell surface molecules of immune cells. In certain embodiments, the CH2 region of a multispecific antibody of the present disclosure is engineered to reduce the ADCC and / or CDC activity of the antibody. In certain embodiments, the CH3 region of a multispecific antibody is engineered to facilitate heterodimerization of the heavy chains.

[0096] Antibodies with reduced effector function are preferably IgG antibodies comprising a modified CH2 / lower hinge region, for example, to reduce Fc receptor interaction or to reduce C1q binding. In certain embodiments, the multispecific IgG antibody has a variant CH2 and / or lower hinge domain to reduce interaction of the multispecific IgG antibody with Fc-gamma receptors. In certain embodiments, a CH2 region of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 114 with 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, a hinge region of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 113 with 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0097] In certain embodiments, the CH3 region of a multispecific antibody is engineered to facilitate heterodimerization of the heavy chains. In certain embodiments, these variations are present to produce essentially only multispecific full-length IgG molecules with amino acid substitutions at positions 351 and 366 in the first CH3 domain (the "KK mutant" heavy chain), e.g., L351K and T366K (EU numbering), and amino acid substitutions at positions 351 and 368 in the second CH3 domain (the "DE mutant" heavy chain), e.g., L351D and L368E, or vice versa. Homodimerization of DE mutant heavy chains (DE-DE homodimers) or KK mutant heavy chains (KK-KK homodimers) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains. In certain embodiments, the multispecific antibodies of the present disclosure comprise a CH3 domain set forth in SEQ ID NOs: 115 and 116 with 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, provided that the DE / KK mutants are unchanged.

[0098] In certain embodiments where the cancer is selected from any solid tumor with high MSI alterations; endometrial cancer, particularly MSI-high endometrial cancer; lung cancer, particularly non-small cell lung cancer (NSCLC), more particularly NSCLC with high PD-L1 expression; breast cancer, particularly metastatic breast cancer, and triple-negative breast cancer (TNBC), the multispecific antibody of the present disclosure is administered at a dose of 10 to 1200 mg, preferably 10 to 600 mg, 25 to 600 mg, 10 to 300 mg, or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 25 to 75 mg. In certain embodiments, where the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, e.g., PD-L1-positive cervical cancer or cervical cancer with high PD-L1 expression; endometrial cancer, e.g., MSI-high endometrial cancer; lung cancer, e.g., non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; brain cancer, e.g., glioblastoma; and breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody of the present disclosure is administered at a dose of 10 to 1200 mg, or 10 to 600 mg, or 25 to 600 mg, or 10 to 300 mg, or 25 to 300 mg, or 10 to 75 mg, or 10 to 50 mg, or 25 to 150 mg, or 25 to 100 mg, or 25 to 75 mg, or 25 to 50 mg. In certain embodiments, where the cancer is selected from any solid tumor with high MSI alterations; endometrial cancer, particularly MSI-high endometrial cancer; lung cancer, particularly non-small cell lung cancer (NSCLC), more particularly NSCLC with high PD-L1 expression; and breast cancer, particularly metastatic breast cancer and triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 50 to 100 mg or 75 to 125 mg.In certain embodiments, where the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, e.g., PD-L1-positive cervical cancer or cervical cancer with high PD-L1 expression; endometrial cancer, e.g., MSI-high endometrial cancer; lung cancer, e.g., non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; brain cancer, e.g., glioblastoma; and breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 25 to 50 mg, 25 to 75 mg, 50 to 100 mg, or 75 to 125 mg. In certain embodiments, where the cancer is selected from any solid tumor with high MSI alterations; endometrial cancer, such as MSI-high endometrial cancer; lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and breast cancer, in particular metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 25 mg, 50 mg, 75 mg, or 100 mg. In certain embodiments where the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, e.g., PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; endometrial cancer, e.g., MSI-high endometrial cancer; lung cancer, e.g., non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, NSCLC with high expression of PD-L1; brain cancer, e.g., glioblastoma; and breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, or 100 mg.

[0099] In certain embodiments where the cancer is selected from any solid tumor with high MSI alterations; endometrial cancer, in particular MSI-high endometrial cancer; lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high PD-L1 expression; breast cancer, in particular metastatic breast cancer, and triple-negative breast cancer (TNBC), the multispecific antibody according to the uses or methods of the present disclosure is administered in a flat dose of 10 to 1200 mg, preferably 10 to 600 mg, 25 to 600 mg, 10 to 300 mg, or 25 to 300 mg, more preferably 25 to 150 mg or 25 to 100 mg, and most preferably 50 to 100 mg. In certain embodiments, where the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, e.g., PD-L1-positive cervical cancer or cervical cancer with high PD-L1 expression; endometrial cancer, e.g., MSI-high endometrial cancer; lung cancer, e.g., non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; brain cancer, e.g., glioblastoma; and breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody according to the uses or methods of the present disclosure is administered in a flat dose of 10 to 1200 mg, or 10 to 600 mg, or 25 to 600 mg, or 10 to 300 mg, or 25 to 300 mg, or 25 to 150 mg, or 25 to 100 mg, or 25 to 75 mg, or 25 to 50 mg. In certain embodiments where the cancer is selected from any solid tumor with high MSI alterations; endometrial cancer, such as MSI-high endometrial cancer; lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly NSCLC with high expression of PD-L1; and breast cancer, in particular metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 50 mg, 75 mg, or 100 mg.In certain embodiments where the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, e.g., PD-L1-positive cervical cancer or cervical cancer with high expression of PD-L1; endometrial cancer, e.g., MSI-high endometrial cancer; lung cancer, e.g., non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, NSCLC with high expression of PD-L1; brain cancer, e.g., glioblastoma; and breast cancer, e.g., metastatic breast cancer or triple-negative breast cancer (TNBC), the multispecific antibody is administered at a dose of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, or 100 mg.

[0100] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure is administered in a flat dose.

[0101] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 50-150 mg, preferably 75-150 mg, more preferably 100-150 mg, or in a flat dose. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 50-150 mg, or 75-150 mg, or 100-150 mg, or in a flat dose.

[0102] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 75 to 125 mg, or in a flat dose.

[0103] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 50-100 mg, preferably 75-100 mg, or a flat dose. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 50-100 mg, or 75-100 mg, or a flat dose.

[0104] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 50-75 mg, or in a flat dose.

[0105] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 10-50 mg, preferably 25-50 mg, or a flat dose. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered in a dose of 10-50 mg, or 25-50 mg, or a flat dose.

[0106] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered once a week, once every two weeks, or once every three weeks. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered once every two weeks. In certain embodiments, when a multispecific antibody according to the uses or methods of the present disclosure is administered once a week, the multispecific antibody is administered at a dose of 10 to 100 mg, preferably 15 to 75 mg. In certain embodiments, when a multispecific antibody according to the uses or methods of the present disclosure is administered once a week, the multispecific antibody is administered at a dose of 10 to 100 mg, or 15 to 75 mg, for example, 15 to 50 mg, or 15 to 40 mg, or 15 to 30 mg, or 15 to 25 mg.

[0107] In certain embodiments, the multispecific antibody according to the uses or methods of the present disclosure is administered in a flat dose once every two weeks.

[0108] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered once every two weeks at a dose of 25, 30, 35, 40, 45, or 50 mg, or as a flat dose. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered once every two weeks at a dose of 25 mg, or as a flat dose. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered once every two weeks at a dose of 50 mg, or as a flat dose.

[0109] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered as a monotherapy.

[0110] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously.

[0111] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered over a period of 30 minutes to 4 hours, preferably 1 to 3 hours, and most preferably 2 hours.

[0112] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered over a period of 30 minutes to 4 hours, preferably 1 to 3 hours, and most preferably 2 hours.

[0113] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure is administered intravenously in a flat dose over 2 hours every 14 days in a 28 day cycle.

[0114] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 25 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 30 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 40 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 50 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 60 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered intravenously at a flat dose of 70 mg over two hours every 14 days in a 28-day cycle. In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure is administered intravenously in a flat dose of 75 mg over 2 hours every 14 days in a 28 day cycle.

[0115] In certain embodiments, the multispecific antibodies according to the uses or methods of the present disclosure are formulated as a liquid at a concentration of 1 mg / mL to 100 mg / mL, preferably 20 mg / mL. In certain embodiments, the multispecific antibodies according to the uses or methods of the present disclosure are formulated as a liquid at a concentration of 1 mg / mL to 100 mg / mL, or at or around 20 mg / mL.

[0116] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered before, simultaneously with, or more preferably after, an antihistamine, a nonsteroidal anti-inflammatory drug (NSAID), a narcotic, intravenous fluids, an antipyretic, a bronchodilator, oxygen, a corticosteroid (IV / oral), a vasoconstrictor, or any combination thereof, to reduce infusion-related reactions. If an infusion-related reaction occurs, subjects may be selected for premedication with the indicated substances to prevent and reduce its incidence and severity.

[0117] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure is administered after the subject has been pre-treated with a standard of care therapy, such as chemotherapy, immunotherapy, or targeted therapy, for advanced metastatic disease.

[0118] In certain embodiments, a multispecific antibody according to the uses or methods of the disclosure is administered to a subject who has not been treated with an anti-PD-L1 agent, such as an anti-PD-L1 antibody or a T-cell agonist.

[0119] In certain embodiments, the cancer is selected from endometrial cancer, particularly MSI-rich endometrial cancer; lung cancer, particularly non-small cell lung cancer (NSCLC), more particularly NSCLC with high PD-L1 expression; and breast cancer, particularly metastatic breast cancer and triple-negative breast cancer (TNBC). In certain embodiments, the cancer is selected from any solid tumor with high MSI alterations; cervical cancer, for example, PD-L1-positive cervical cancer or cervical cancer with high PD-L1 expression; endometrial cancer, MSI-rich endometrial cancer; lung cancer, for example, non-small cell lung cancer (NSCLC), or PD-L1-positive NSCLC, or NSCLC with high PD-L1 expression; brain cancer, for example, glioblastoma; and breast cancer, for example, metastatic breast cancer or triple-negative breast cancer (TNBC).

[0120] Any solid tumor with high MSI alterations includes, but is not limited to, for example, MSH-high colorectal cancer, MSI-high gastric cancer, MSI-high breast cancer, MSI-high prostate cancer, MSI-high bladder cancer, MSI-high ovarian cancer, MSI-high endometrial cancer, MSI-high endometrial cancer, pancreatic ductal adenocarcinoma, follicular thyroid cancer, and adrenocortical carcinoma.

[0121] PD-L1 expression is determined by a clinician skilled in the art; see the exemplary methods in de Ruiter et al. (2021), incorporated herein in its entirety. High expression as determined by any one of these methods constitutes high expression for purposes of this disclosure. When assessed by tumor proportion score (TPS), a TPS score of 1% or greater is a PD-L1 positive cancer. When assessed by combined positive score (CPS), a score of 1% or greater is a PD-L1 positive cancer. A cancer is PD-L1 high if it has a PD-L1 expression score of 10% or greater, 20% or greater, 30% or greater, 40% or greater, or 50% or greater, as determined using the tumor proportion score (TPS). A cancer is PD-L1 high if it has a PD-L1 expression score of 5% or greater, 15% or greater, or 20% or greater, as determined using the combined positive score (CPS). When assessed by multiple such methods, if any one or more methods identify the tumor as PD-L1 positive or PD-L1 high, it meets the criteria of this disclosure.

[0122] In certain embodiments, the subject has not received prior immune checkpoint inhibitor treatment. In certain embodiments, the subject has not received prior immune checkpoint inhibitor treatment and is being treated for MSI-high cancer. In certain embodiments, the subject has not received prior immune checkpoint inhibitor treatment and is being treated for endometrial cancer, particularly MSI-high endometrial cancer. In certain embodiments, the subject has not received prior immune checkpoint inhibitor treatment and is being treated for lung cancer, particularly non-small cell lung cancer. In certain embodiments, the subject has not received prior immune checkpoint inhibitor treatment and is being treated for breast cancer, particularly metastatic breast cancer and triple-negative breast cancer (TNBC).

[0123] In certain embodiments, the multispecific antibodies according to the uses or methods of the present disclosure inhibit CD8 + Increases the number of T cells.

[0124] In certain embodiments, the multispecific antibodies according to the uses or methods of the present disclosure are significantly less toxic than the combination of urelumab and atezolizumab, which is preferably determined in the same studies, particularly in immunocompromised mice bearing breast cancer, more particularly human MDA-MB-231 tumors.

[0125] In certain embodiments, a multispecific antibody according to the uses or methods of the present disclosure does not induce graft-versus-host disease.

[0126] The term "antibody," as used herein, preferably refers to a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, where such domains are derived from or share sequence homology with the variable region of the antibody. Antibodies for therapeutic use are preferably as close as possible to the native antibody of the subject to be treated (e.g., a human antibody for a human subject). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. Antibodies, such as the multispecific antibodies of the present disclosure, typically comprise the constant domain (Fc portion) of a native antibody, which can be engineered as described elsewhere herein, e.g., to reduce ADCC and / or CDC activity.

[0127] A "multispecific antibody" refers to an antibody that comprises at least two binding sites with different antigen or epitope specificities. In certain embodiments, one or more of the antigen binding sites comprises an immunoglobulin VH / VL pair. In certain embodiments, each of the antigen binding sites comprises an immunoglobulin VH / VL pair.

[0128] In certain embodiments, a multispecific antibody according to the present disclosure has no more than two antigen-binding sites. This means that the antigen-binding portion of such a multispecific antibody consists of two antigen-binding sites without the presence of an additional antigen-binding site. In certain embodiments, each of the two antigen-binding sites contains an immunoglobulin VH / VL pair.

[0129] In certain embodiments, the VLs in each VH / VL pair are similar. In certain embodiments, the VLs in each VH / VL pair are identical. In certain embodiments, the multispecific antibody is a full-length antibody having one heavy / light (H / L) chain combination that binds to the extracellular portion of CD137 and one H / L chain combination that binds to the extracellular portion of a member of the B7 family. In certain embodiments, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In certain embodiments, the light chains in the first and second H / L chain combinations are identical.

[0130] In certain embodiments, the multispecific antibody is a bispecific antibody.

[0131] The term "bispecific antibody" means that one part of an antibody binds to one epitope on an antigen, while a second part binds to either a different epitope on the same antigen or a different antigen. The different epitopes typically exist on different antigens. However, the different epitopes can also exist on the same antigen. Depending on the expression levels, (sub)cellular localization, and stoichiometry of the two antigens recognized by the bispecific antibody, both Fab arms of the antibody may or may not simultaneously bind to their epitopes. One arm of a bispecific antibody typically contains the variable domain of one antibody, and the other arm contains the variable domain of another antibody (i.e., one arm of a bispecific antibody is formed by one heavy chain paired with one light chain, and the other arm is formed by a different heavy chain paired with a light chain). In certain embodiments, the heavy chain variable regions of bispecific antibodies of the present disclosure are different from each other, but the light chain variable regions are the same in bispecific antibodies of the present disclosure. Bispecific antibodies in which different heavy chain variable regions are associated with the same or a common light chain variable region are also referred to as bispecific antibodies with a common light chain variable region (cLcv). In certain embodiments, the light chain constant region is also the same. Such bispecific antibodies are referred to as having a common light chain (cLc).

[0132] Certain preferred embodiments are immunoglobulins having an IgG format, offering the advantage that the half-life of bivalent binding molecules / antibodies / variants according to the invention is typically longer compared to multivalent compounds. Furthermore, the immunogenicity of bivalent binding molecules according to the invention is typically lower compared to multivalent compounds. Molecules / antibodies / variants according to these embodiments preferably maintain the structure of native IgG and therefore maintain all the benefits associated with that structure of native IgG.

[0133] "Variants" of antibodies or multispecific antibodies described herein include functional portions, derivatives, and / or analogs of antibodies or multispecific antibodies. The variant may be a fragment of an antibody, e.g., a Fab fragment. The variant may be a single-chain variable fragment (scFv). The variant maintains the binding specificity of the antibody. The functional portions, derivatives, and / or analogs maintain the binding specificity of the antibody. The binding specificity is defined by the ability to bind to the extracellular portions of the first membrane protein and the second membrane protein, as described herein. A variant may have amino acid insertions, deletions, substitutions, or combinations thereof relative to a given amino acid sequence (e.g., SEQ ID NO:), and may have up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 0, 1, 2, 3, 4, or 5, more preferably 0, 1, 2, 3, or 4, more preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, and most preferably 0 or 1, amino acid insertions, deletions, substitutions, or combinations thereof relative to the amino acid sequence of a given SEQ ID NO.

[0134] As used herein, the term "antigen-binding site" refers to the site of a binding molecule or antibody that specifically binds to an epitope of an antigen. Such antigen-binding sites are preferably derived from or share sequence homology with the variable domains of antibodies, particularly the CDR regions thereof. In some preferred embodiments, said antigen-binding site is an immunoglobulin variable domain formed by an immunoglobulin VH / VL pair. In other embodiments, said antigen-binding site is derived from an antibody mimetic, such as, for example, an affibody molecule, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a finomer, a Kunitz domain peptide, or a monobody, as previously described herein.

[0135] The term "full-length" according to the present invention is defined to include essentially complete antibodies that do not contain one or more artificially added moieties having a size of more than 20 amino acid residues, such as additional antigen-binding or activation sites, or additional ligands or additional ligand-binding sites. However, a full-length antibody does not necessarily have all the functions of an intact antibody. For the avoidance of doubt, a full-length antibody contains two heavy chains and two light chains. Each chain contains a constant (C) and a variable (V) region, which can be divided into domains designated CH1, CH2, CH3, and VH for the heavy chain, and CL and VL for the light chain. The heavy chain domains are preferably present in the order of natural antibodies (VHCH1-CH2-CH3; meaning that the VH domain is adjacent to the CH1 domain, followed by the CH2 domain, which is then followed by the CH3 domain). The light chain domains are also preferably present in the order of natural antibodies (VL-CL; meaning that the VL domain is adjacent to the CL domain). Antibodies bind to antigens via the variable domains contained in the Fab fragment portion. Antibodies can interact with molecules and cells of the immune system via their constant domains, primarily via the Fc portion.

[0136] In certain embodiments, full-length IgG antibodies are preferred due to their typically favorable half-life and the desire to remain close to fully self (human) molecules for immunogenicity reasons, hi certain embodiments, the multispecific antibodies of the present disclosure are full-length IgG1, full-length IgG2, full-length IgG3, or full-length IgG4 antibodies.

[0137] Full-length antibodies encompass antibodies in which mutations may exist that provide desired characteristics or that may simply be substitutions for those in the original chain. Such mutations typically do not involve the deletion of a substantial portion of any region. However, antibodies in which one or several amino acid residues have been inserted, deleted, or substituted, or a combination thereof, without essentially altering the antigen-binding properties of the resulting antibody, are encompassed by the term "full-length antibody." For example, an IgG antibody can have 1 to 20 amino acid residue insertions, substitutions, deletions, or a combination thereof within the constant region. [Example]

[0138] Example 1: Multispecific antibody that binds to PD-L1 and CD137. Multispecific antibodies comprising the heavy chain variable regions described in Table 1 were obtained as described in WO2018 / 056821. [Table 1] [Table 2]

[0139] Example 2 - Ex vivo activity on T cell function in endometrial tumor xenografts To investigate the activity of multispecific antibodies that bind CD137 and PD-L1 under therapeutically relevant conditions, fresh tumor xenografts containing tumor-specific effector T cells and regulatory T cells (Tregs) were evaluated.

[0140] Five surgically resected primary endometrial tumors were dissociated into single-cell suspensions. Tumor samples were then analyzed for cell count by flow cytometry or incubated with a multispecific antibody or reference antibody that binds to CD137 and PD-L1 in the presence of a soluble anti-CD3 antibody to measure IFNγ production. Any multispecific antibody that binds to CD137 and PD-L1 described herein can be used, including those containing the VHs of MF6797 and MF7702, as exemplified herein. Reference antibodies included monospecific urelumab analogs, monospecific atezolizumab analogs, and combinations of urelumab and atezolizumab analogs. The atezolizumab analogs were produced based on the information disclosed in WO2010 / 077634. ​​The information used to produce the urelumab analogs was obtained from WO2005 / 035584.

[0141] Tumor samples were stained with an 18-color flow cytometry phenotyping panel. A dead cell stain was used to distinguish live from dead cells (Biolegend, Cat. No. 423110). T cells were first stained with CD45 + Cells (BD Biosciences, Cat. No. 560178) were isolated and then CD14 + CD3+ cells (Thermo Fisher Scientific, Cat. No. 15-0149-42) and CD19+ cells (Biolegend, Cat. No. 302210) were then depleted. + T cells were then identified by selecting CD4 + Cells (BD Biosciences, Catalog No. 564305) and CD8 + CD4 cells (BD Biosciences, Cat. No. 564804) were isolated. + Cells were stained with double positive FoxP3 staining (Thermo Fisher Scientific, Cat. No. 25-4777-42) and CD25 + Treg cells were further separated by CD4+ Treg - , Tregs + , and CD8 + T cell subsets were classified by their positive expression of CD226 (Biolegend, Catalog No. 338330), ICOS (Thermo Fisher Scientific, Catalog No. 62-9948-42), CTLA-4 (BD Biosciences, Catalog No. 555853), CD137 (BD Biosciences, Catalog No. 745256), OX40 (Biolegend, Catalog No. 350018), Lag-3 (Biolegend, Catalog No. 369312), Tim-3 (BD ​​Biolegend, Catalog No. 565564), IL-10 (Biolegend, Catalog No. 501411), GITR (BD Biosciences, Catalog No. 747661), and PD-L1 (BD Biosciences, Catalog No. 565188). Stained samples were run on an LSR Fortessa X-20 cell analyzer (BD Biosciences) and gates were drawn using fluorescence minus one (FMO) controls.

[0142] CD3 + T cell percentage, CD4 + , CD8 + The proportions of Treg and Treg subsets, as well as the levels of IFNγ production, were heterogeneous among tumor samples (Figure 1; top panel). Treatment with the multispecific antibody increased IFNγ production in all tumor samples compared to the negative control antibody and to the comparators urelumab and atezolizumab analogs, as well as their combination. This effect was also observed in the context of high Treg numbers (Figure 1; bottom panel).

[0143] Example 3 - In vivo efficacy in immunodeficient mice bearing human A549 tumors The efficacy of multispecific antibodies that bind to CD137 and PD-L1 in vivo was evaluated in a mouse xenograft model. Any of the multispecific antibodies described herein that bind to CD137 and PD-L1 can be used, including those containing the VHs of MF6797 and MF7702, as exemplified herein. The binding arms of this multispecific antibody do not cross-react with the mouse homologs of PD-L1 and CD137. Therefore, a humanized mouse xenograft model was used. In this model, human transgenic Ly95 T cells, which resemble patient tumor-infiltrating lymphocytes (TILs), were adoptively transferred into immunodeficient mice bearing human A549 tumors. This allows the effects of antibody treatment on such TILs to be studied in a nonhuman model that mimics the human tumor microenvironment. The NSCLC cell line A549 was modified for this study to express the NY-ESO antigen in the appropriate HLA context and stably express high levels of PD-L1. Ly95 cells express PD-1 and CD137.

[0144] First, NSG mice (6-8 weeks old; Jackson Laboratory, Bar Harbor, ME) were treated with 5 × 10 mice suspended in 100 μL of serum-free medium and an equal volume of Matrigel membrane matrix (Corning). 6 A549-A2-ESO tumor cells were inoculated subcutaneously. After tumor establishment (average volume 150 mm), 3 ), mice were randomized into six groups of seven mice, whereby one group received a single intravenous tail vein injection of PBS alone, five groups received 10 × 10 6 1000 mice were injected with PBS containing 1000 human T cells expressing a NY-ESO1-reactive Ly95 TCR construct. Five groups that received adoptive transfer of tumor-specific transgenic Ly95 T cells were then treated intraperitoneally every 5 days with PBS, atezolizumab (5 mg / kg), a urelumab analog (5 mg / kg), an equimolar mixture of atezolizumab and a urelumab analog (5 mg / kg), or a multispecific antibody (5 mg / kg). Tumor volumes were recorded twice weekly over a 4-week period using a research log system.

[0145] In mice implanted with A549PD-L1hi cells, treatment with Ly95 cells plus urelumab analogs, atezolizumab, or a combination of urelumab analogs and atezolizumab did not significantly alter tumor growth compared to controls (Figure 2A). All mice treated with the multispecific antibodies were able to control tumor growth (Figure 2A). Importantly, multispecific antibody-dependent tumor growth inhibition was significantly reduced by adoptively transferred huCD3 into the tumor-to-blood compared to control-treated mice. + This was clearly associated with a skewed distribution of Ly95 cells (Fig. 2B) and an increase in NY-ESO antigen-specific T cells within the tumor (Fig. 2C).

[0146] Example 4 - In vivo efficacy in immunodeficient mice bearing human MDA-MB-231 tumors To assess the impact of treatment with a multispecific antibody that binds CD137 and PD-L1 in the context of heterogeneous T cell populations, we administered human CD34 T cells to NSG mice. + Hematopoietic stem cells were transplanted, followed by the PD-L1-expressing human MDA-MB-231 breast cancer line. Any of the multispecific antibodies described herein that bind to CD137 and PD-L1 can be used, including those comprising the VHs of MF6797 and MF7702, as exemplified herein.

[0147] To generate human stem cell-engrafted NSG mice, immunodeficient NSG mice (6–8 weeks old; Jackson Laboratory, Bar Harbor, ME) were administered 15 mg / kg intraperitoneal busulfan (Busilvex, Pierre Fabre) and, 24 hours later, 1 × 10 5 Human CD34 +Intravenous injections containing umbilical cord blood cells (purchased from STEMCELL Technologies) were performed as previously described (Ishikawa F. et al. Development of functional human blood and immune systems in NOD / SCID / IL2 receptor γ chain null mice. Blood. 2005 Jan 9;106(5):1565-73). + Only mice with >25% cells and T cell levels >80 counts / μL were included. Mice received a total of 3 × 10 cells suspended in 100 μL of serum-free medium and an equal volume of Matrigel matrix (Corning). 6 MDA-MB-231 tumor cells were inoculated subcutaneously. The tumors were approximately 80-100 mm 3 When the tumor size reached 100 μg / mL, mice were randomized into the following groups (n=7 per group): 1) Fc-silenced IgG1 control (5 mg / kg); 2) atezolizumab (5 mg / kg); 3) urelumab analog (5 mg / kg); 4) an equimolar mixture of atezolizumab and urelumab analog (5 mg / kg); 5) pembrolizumab (5 mg / kg); and 6) a multispecific antibody binding to CD137 and PD-L1 (0.5 and 5 mg / kg). Antibodies were diluted in PBS (Life Technologies) and administered intraperitoneally. Animals were dosed intraperitoneally every 5 days for 31 days. Tumors were measured using calipers and calculated using the formula: l (length) × w 2 Tumor volumes were calculated by fitting to an ellipsoid using (width) x 1 / 2. Statistical significance was determined by one-way ANOVA. Body weight was also monitored throughout the study.

[0148] In animals receiving control IgG, tumors grew progressively over the first 40 days, after which more rapid growth kinetics were observed in most animals (Figure 3Aa). Groups treated with the CD137 agonist, urelumab analog, showed tumor growth patterns very similar to those of controls (Figure 3Ac). In groups treated with pembrolizumab (Figure 3F), atezolizumab (Figure 3Ad), or the combination of atezolizumab and urelumab analog (Figure 3Ae), there was evidence of tumor growth control in some mice, but by the end of the observation period, tumors in all surviving mice were rapidly growing. In contrast, all animals treated with the multispecific antibody experienced tumor control, including two complete responses, and showed slower growth kinetics at the end of the treatment period (Figure 3Ab). A cohort of mice treated with a 10-fold lower dose of the multispecific antibody (0.5 mg / kg) also showed similar inhibition of tumor growth (Figure 3Ab). Consistent with the NY-ESO model, analysis of TILs in this model demonstrated that treatment with multispecific antibodies significantly increased intratumoral CD8 + Treatment with urelumab analogs resulted in an increase in the frequency of CD8 T cells in the tumor (Figure 3B; upper graph). + This reduces the number of CD4 + T cells and PD-L1 + This was associated with increased monocyte frequencies. These results indicate that the potent T cell agonism observed in vitro with the multispecific antibody also translated into PD-L1-dependent tumor suppression in vivo.

[0149] We also noted that some mice in the pembrolizumab, atezolizumab, or urelumab analog groups, and all mice in the combination treatment groups, exhibited rough fur and skin, hunched posture, and in some cases required euthanasia (3–5 mice in each group) due to excessive weight loss (Figure 4). Animals in the control or multispecific antibody groups showed no signs of stress (although one animal in the multispecific antibody group was euthanized for unrelated reasons). Notably, we observed that the combination treatment with urelumab analog and atezolizumab (Figure 4e) was significantly more toxic than treatment with the control or multispecific antibody (Figure 4b).

[0150] Example 5 - Phase 1 Dose-Escalation Single-Agent Study for Dose-Finding, Safety, and Preliminary Efficacy of a Multispecific Antibody Targeting CD137 and PD-L1 in Participants with Advanced or Metastatic Malignancies Treatment group and duration: An open-label, randomized Phase 1 study has been initiated to determine the safety, tolerability, and preliminary efficacy of a multispecific antibody targeting CD137 and PD-L1 in adult participants with advanced or metastatic malignancies.

[0151] The following multispecific antibodies are suitable for use in the studies and methods of the invention: MF6797xMF7702, MF6763xMF5442, MF6754xMF5561, MF6785xMF5439, and MF6785xMF5442, preferably MF6797xMF7702. Each multispecific antibody comprises two VHs designated by MF numbers capable of binding to CD137 and PD-L1, respectively, an Fc region with KK / DE CH3 heterodimerization domains set forth in SEQ ID NO:115 and SEQ ID NO:116, respectively, a CH2 domain set forth in SEQ ID NO:114, a CH1 domain set forth in SEQ ID NO:112, and a common light chain set forth in SEQ ID NO:109.

[0152] A dose escalation study was conducted to determine the MTD and / or RDE of a multispecific antibody in participants with advanced or recurrent / metastatic solid tumors. Participants received escalating doses of one of the exemplary multispecific antibodies described above, further referred to herein as the "study antibody," every two weeks until the MTD or RDE was reached. Each treatment cycle was 28 days in duration. A total of 10 doses were planned: 0.4 mg, 1.2 mg, 3.5 mg, 10 mg, 25 mg, 75 mg, 150 mg, 300 mg, 600 mg, and 1200 mg.

[0153] Overall study design This is an open-label, Phase 1, dose-escalation study to determine the safety, tolerability, and preliminary efficacy of the investigational antibody in adult participants with advanced or metastatic malignancies, and will be conducted in two parts. The investigational antibody will be administered intravenously as a flat dose over two hours every 14 days in a 28-day cycle.

[0154] Part 1: Dose escalation Part 1 is a dose escalation to determine the MTD and / or RDE of the investigational antibody administered every 14 days to participants with advanced or metastatic solid tumors, regardless of PD-L1 expression. During dose escalation, cohorts of participants will be treated with the investigational antibody until the MTD is reached or a lower recommended dose is established. Dose escalation will be guided by adaptive BLRM followed by titration according to overdose control principles.

[0155] During dose escalation, additional cohorts of up to six participants may be enrolled at the next dose level or at any planned dose level below the MTD or at intermediate dose levels to better characterize safety, PK, and / or pharmacodynamic activity. A maximum of five participants with a given tumor type may be enrolled across all dose levels in Part 1, unless the medical monitor approves additional enrollment for that tumor type.

[0156] Enrollment in Part 1 will begin at dose level 1 (0.4 mg IV test antibody) with the option to escalate or deescalate based on defined criteria. Dose-limiting toxicities occurring by Day 28 will guide dose escalation / deescalation and determination of the RDE or MTD. However, delayed immune-mediated toxicities up to 90 days after initiation of study treatment will be considered when assessing safety; therefore, a lower RDE or MTD may be subsequently determined based on relevant toxicities evident after Day 28. If the lowest dose level is deemed unsafe, enrollment in Part 1 will be halted.

[0157] Part 2: Dose Confirmation / Safety Extension Part 2 is a dose expansion to confirm the dose of the investigational antibody through further evaluation of safety, tolerability, PK, preliminary antitumor activity, and functional target engagement.

[0158] Participants with advanced or metastatic tumors, regardless of PD-L1 expression, will be enrolled as follows: Participants with relapsed or refractory NSCLC who received anti-PD-1 therapy; Participants with refractory MSI-H / dMMR tumors who received anti-PD-1 therapy; Participants with immunotherapy-naive TNBC; Participants with tumor histology in Part 1 where preliminary efficacy was observed with the study antibody.

[0159] Up to two confirmatory dose levels may be considered, selected based on PK, antitumor, pharmacodynamic activity including receptor modulation, safety, and tolerability.

[0160] Initial enrollment will be limited to 10 or 20 participants per indication per confirmatory dose level. If at least one participant has a response at one or both confirmatory dose levels, the indication may be expanded to a total of 40 participants across dose levels. If three or more indications enrolled in Part 2 show clinical activity (i.e., one or more of the first 10 participants have a response), each indication may be expanded to a maximum of 40 participants. The decision to expand each indication will be determined by the Clinical Trial Steering Committee.

[0161] If there is insufficient evidence of clinical activity in Part 1, as determined by the clinical trial steering committee, only one dose will be evaluated in Part 2. This cohort will include a minimum of 20 participants with PD-L1-positive tumors as assessed by the Ventana PD-L1 SP263 assay with a CPS cutoff of 1% or greater. If 20 participants are found to have PD-L1-negative tumors before full enrollment in Part 2, subsequent participants with PD-L1-negative tumors will not be eligible to enroll in Part 2.

[0162] Inclusion criteria Participants are eligible to participate in this study only if all of the following criteria apply:

[0163] To be eligible, participants with NSCLC, MSI-H / dMMR tumors, or TNBC must meet all criteria described herein for "all participants" as well as any applicable tumor-specific criteria described below.

[0164] All participants - Possess the ability and willingness to sign a written ICF for the study; - be 18 years of age or older at the time of signing the informed consent; - Willing and able to comply with and follow all protocol requirements, including all scheduled visits and protocol procedures; - Having a life expectancy of 12 weeks or more; -ECOG performance status of 0 or 1; -Part 1: Histologically or cytologically confirmed advanced metastatic solid tumors that are not considered suitable for surgery or other curative treatment or procedures (if applicable); Part 2: Participants must have a diagnosis of one of the following tumor types and meet applicable tumor-specific criteria: NSCLC, MSI-H / dMMR tumor, TNBC, or other malignancies that have shown preliminary efficacy after treatment with the investigational antibody; - Measurable disease according to RECIST v1.1 or Lugano criteria; Note: Tumor lesions located in previously irradiated areas or areas that have received other local therapies are considered measurable if clear progression in the lesion has been demonstrated; - Received previous standard treatment for advanced metastatic disease applicable to the tumor type; - Received up to four prior systemic treatment regimens (including chemotherapy, immunotherapy, and targeted therapy regimens) for progressive or recurrent / metastatic disease; - Received up to one prior anti-PD-1 therapy, including an immunotherapy regimen, in the advanced / metastatic setting. NOTE: This criterion does not apply to participants with immunotherapy-naive TNBC in Part 2. - Willingness to undergo pre- and intra-treatment tumor biopsies to obtain tumor tissue. NOTE: If a participant is scheduled to undergo a tumor biopsy for the purposes of this study and it is subsequently determined that tumor tissue cannot be safely obtained, the participant may still be enrolled in the study; -Part 2 only: Participants must have evaluable PD-L1 from a fresh tumor biopsy during screening; - Pregnancy or male willingness to avoid childbearing based on the following criteria: a. Men must agree to take appropriate precautions to avoid conception (with at least 99% certainty) from screening through 90 days after the last dose of study antibody and must refrain from donating sperm during this period. Permitted methods that are at least 99% effective in preventing pregnancy must be communicated to participants and their understanding confirmed; b. Females of childbearing potential must have a negative serum pregnancy test at screening and before the first dose on Day 1 and must agree to take appropriate precautions to avoid pregnancy (with at least 99% certainty) from screening through 90 days after the last dose of study antibody. Acceptable methods that are at least 99% effective in preventing pregnancy must be communicated to participants and their understanding confirmed. Females of childbearing potential must refrain from donating oocytes from 30 days before the first dose of study antibody through 90 days after the last dose of study antibody. c. Women of non-childbearing potential (i.e., surgically sterile by hysterectomy and / or bilateral oophorectomy, or women who have been amenorrheic for 12 months or more and are at least 50 years old) are eligible for enrollment.

[0165] Tumor-specific criteria for participants with histologically or cytologically confirmed TNBC - Histologically or cytologically confirmed HER2-negative / ER-negative / PR-negative breast cancer of documented origin, defined as both of the following: a. Estrogen receptor (ER) and progesterone receptor (PgR) negative: Less than 1% of tumor cell nuclei are immunoreactive in the presence of evidence that the sample can express ER or PgR (positive endogenous control); Human epidermal growth factor receptor 2 (HER2) negative per American Society of Clinical Oncology-College of American Pathologists (ASCO / CAP) guidelines: immunohistochemistry (IHC) 0 or 1 or fluorescent in situ hybridization (FISH) negative (or equivalent negative test). Subjects with IHC 2 must also be negative by fluorescent in situ hybridization (FISH) (or equivalent negative test). -Unresectable advanced or metastatic TNBC according to the current AJCC staging system, in which local therapy has failed; Must have received prior treatment with a taxane-containing chemotherapy regimen in the advanced or metastatic setting. Part 2 participants only: Have not received prior immunotherapy containing PD-1, PD-L1, CTLA-4, or other immune checkpoint inhibitors, either alone or as part of a combination therapy.

[0166] Tumor-specific criteria for participants with histologically or cytologically confirmed NSCLC Histologically or cytologically confirmed diagnosis of NSCLC (non-squamous or squamous). If the tumor is of exclusively non-squamous histology, documentation of test results for mutations or gene rearrangements in EGFR, ALK, BRAF, and ROS1 (molecular testing is not currently part of diagnostic guidelines for predominantly squamous histology). If mutations or gene rearrangements are present, the participant is likely to progress on or not tolerate targeted therapy. -Unresectable advanced or metastatic NSCLC according to the current AJCC staging system, in which local therapy has failed; Part 2 participants only: Must have documented progression on anti-PD-1 therapy as defined by meeting one of the following criteria: a. Primary refractory: Previously received anti-PD-1 therapy (alone or as part of a combination) in the advanced or metastatic setting for at least 12 weeks with PD as the best response to treatment. b. Secondary resistance: Previously received anti-PD-1 therapy (alone or as part of a combination) in the advanced or metastatic setting and achieved CR, PR, or SD, but required confirmation of PD while receiving subsequent anti-PD-1 therapy (PD confirmed after at least 4 weeks [≥ 28 days]).

[0167] Tumor-specific criteria for participants with solid tumor indications with confirmed MSI-H or dMMR status MSI-H or dMMR solid tumor, as determined by a local laboratory using IHC or polymerase chain reaction and must have tissue available for central diagnostic confirmation; -Only participants in Part 2 who are deemed refractory to primary anti-PD-1 therapy are considered refractory, defined as: having received prior anti-PD-1 therapy (alone or as part of a combination) in the advanced or metastatic setting for at least 12 weeks and having documented PD as their best response to treatment.

[0168] result Several patients enrolled in the study. Clinical activity was observed at least in the subgroup of patients with MSI-rich endometrial cancer. In this subgroup, 40-year-old patients diagnosed with MSI-rich endometrial cancer with PD-L1 expression between 10% and 50% received adjuvant treatment with carboplatin plus taxol, cisplatin plus 5FU, epirubicin / cyclophosphamide, tamoxifen, and adriamycin prior to treatment with the study drug. Total target lesions were 51 mm at the start of treatment. The study drug was administered at 75 mg. Total target lesions decreased to 42 mm after 8 weeks of treatment.

[0169] Clinical activity was also observed in a subgroup of patients with glioblastoma. In this subgroup, a 35-year-old patient diagnosed with glioblastoma underwent radiation therapy, surgery, and temozolomide prior to treatment with the study drug, which was administered at 25 mg. The patient demonstrated stable disease, with baseline target lesion dimensions of 9.88 mm x 14.62 mm, which decreased to 4.87 mm x 6.01 mm after 19 months of treatment.

[0170] Clinical activity was further observed in a subgroup of patients with cervical cancer. In this subgroup, a 70-year-old patient diagnosed with cervical cancer with PD-L1 expression of 1% on tumor cells and 30% on tumor immune cells received prior treatment with radiation therapy, surgery, carboplatin, taxol, and Avastin prior to treatment with study drug. The total target lesion size was 40 mm at the start of treatment. Study drug was administered at 50 mg. The total target lesion size decreased to 26 mm after 16 weeks of treatment. The patient achieved a partial response (35% reduction) per RECIST v1.1 as determined by the investigator, even after discontinuing study drug treatment.

[0171] array SEQ ID NO: 1: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRRAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWGVIGGHYMDVWGKGTTVTVSS SEQ ID NO: 2: HCDR1 according to Kabat from SEQ ID NO: 1 NFAMN SEQ ID NO: 3: HCDR2 according to Kabat from SEQ ID NO: 1 WINTNTGNPTYAQGFTG SEQ ID NO: 4: HCDR3 according to Kabat from SEQ ID NO: 1 DWGVIGGHYMDV SEQ ID NO: 5: Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSDGYGPKAFDYWGQGTLVTVSS SEQ ID NO: 6: HCDR1 according to Kabat SYGIS SEQ ID NO: 7: HCDR2 according to Kabat WISAYNGNTNYAQKLQG SEQ ID NO: 8: HCDR3 according to Kabat DSDGYGPKAFDY SEQ ID NO: 9: Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSSSTAYLQWSSLKASDTAMYYCASFYTGIVGATGAFDVWGQGTTVTVSS SEQ ID NO: 10: HCDR1 according to Kabat SYWIG SEQ ID NO: 11: HCDR2 according to Kabat IIYPDDSDTRYSPSFQG SEQ ID NO: 12: HCDR3 according to Kabat FYTGIVGATGAFDV SEQ ID NO: 13: Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSDAISWVRQAPGQGLEWMGGMIPILGTANYAQKFQGRVTITADRSSTSTAYMELSSLRSEDTAVYYCVRGATYYYGSGTYYSINWFDPWGQGTLVTVSS SEQ ID NO: 14: HCDR1 according to Kabat SDAIS SEQ ID NO: 15: HCDR2 according to Kabat GMIPILGTANYAQKFQG SEQ ID NO: 16: HCDR3 according to Kabat GATYYYGSGTYYSINWFDP SEQ ID NO: 17: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCRASGYTFTNFAMTWVRQAPGQGPEYMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWASVMVRGDLDYWGQGTLVTVSS SEQ ID NO: 18: HCDR1 according to Kabat NFAMT SEQ ID NO: 19: HCDR3 according to Kabat DWASVMVRGDLDY SEQ ID NO: 20: Heavy chain variable region QVQLVQSGAEVKKPGASVKVSCKVSGYTLSELSIHWVRQAPGKGVEWMGGFYPEDVEPIYARKFQGRVTMTEDTSTDTAYMELNSLRSEDTAVYYCAAEGFDNYGSGIRGNWFDPWGQGTLVTVSS SEQ ID NO: 21: HCDR1 according to Kabat ELSIH SEQ ID NO: 22: HCDR2 according to Kabat GFYPEDVEPIYARKFQG SEQ ID NO: 23: HCDR3 according to Kabat EGFDNYGSGIRGNWFDP SEQ ID NO: 24: Heavy chain variable region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQSPGKGLEWMGSFYPEDGETIYAQKFQGRITMTEDTSADTAYMELSSLRSEDTAVYYCATEGVGVIRGNWFDPWGQGTLVTVSS SEQ ID NO: 25: HCDR1 according to Kabat ELSMH SEQ ID NO: 26: HCDR2 according to Kabat SFYPEDGETIYAQKFQG SEQ ID NO: 27: HCDR3 according to Kabat EGVGVIRGNWFDP SEQ ID NO: 28: Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVTVSS SEQ ID NO: 29: HCDR2 according to Kabat IIFPDDSDTRYSPSFQG SEQ ID NO: 30: HCDR3 according to Kabat LGGYSGYAEDFVDF SEQ ID NO: 31: Heavy chain variable region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTKLSMHWVRQAPGKGLEWMGGFEPEDGETINAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATDLRLGASYYYSYMDVWGRGTMVTVSS SEQ ID NO: 32: HCDR1 according to Kabat KLSMH SEQ ID NO: 33: HCDR2 according to Kabat GFEPEDGETINAQKFQG SEQ ID NO: 34: HCDR3 according to Kabat DLRLGASYYYSYMDV SEQ ID NO: 35: Heavy chain variable region QITLKESGPTLVKPTQTLTLSCTFSGFSLSTSGMSVGWIRQPPGKALEWLALIYWNDDKYFSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCAHTLWGSDDVFDVWGQGTMVTVSS SEQ ID NO: 36: HCDR1 according to Kabat TSGMSVG SEQ ID NO: 37: HCDR2 according to Kabat LIYWNDDKYFSPSLKS SEQ ID NO: 38: HCDR3 according to Kabat TLWGSDDVFDV SEQ ID NO: 39: Heavy chain variable region EVQLVQSGAEVKKPGESLKISCKVSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWHTLKASDTAMYYCARHQGYSFSGSHIDDYWGQGTLVTVSS SEQ ID NO: 40: HCDR1 according to Kabat NYWIG SEQ ID NO: 41: HCDR2 according to Kabat IIYPGDSDTRYSPSFQG SEQ ID NO: 42: HCDR3 according to Kabat HQGYSFSGSHIDDY SEQ ID NO: 43: Heavy chain variable region EVQLVQSGAEVRKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTVYLQWSSLKASDTAMYYCARHAGFIITSQNIDDYWGQGTLVTVSS SEQ ID NO: 44: HCDR1 according to Kabat TYWIG SEQ ID NO: 41: HCDR2 according to Kabat IIYPGDSDTRYSPSFQG SEQ ID NO: 45: HCDR3 according to Kabat HAGFIITSQNIDDY SEQ ID NO: 46: Heavy chain variable region EVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRQAPGQGLEWMGWINTNTGNPTYAQDFTGRFVFSLDTSGNTAYLQISSLKAEDTAVYYCARDWGLVAIGYFDYWGQGTLVTVSS SEQ ID NO: 47: HCDR2 according to Kabat WINTNTGNPTYAQDFTG SEQ ID NO: 48: HCDR3 according to Kabat DWGLVAIGYFDY SEQ ID NO: 49: Heavy chain variable region QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVNWIRQPPGEALEWLALIYWNDDTYYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCAHEGIIGFLGGNWFDPWGQGTLVTVSS SEQ ID NO: 50: HCDR1 according to Kabat TTGVGVN SEQ ID NO: 51: HCDR2 according to Kabat LIYWNDDTYYSPSLKS SEQ ID NO: 52: HCDR3 according to Kabat EGIIGFLGGNWFDP SEQ ID NO: 53: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS SEQ ID NO: 54: HCDR1 according to Kabat SHAMN SEQ ID NO: 55: HCDR2 according to Kabat WINPNTGNPTYAQGFTG SEQ ID NO: 56: HCDR3 according to Kabat DRKYVTNWVFAEDFQH SEQ ID NO: 57: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS SEQ ID NO: 58: HCDR3 according to Kabat DRGYMSNWVFAEYFPH SEQ ID NO: 59: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCATDRGYISSWVFAEDFQHWGQGTLVTVSS SEQ ID NO: 60: HCDR1 according to Kabat SYAMN SEQ ID NO: 61: HCDR3 according to Kabat DRGYISSWVFAEDFQH SEQ ID NO: 62: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCTASGYTFTSYAMNWVRQAPGQRLEWMACVNPNTGSPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGHGTLVTVSS SEQ ID NO: 63: HCDR2 according to Kabat CVNPNTGSPTYAQGSTG SEQ ID NO: 64: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWMNPNTGNPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS SEQ ID NO: 65: HCDR1 according to Kabat NYAMN SEQ ID NO: 66: HCDR2 according to Kabat WMNPNTGNPTYAQGSTG SEQ ID NO: 67: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS SEQ ID NO: 68: HCDR1 according to Kabat NYAIN SEQ ID NO: 69: Heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGDTFNTYSITWVRQAPGQGLEWMGSIVPIFGTINNAQKFQGRVTITADKSANTAYMELSSLRSEDTAVYYCARDNTMVRGVDYYYMDVWGKGTMVTVSS SEQ ID NO: 70: HCDR1 according to Kabat TYSIT SEQ ID NO: 71: HCDR2 according to Kabat SIVPIFGTINNAQKFQG SEQ ID NO: 72: HCDR3 according to Kabat DNTMVRGVDYYYMDV SEQ ID NO: 73: Heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGIFSTYAISWVRQAPGQGLEWMGGIIPIFDTPNYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCAKNVRGYSAYDLDYWGQGTLVTVSS SEQ ID NO: 74: HCDR1 according to Kabat TYAIS SEQ ID NO: 75: HCDR2 according to Kabat GIIPIFDTPNYAQKFQG SEQ ID NO: 76: HCDR3 according to Kabat NVRGYSAYDLDY SEQ ID NO: 77: Heavy chain variable region EVQLVQSGAEVKNPGSSVKVSCKATGGTFNTYGTNWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTTTAYMEVSSLRSEDTAVYYCARGGADMGTLDYWGQGTLVTVSS SEQ ID NO: 78: HCDR1 according to Kabat TYGTN SEQ ID NO: 79: HCDR2 according to Kabat GIIPIFGTANYAQKFQG SEQ ID NO: 80: HCDR3 according to Kabat GGADMGTLDY SEQ ID NO: 81: Heavy chain variable region EVQLVQSGAEVMRPGSSVKVSCKASGGIFTYTIIWVRQAPGQGLEWMGGIIPIFDTPNFAQKFQGRLTITADKSTNTAYMELTSLRSEDTAVYYCAREGCNHGVCYPYWGQGTLVTVSS SEQ ID NO: 82: HCDR1 according to Kabat TYTII SEQ ID NO: 83: HCDR2 according to Kabat GIIPIFDTPNFAQKFQG SEQ ID NO: 84: HCDR3 according to Kabat EGCNHGVCYPY SEQ ID NO: 85: Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADKSTSTVYMELSSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS SEQ ID NO: 86: HCDR1 according to Kabat SYGIT SEQ ID NO: 87: HCDR2 according to Kabat GIIPIFGTTNYAQKFQG SEQ ID NO: 88: HCDR3 according to Kabat RRGYSNPHWLDP SEQ ID NO: 89: Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGILWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADISTSTAYMELSSLRSEDTAVYYCARGGGNYYEFVYWGQGTLVTVSS SEQ ID NO: 90: HCDR1 according to Kabat TYGIL SEQ ID NO: 91: HCDR3 according to Kabat GGGNYYEFVY SEQ ID NO: 92: Heavy chain variable region EVQLVQSGAEVKKPGSSVRVSCKASGGTFNTYAINWVRQAPGQGLEWVGRIIPIFDTANYAQKFQGRVTISADKSTTTAYMELSSLRSEDTAVFYCAKDETGYSSSNFQHWGQGTLVTVSS SEQ ID NO: 93: HCDR1 according to Kabat TYAIN SEQ ID NO: 94: HCDR2 according to Kabat RIIPIFDTANYAQKFQG SEQ ID NO: 95: HCDR3 according to Kabat DETGYSSSNFQH SEQ ID NO: 96: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS SEQ ID NO: 97: Heavy chain variable region QVQLVQSGAEVKRPGSSVKVSCKASGGTFNTYSITWVRQAPGQGLEWMGIIPVFGTSKYAQKFQDRVTITADKSTNTAYMELSSLRSEDTAVYYCARDPSFSSSSGWFDPWGQGTLVTVSS SEQ ID NO: 98: HCDR2 according to Kabat GIIPVFGTSKYAQKFQD SEQ ID NO: 99: HCDR3 according to Kabat DPSFSSSSGWFDP SEQ ID NO: 100: Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYAINWVRQAPGQGLEWMGGIIPIFDTANYAQRFQGRVTITADKSTSTAYMELSSLRSEDTAVYFCAKDQTGYSSTLFDYWGQGTLVTVSS SEQ ID NO: 101: HCDR2 according to Kabat GIIPIFDTANYAQRFQG SEQ ID NO: 102: HCDR3 according to Kabat DQTGYSSTLFDY SEQ ID NO: 103: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS SEQ ID NO: 104: Heavy chain variable region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGWIIPIFDTGNYAQKIQGRVTITADKSTSTAYMELTSLRSEDTAVYYCARHDYTNTVDAFDIWGQGTMVTVSS SEQ ID NO: 105: HCDR2 according to Kabat WIIPIFDTGNYAQKIQG SEQ ID NO: 106: HCDR3 according to Kabat HDYTNTVDAFDI SEQ ID NO: 107: Heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPVFGTTNYAQKFQGRVTITADKSTSTVFMELNSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS SEQ ID NO: 108: HCDR2 according to Kabat GIIPVFGTTNYAQKFQG SEQ ID NO: 109: Amino acid sequence of human common light chain IGKV1-39 / jk1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 110: Amino acid sequence of the consensus light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 111: Amino acid sequence of the consensus light chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 112: Amino acid sequence of CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 113: Amino acid sequence of hinge EPKSCDKTHTCPPCP SEQ ID NO: 114: Amino acid sequence of CH2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 115: Amino acid sequence of CH3 with KK mutation GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 116: Amino acid sequence of CH3 with DE mutation GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 117: Amino acid sequence of CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFS SAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCK QGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDWCG PSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSWKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

Claims

[Claim 1] 1. A multispecific antibody comprising an antigen-binding site that binds to the extracellular portion of CD137 and an antigen-binding site that binds to the extracellular portion of a second membrane protein, for use in a method of treating cancer in a subject in need thereof, wherein said multispecific antibody is administered at a dose of 25 to 300 mg, preferably 25 to 150 mg or 25 to 100 mg, more preferably 50 to 100 mg.

Images