Combination therapy for treating cancer

Abstract

The present invention relates to combination therapies for the treatment of cancer and to the field of binding molecules. In particular, it relates to the field of therapeutic binding molecules for the treatment of diseases involving abnormal cells, such as cancer cells. In particular, it relates to multispecific antibodies that bind to extracellular portions of two or more different membrane-associated proteins and thereby modulate the biological activity of cell expression, and the use of such antibodies in combination therapy.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of January 24, 2023, application number 202380018389.X, and invention name “Combination therapy for treating cancer”. Technical Field

[0002] The present invention relates to the field of binding molecules. In particular, it relates to the field of therapeutic binding molecules for treating diseases involving abnormal cells (such as cancer cells). In particular, it relates to multispecific antibodies that bind to the extracellular parts of two or more different membrane-associated proteins and thereby modulate the biological activity expressed by the cells, and the use of such antibodies in combination therapy. Background Art

[0003] Cancer currently remains the leading cause of morbidity and mortality in the world, despite many advances in treating the disease and an increased understanding of the molecular events that lead to cancer.

[0004] Traditionally, most cancer drug discovery has focused on drugs that block basic cellular functions and kill dividing cells. However, in the case of advanced cancer, chemotherapy rarely results in a complete cure, no matter how aggressively the drug is administered, even to the point where patients experience life-threatening side effects from treatment. In most cases, tumors in patients stop growing or temporarily shrink (called remission) only to begin proliferating again, sometimes more rapidly (called relapse), and become increasingly difficult to treat. Recently, the focus of cancer drug development has shifted from broad cytotoxic chemotherapy to less toxic targeted cytostatic therapies. Treatment of advanced cancer has been clinically validated in leukemia and some other cancers. However, in most cancers, targeted approaches have still proven insufficient to completely eliminate the cancer in most patients.

[0005] Targeting of cancer has been achieved using a variety of different approaches, including, for example, small molecules that target signaling proteins that cancer relies on for survival and / or growth; vaccines with tumor-specific proteins; cell therapies using immune cells that actively kill tumor cells, and antibodies that target cytotoxic molecules to tumors; and interfering with signals and / or (re)directing the host's immune system to tumor cells.

[0006] (Re)direction of the immune system can be achieved in a variety of ways. One method is to activate T-cell co-stimulatory 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 prolonged survival of CD8+T-cells. 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 homolog ligand programmed cell death 1 ligand 1 (PD-L1), which can be expressed on tumor cells. After PD-L1 binds to PD-1, signaling leads to weakened T-cell receptor (TCR) signals and T-cell exhaustion. This is a mechanism used by tumors to escape and / or suppress the immune system.

[0007] This immunosuppression can be blocked by immune checkpoint inhibitor therapy (ICI), such as antagonistic antibodies against PD-1 or PD-L1. ICI treatment has shown significant and durable responses in a subgroup of cancer patients associated with activated CD8+ T-cell infiltration and proliferation. Combinations of ICIs (such as anti-PD-1 and anti-CTLA-4) have been shown to further improve efficacy, but at the expense of toxicity, as most patients experience grade 3 or 4 treatment-related adverse events.

[0008] Dual targeting of the PD-1 / PD-L1 axis and CD137 may be beneficial for optimal engagement of specific antitumor immunity. The two most advanced therapeutic CD137 agonist antibodies currently in clinical trials are usurelumab (IgG4) and utolumab (IgG2). Development of usurelumab has been discontinued because systemic activation of the CD137 pathway in patients resulted in dose-dependent hepatitis. Safe administration of usurelumab requires reduced doses; 0.1 mg / kg was selected for combination studies with PD-1 inhibitors. Utoluumab was well tolerated by patients but had only modest antitumor activity as a monotherapy and no clear synergy with PD-1 blockade in combination therapy.

[0009] Therefore, there remains a need to provide healthcare professionals with more and better options for treating cancer, especially advanced or metastatic solid tumors. Summary of the invention

[0010] The present invention relates to a combination therapy, wherein 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 is used together with a PD-L1 or PD-1 inhibitor in a method of treating cancer in a subject in need thereof.

[0011] The present invention provides devices and methods for (re)directing components of the immune system to treat cancer, particularly advanced or metastatic solid tumors.

[0012] In certain aspects, the present invention 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 treatment further comprises administering a PD-L1 or PD-1 inhibitor.

[0013] In certain aspects, the present invention 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 and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

[0014] In certain aspects, the present invention provides a combination 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, and a PD-L1 or PD-1 inhibitor, for use in a method of treating cancer in a subject in need thereof.

[0015] In certain aspects, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof 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 and a PD-1 or PD-L1 inhibitor.

[0016] In certain aspects, the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

[0017] In certain aspects, the cancer is an advanced or metastatic solid tumor, such as selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma, such as the cancer is NSCLC. In certain aspects, the melanoma is selected from skin, acral or mucosal melanoma. In certain aspects, the cancer is Merkel cell carcinoma (MCC) (also known as neuroendocrine carcinoma of the skin or trabecular carcinoma).

[0018] In certain aspects, the cancer has relapsed after PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression.

[0019] In certain aspects, the multispecific antibody is administered prior to a PD-1 or PD-L1 inhibitor.

[0020] In certain aspects, the multispecific antibody is a bispecific antibody.

[0021] In certain aspects, the antigen binding site of the multispecific antibody that binds to the extracellular portion of the second membrane protein binds to PD-L1.

[0022] In certain aspects, the present invention provides a multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising:

[0023] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0024] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0025] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0026] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0027] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0028] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0029] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0030] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0031] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0032] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0033] The multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

[0034] In certain aspects, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to a subject in need thereof a multispecific antibody comprising a binding domain that binds to CD137, the binding domain comprising:

[0035] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0036] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0037] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0038] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0039] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0040] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0041] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0042] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0043] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0044] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0045] and PD-1 or PD-L1 inhibitors.

[0046] In certain aspects, the present invention provides a multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising:

[0047] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0048] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0049] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0050] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0051] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0052] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0053] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0054] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0055] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0056] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0057] wherein the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately; and

[0058] Wherein the cancer is selected from advanced or metastatic solid tumors.

[0059] In certain aspects, the present invention provides a pharmaceutical composition or kit comprising 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, and instructions for use of the multispecific antibody in combination with a PD-1 or PD-L1 inhibitor, particularly useful for treating cancer.

[0060] In certain aspects, the present invention provides a combination 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 and a PD-1 or PD-L1 inhibitor for use in treating cancer in a subject in need thereof.

[0061] In certain aspects, the present invention provides a PD-1 or PD-L1 inhibitor for use in treating cancer in a subject in need thereof, wherein the PD-L1 inhibitor is for simultaneous or sequential administration with 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.

[0062] In certain aspects, the present invention 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 treating cancer in a subject who has been or is to be administered a PD-1 or PD-L1 inhibitor.

[0063] In certain aspects, the present invention provides a PD-1 or PD-L1 inhibitor for treating cancer in a patient, wherein the multispecific antibody, which comprises 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, has been administered or is to be administered.

[0064] In certain aspects, the multispecific antibody is administered at a dose of between 25-300 mg, such as 25-150 mg or 25-100 mg, or such as between 25-50 mg or 50-100 mg.

[0065] In certain aspects, the PD-1 inhibitor is pembrolizumab and is administered at a dose of between 200-600 mg, such as between 300-500, or such as about 400 mg.

[0066] In certain aspects, the cancer is an advanced or metastatic solid tumor.

[0067] In certain aspects, the multispecific antibody, PD-L1 or PD-1 inhibitor is an isolated antibody.

[0068] In certain aspects, the subject is a human subject.

[0069] In certain aspects, the subject has not received prior treatment with a CD137 agonist or prior treatment with a CAR T cell therapy, or prior treatment including targeted small molecule therapy or radiation therapy. DETAILED DESCRIPTION

[0070] In certain aspects, the present invention 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 used for simultaneous, sequential or separate administration with a PD-1 or PD-L1 inhibitor. In certain aspects, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering a multispecific antibody and a PD-1 or PD-L1 inhibitor to the subject in need thereof, the 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.

[0071] In certain aspects, the second membrane protein is PD-L1, and the antigen binding site that binds the extracellular portion of the second membrane protein binds to PD-L1.

[0072] CD137 can be expressed by activated T-cells. It is also present in other cells, such as dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The protein is known for its co-stimulatory activity in activated T-cells. CD137 has many different names, such as: 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, mouse Ly63 homolog; induced by lymphocyte activation (ILA); mouse homolog of 4-1BB; receptor protein 4-1BB; T cell antigen ILA; and 4-1BB. The external IDs for CD137 are HGNC:11924; Entrez Gene:3604; Ensembl:ENSG00000049249; OMIM:602250; UniProtKB:Q07011. CD137 is an inducible receptor that is most often upregulated on activated CD8+ T cells. CD137 signaling enhances T cell function by activating NF-κB [Arch et al., 1998]. Other cellular immune cell types, including CD4+ T cells, monocytes, B cells, dendritic cell (DC) subsets and granulocytes, and NK cells, express CD137 at varying levels [Shao et al., 2011]. In monocytes, CD137 can be induced by activation with lipopolysaccharide (LPS) and IL-1b. In B lymphocytes, CD137 expression is induced by antibodies against cell surface immunoglobulins and by EBV transformation. In DCs, ligation of CD137 induces their maturation by upregulating B7 co-stimulatory molecules (CD80 and CD86), in addition to enhancing their production of inflammatory cytokines (IL-6 and IL-12) and their survival [Makkouk et al., 2015]. The natural function of CD137 ligation to neutrophils is to increase phagocytosis of bacterial and parasitic infections. In addition, ligation of CD137 blocks anti-apoptotic signals mediated by the IL-3 / IL-5 / GM-CSF receptors of neutrophils and eosinophils in vitro, thus preventing granulocyte accumulation [Simon, 2001; Vinay et al., 2011]. In non-lymphoid cells such as chondrocytes, endothelial cells and tumor cells, the expression of CD137 is driven by cytokine stimulation, such as IL-1b for chondrocytes, inflammatory cytokines TNFα / IFNγ / IL-1b for endothelial cells and IFNγ for tumor cells. The ligand that stimulates CD137 (CD137L) is expressed on activated antigen-presenting cells. CD137 exists in membranes as monomers and dimers [Pollok et al., 1993].

[0073] The B7 family comprises a number of structurally related cell surface proteins that bind to receptors on lymphocytes that regulate immune responses. Activation of lymphocytes is initiated by binding to cell surface antigen-specific T-cell receptors or B-cell receptors. Additional signals transmitted simultaneously by B7 ligands further determine the immune response of these cells. These so-called "co-stimulatory" or "co-inhibitory" signals are transmitted by B7 family members via receptors of the CD28 family on lymphocytes. Binding of B7 family members to co-stimulatory receptors can enhance immune responses, whereas binding to co-inhibitory receptors can attenuate immune responses. Currently, the following members are considered to be part of this family: B7.1 (CD80), B7.2 (CD86), inducible co-stimulatory 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. The influence of members on the regulation of immune responses has been shown in the development of immunodeficiency and autoimmune diseases in mice carrying mutations in B7-family genes. Manipulation of the signals transmitted by B7 ligands has shown potential for the treatment of autoimmune, inflammatory diseases and cancer.

[0074] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses in specific events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis. PD-L1 is expressed in many types of cancer, especially 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 cell membrane of cancer cells, but not all cancers or all cells within tumors express PD-L1 (Dong et al., 2002). Various tumor microenvironment cells promote immunosuppression 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 innate immune resistance (Akbay et al., 2013). In 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 a variety of pro-inflammatory molecules, including type I and 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).

[0075] Programmed cell death 1 protein (PD-1) is a cell surface receptor that belongs to the CD28 receptor family and is expressed on T cells and progenitor-B cells. It is currently known that PD-1 binds to two ligands, PD-L1 and PD-L2. PD-1, as an immune checkpoint, plays an important role in downregulating the immune system by inhibiting the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is thought to be achieved through a dual mechanism of promoting apoptosis (programmed cell death) of antigen-specific T cells in the lymph nodes while reducing apoptosis of regulatory T cells (suppressor T cells). PD-1 also has many different aliases, such as 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. A new class of drugs that block the activity of PD-1, called PD-1 inhibitors, can activate the immune system to attack tumors and are therefore successfully used to treat certain types of cancer.

[0076] Binding of PD-L1 to PD-1 or B7.1 (CD80) transmits an inhibitory signal, thereby reducing the proliferation of T cells expressing PD-1. PD-1 is thought to be able to control the accumulation of foreign antigen-specific T cells through apoptosis. PD-L1 is expressed by a variety of cancer cells, and this expression is thought to be at least partially responsible for suppressing the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins, known by various other names, such as 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 Gene:29126; Ensembl:ENSG00000120217; OMIM:605402; UniProtKB:Q9NZQ7.

[0077] PD-L2 is the second ligand for PD-1. Binding of PD-L2 to PD-1 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 on antigen-presenting cells is upregulated by interferon gamma treatment. It is expressed in some normal tissues and a variety of tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. This protein has many other names, such as 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 IDs for PD-L2 are HGNC:18731; Entrez Gene:80380; Ensembl:ENSG00000197646; OMIM:605723; and UniProtKB:Q9BQ51.

[0078] PD-1 inhibitors are known in the art. They include antibodies that bind to and block PD-1. Pembrolizumab is a humanized antibody used in cancer immunotherapy to treat melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, gastric cancer and cervical cancer. It is administered slowly by intravenous injection. Pembrolizumab is a therapeutic antibody that binds to and blocks PD-1 located on lymphocytes. This receptor is a so-called "immune checkpoint" and is therefore normally responsible for preventing the immune system from attacking the body's own tissues. Normally, the PD-1 receptor on activated T cells binds to the PD-L1 or PD-L2 ligands present on normal cells in the body, inactivating any potential cell-mediated immune response against these cells. Many cancers produce proteins such as PD-L1, which also bind to the PD-1 receptor, thus shutting down the body's ability to kill the cancer. Pembrolizumab works by inhibiting the lymphocyte PD-1 receptor, blocking the ligand that inactivates it and preventing the immune response. This enables the immune system to target and destroy cancer cells, but it also blocks a key mechanism that prevents the immune system from attacking itself.

[0079] Tumors often harbor mutations that impair DNA mismatch repair. This in turn often results in microsatellite instability, causing the tumor to produce many mutant proteins that can act as tumor antigens and trigger an immune response against the tumor. By preventing the self-checkpoint system from blocking T cells,

[14]

[30] pembrolizumab appears to promote the immune system to clear any such tumors.

[0080] Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved it for use in any unresectable or metastatic solid tumor with certain genetic abnormalities (mismatch repair deficiency or microsatellite instability).

[0081] In some aspects, the multispecific antibody according to the use or method of the invention binds to a second membrane protein that is not a member of the TNF receptor superfamily. In some aspects, the second membrane protein is a member of the B7 family. In some aspects, the second membrane protein is PD-L1 or PD-L2, in some aspects PD-L1.

[0082] In certain aspects, the multispecific antibody according to the uses or methods of the invention comprises an antigen binding site that binds to the PD-1 binding domain of PD-L1.

[0083] In certain aspects, the multispecific antibody according to the uses or methods of the invention binds to a second membrane protein that is not expressed to a significant extent on T cells.

[0084] In certain aspects, the multispecific antibody according to the uses or methods of the invention binds to a second membrane protein that is expressed on an antigen presenting cell, a tumor cell, a virus infected cell or a parasite infected cell.

[0085] In some aspects, the multispecific antibody according to the uses or methods of the invention binds to a second membrane protein, which is a membrane protein present in one or more regions on the cell membrane. In some aspects, the region is a cluster, domain, microdomain or compartment on the cell membrane, in some aspects an immunological synapse.

[0086] In some aspects, the multispecific antibody according to the use or method of the invention binds to a second membrane protein, which is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins. In some aspects, the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

[0087] In certain aspects, the multispecific antibody for use or method according to the invention comprises an antigen binding site that binds to the CD137L binding domain of CD137.

[0088] In certain aspects, the multispecific antibodies for use or methods according to the invention comprise an antigen binding site that blocks the binding of a ligand to CD137, or an extracellular ligand binding to CD137 blocks the binding site, and in certain aspects, CD137L blocks the binding site.

[0089] In certain aspects, the multispecific antibody for use or method according to the invention comprises a variable domain that binds to the extracellular part of CD137, which is defined as a variable domain that, when in the form of a bivalent monospecific antibody comprising two said variable domains that bind to CD137, does not stimulate CD137 activity on cells, or stimulates CD137 activity on cells at a reduced level compared to one of said variable domains as part of a bispecific antibody with a second variable domain that binds a tumor associated antigen (such as a B7 family member, such as PD-L1).

[0090] In certain aspects, the variable domains binding to the extracellular part of CD137 are defined such that when in the form of a bivalent monospecific antibody comprising two said variable domains binding to CD137 (such as IgG, in particular IgG1 or human IgG1), they do not stimulate the activity of CD137 on cells, or stimulate CD137 activity on cells at a reduced level compared to when one of said variable domains is part of a bispecific antibody having a second variable domain binding to a tumor associated antigen (such as IgG, in particular IgG1 or human IgG1). The skilled person will recognize that when comparing the CD137 stimulating activity between the above-mentioned bivalent monospecific antibodies and bispecific antibodies, the antibodies are of the same format (such as IgG, in particular IgG1 or human IgG1), wherein the difference between the compared antibodies is, on the one hand, a monospecific antibody comprising the same variable domains that bind to CD137 in a bivalent format, and on the other hand a bispecific antibody comprising one of the CD137 variable domains and a variable domain that binds to a tumor-associated antigen, such as a variable domain comprising the amino acid sequence of the following VH: 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); MF5377 (SEQ ID NO: 73); MF5382 (SEQ ID NO: 74). 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). In certain aspects, the variable domain that binds to the extracellular portion of CD137 is non-stimulatory, when the CDR sequence of the following VH is included in the bivalent monospecific antibody format, such as: MF6754, MF6808, MF6763, MF6785 or MF6797.

[0091] In certain aspects, the multispecific antibody for use or method according to the invention comprises a variable domain that binds to an extracellular portion of CD137, which, when combined with a multispecific antibody having a second variable domain that binds to PD-L1, is capable of stimulating CD137 activity on cells when the multispecific antibody is in the presence of a first cell expressing CD137 and a second cell expressing PD-L1.

[0092] In certain aspects, the multispecific antibody according to the use or method of the invention is capable of binding to CD137 and PD-L1 simultaneously.

[0093] The multispecific antibodies of the present invention that bind to the extracellular portion of CD137 and a second membrane protein (particularly a membrane protein of a member of the B7 family) provide the advantage of being particularly good at promoting the desired immune response, because the B7 family members transmit "co-stimulatory" or "co-inhibitory" signals to lymphocytes, thereby enhancing or weakening the immune response. Therefore, by targeting a second transmembrane protein, especially a transmembrane protein of a B7 family member, the stimulatory signal can be enhanced and / or the inhibitory signal can be offset, thereby inducing or enhancing the desired immune response, for example, against abnormal cells such as cancer cells. Therefore, according to the present invention, the multispecific antibody is used to treat cancer in a subject in need thereof, when the immune response required to fight against abnormal cells found in cancer is induced.

[0094] In certain aspects, the multispecific antibodies of the present invention have an antigen binding site that can bind to the extracellular portion of CD137, and a second antigen binding site that can bind to the extracellular portion of a second membrane protein (which is not a member of the TNF receptor superfamily, but in certain aspects is a member of the B7 family, or such as PD-L1). This provides the following advantages, namely, at least partially avoiding the cis activation of (immune) cells (such as T cells expressing several different members of the TNF receptor superfamily), thereby reducing potential adverse side effects and toxicity due to non-specific T cell activation. Prior art methods may result in cis activation of T cells, which means that there is no second target, and may involve the risk of excessive T cell reactions, such as causing cytokine storms. Therefore, the possibility of adverse side effects of such prior art methods is increased, compared with the binding molecule according to the present invention having an antigen binding site that can bind to CD137 and an antigen binding site that can bind to the extracellular portion of a second membrane protein.

[0095] In certain aspects, the present invention relates to a multispecific antibody targeting both PD-L1 and CD137 to partially avoid cis activation of (immune) cells such as T cells. In certain aspects, the variable domain that binds to the extracellular part of CD137 is the following domain, which, when in the form of a bivalent monospecific antibody comprising two variable domains that bind to CD137, does not stimulate CD137 activity on cells, or stimulates CD137 activity on cells at a reduced level compared to one of the variable domains as a part of a bispecific antibody having a second variable domain that binds to a tumor-associated antigen (such as a B7 family member, such as PD-L1). Suitable CD137 binding arms are disclosed in WO 2018 / 056821.

[0096] In some aspects, the multispecific antibody of the uses or methods of the present invention may be an agonist CD137 antibody, such as an antibody capable of stimulating CD137 activity. In some aspects, the multispecific antibody of the uses or methods of the present invention may be an antagonist CD137 antibody, such as an antibody capable of reducing CD137 activity.

[0097] In certain aspects, the multispecific antibodies of the uses or methods of the invention may be agonistic B7 antibodies, such as antibodies capable of stimulating the activity of B7 family members. In certain aspects, the multispecific antibodies of the uses or methods of the invention may be antagonistic B7 antibodies, such as antibodies capable of reducing the activity of B7 family members.

[0098] In certain aspects, the multispecific antibody of the uses or methods of the present invention may be an agonist PD-L1 antibody, such as an antibody capable of stimulating PD-L1 activity. In certain aspects, the multispecific antibody of the uses or methods of the present invention may be an antagonist PD-L1 antibody, such as an antibody capable of reducing PD-L1 activity.

[0099] In some aspects, the multispecific antibodies according to the uses or methods of the invention can stimulate the activity of CD137 when bound to a B7 family member. In some aspects, the multispecific antibodies can stimulate the activity of CD137 when bound to CD137 and PD-L1. In some aspects, the multispecific antibodies induce or activate CD137 signaling only in the presence of PD-L1 expressing cells.

[0100] In certain aspects, the multispecific antibody according to the use or method of the invention comprises an antigen binding site consisting of an immunoglobulin variable domain that binds to CD137 and an immunoglobulin variable domain that binds to the extracellular part of a second membrane protein.

[0101] In certain aspects, the variable domain comprised in the multispecific antibody for use or method according to the invention, which binds to the extracellular part of CD137 and at least partially blocks the binding of CD137 ligand to CD137, is a variable domain comprising the amino acid sequence of the following VH: 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: 44). NO:46); or MF6797 (SEQ ID NO:49).

[0102] In certain aspects, the variable domain comprised in the multispecific antibody for use or method according to the invention, which binds to the extracellular part of CD137 and at least partially blocks the binding of CD137 ligand to CD137, is a variable domain comprising the amino acid sequence of the CDR1, CDR2 and CDR3 sequences of the following VH: 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: 44). ID NO:46); or MF6797 (SEQ ID NO:49).

[0103] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, comprises a heavy chain variable region with a CDR3 region, and comprises the amino acid sequence of the CDR3 region of the following variable heavy chain region: MF6808 (SEQ ID NO: 13); MF6754 (SEQ ID NO: 23); MF6763 (SEQ ID NO: 27); MF6785 (SEQ ID NO: 34); or MF6797 (SEQ ID NO: 52).

[0104] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, comprises a heavy chain variable region with a CDR2 region, and comprises the amino acid sequence of the CDR2 region of the following variable heavy chain region: MF6808 (SEQ ID NO: 13); MF6754 (SEQ ID NO: 22); MF6763 (SEQ ID NO: 26); MF6785 (SEQ ID NO: 33); or MF6797 (SEQ ID NO: 51).

[0105] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, comprises a heavy chain variable region with a CDR1 region, and comprises the amino acid sequence of the CDR1 region of the following variable heavy chain region: MF6808 (SEQ ID NO: 13); MF6754 (SEQ ID NO: 21); MF6763 (SEQ ID NO: 25); MF6785 (SEQ ID NO: 32); or MF6797 (SEQ ID NO: 50).

[0106] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, comprises a heavy chain variable region having CDR1, CDR2 and CDR3 regions, and comprises the amino acid sequence of CDR1, CDR2 and CDR3 of the variable heavy chain region of one of the following VHs: MF6808; MF6754; MF6763; MF6785; or MF6797. In certain aspects, the sequences of the CDR1, CDR2 and CDR3 are selected from the same VH region.

[0107] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, comprising the amino acid sequence of the variable heavy chain region of MF6808, MF6754; MF6763; MF6785; or MF6797, with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and such as 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid insertions, deletions, substitutions or combinations thereof, if any, are not in the amino acid sequence of the CDR region.

[0108] In certain aspects, the variable domain binding to the extracellular part of CD137 comprised in the multispecific antibody for use or method according to the present invention, or a functional part, derivative and / or analog thereof, comprises a VH region having a CDR3 amino acid sequence, or a CDR1, CDR2 and CDR3 amino acid sequence of one of the following VHs: MF6808; MF6754; MF6763; MF6785; or MF6797. In certain aspects, the variable domain that binds to the extracellular portion of CD137 comprises a VH region having the amino acid sequence of the VH region of MF6808 (SEQ ID NO: 13); MF6754 (SEQ ID NO: 20); MF6763 (SEQ ID NO: 24); MF6785 (SEQ ID NO: 31); or MF6797 (SEQ ID NO: 49), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid insertions, deletions, substitutions, or combinations thereof, if any, are not in the amino acid sequence of the CDR region.

[0109] In certain aspects, the variable domain comprised in the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention, which binds to the extracellular part of PD-L1 and blocks the binding of PD1 to PD-L1, is a variable domain comprising the amino acid sequence of VH as follows: 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); 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: 90); In some aspects, the CDRs from the MF sequence are determined using the Kabat numbering system, as described elsewhere herein.

[0110] In certain aspects, the variable domain comprised in the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention, which binds to the extracellular part of PD-L1 and blocks the binding of PD1 to PD-L1, is a variable domain comprising the amino acid sequence of the CDR1, CDR2 and CDR3 sequences of the following VH: 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); MF5377 (SEQ ID NO: 73); MF5382 (SEQ ID NO: 77); MF5424 (SEQ ID NO: 81); MF5426 (SEQ ID NO: 85); MF5439 (SEQ ID NO: 86). 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).

[0111] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region having a CDR3 region, which comprises the amino acid sequence of the CDR3 region of the variable heavy chain region as follows: 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: 91). NO:95).

[0112] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof for the use or method according to the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region having a CDR3 region, which comprises the amino acid sequence of the CDR3 region of the variable heavy chain region as follows: 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).

[0113] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a heavy chain variable domain that binds to the extracellular part of PD-L1, which comprises a CDR3 region having the following amino acid sequence: 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. In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a heavy chain variable domain that binds to the extracellular part of PD-L1, which comprises a CDR3 region having the following amino acid sequence: SEQ ID NO: 56; SEQ ID NO: 91; SEQ ID NO: 95; or SEQ ID NO: 102, or a variant thereof.

[0114] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain, which comprises a heavy chain variable domain that binds to the extracellular part of PD-L1, comprising a heavy chain variable region with a CDR2 region, which comprises the amino acid sequence of the CDR2 region of the following variable heavy chain region: 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: 83); MF5595 (SEQ ID NO: 83); MF5596 (SEQ ID NO: 83); MF5597 (SEQ ID NO: 83); MF5598 (SEQ ID NO: 83); MF5599 (SEQ ID NO: 83); MF5591 (SEQ ID NO: 83); MF5593 (SEQ ID NO: 83); MF5594 (SEQ ID NO: 83); MF5597 (SEQ ID NO: 83); MF5598 (SEQ ID NO: 83); MF5599 (SEQ ID NO: 83); MF5591 ... ID NO:87); or MF5442 (SEQ ID NO:94).

[0115] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region with a CDR2 region, and comprises the amino acid sequence of the CDR2 region of the following variable heavy chain region: 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).

[0116] In certain aspects, the multispecific antibody or functional part, derivative and / or analogue thereof according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of PD-L1, wherein the antibody comprises a heavy chain variable region that binds to the extracellular part of PD-L1, which comprises a CDR2 region of the amino acid sequence shown below: 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.

[0117] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region with a CDR1 region, which comprises the amino acid sequence of the CDR1 of the variable heavy chain region as shown below: 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: 87). NO:93).

[0118] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof for the use or method according to the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region having a CDR1 region, and comprises the amino acid sequence of the CDR1 region of the variable heavy chain region as shown below: 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).

[0119] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable domain that binds to the extracellular part of PD-L1, comprises a CDR1 region having an amino acid sequence as shown below: 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.

[0120] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of PD-L1, comprises a heavy chain variable region having CDR1, CDR2 and CDR3 regions, and comprises the amino acid sequence of the CDR1, CDR2 and CDR3 regions of the variable heavy chain region of one of the following VHs: 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.

[0121] In certain aspects, a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention comprises a variable domain that binds to the extracellular part of PD-L1, comprising the amino acid sequence of the variable heavy chain region as follows: 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); or MF5442 (SEQ ID NO: 86). NO:92), has up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid insertions, deletions, substitutions or combinations thereof, if any, are not in the amino acid sequence of the CDR region.

[0122] In certain aspects, a particularly preferred combination of multispecific antibodies or functional parts, derivatives and / or analogs according to the uses or methods of the present invention is a combination of variable domains comprising the following specified sequences or variants thereof: 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 MF5553 (SEQ ID NO: 96); MF6754 (SEQ ID NO: 20) and MF5424 (SEQ ID NO: 81); MF6763 (SEQ ID NO: 24) and MF7702 (SEQ ID NO: 67). 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).

[0123] In certain aspects, the multispecific antibody or functional part, derivative and / or analogue according to the use or method of the invention comprises:

[0124] - a CD137 binding variable domain comprising a VH region having an amino acid sequence of CDR3 or the amino acid sequences of CDR1, CDR2 and CDR3 of the VH of MF6797 (SEQ ID NO: 49); and - a PD-L1 binding variable domain comprising a VH region having an amino acid sequence of CDR3 or the amino acid sequences of CDR1, CDR2 and CDR3 of the VH as shown below: 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: 106); NO:96); MF5442 (SEQ ID NO:92); MF5561 (SEQ ID NO:100); or MF5439 (SEQ ID NO:89).

[0125] In certain aspects, the multispecific antibody or functional part, derivative and / or analogue according to the use or method of the invention comprises:

[0126] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of VH of MF6797 (SEQ ID NO: 49) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the amino acid sequence of VH of MF6797 (SEQ ID NO: 49); and

[0127] - a PD-L1 binding variable domain comprising a VH region having an amino acid sequence of a VH as shown below: 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: 101). NO:89), has up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0128] Certain aspects also provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0129] - 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); and

[0130] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5442 (SEQ ID NO: 95).

[0131] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0132] - a CD137 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF6763 (SEQ ID NO: 24); and

[0133] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF5442 (SEQ ID NO: 92).

[0134] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0135] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of VH of MF6763 (SEQ ID NO: 24) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF6763; and

[0136] -PD-L1 binding variable domain, comprising a VH region, having an amino acid sequence of MF5442 (SEQ ID NO: 92), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF5442. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0137] Certain aspects also provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0138] - 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); and

[0139] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF7702 (SEQ ID NO: 56).

[0140] Certain aspects also provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0141] - a CD137 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF6797 (SEQ ID NO: 49); and

[0142] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF7702 (SEQ ID NO: 67).

[0143] Certain aspects also provide a multispecific antibody or a functional part, derivative and / or analogue according to the use or method of the invention, comprising:

[0144] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of VH of MF6797 (SEQ ID NO: 49) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of MF6797; and

[0145] -PD-L1 binding variable domain, comprising a VH region, having the amino acid sequence of MF7702 (SEQ ID NO: 67), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of MF7702. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of a specified MF.

[0146] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0147] - 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); and

[0148] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5561 (SEQ ID NO: 102).

[0149] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0150] - a CD137 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF6754 (SEQ ID NO: 20); and

[0151] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF5561 (SEQ ID NO: 100).

[0152] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0153] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6754 (SEQ ID NO: 20) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 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 as specified; and

[0154] -PD-L1 binding variable domain, comprising a VH region, having the amino acid sequence of MF5561 (SEQ ID NO: 100), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF5561. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0155] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0156] - 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); and

[0157] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5439 (SEQ ID NO: 91).

[0158] Also provided is a bispecific antibody or a functional part, derivative and / or analogue thereof, comprising:

[0159] - a CD137 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF6785 (SEQ ID NO: 31); and

[0160] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF5439 (SEQ ID NO: 89).

[0161] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0162] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785 (SEQ ID NO: 31) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 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 as specified; and

[0163] -PD-L1 binding variable domain, comprising a VH region, having the amino acid sequence of MF5439 (SEQ ID NO: 89), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF5439. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0164] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0165] - 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); and

[0166] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5542 (SEQ ID NO: 95).

[0167] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analog thereof, comprising:

[0168] - a CD137 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF6785 (SEQ ID NO: 31); and

[0169] - A PD-L1 binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the VH of MF5442 (SEQ ID NO: 92).

[0170] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising:

[0171] - a CD137 binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785 (SEQ ID NO: 31) with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF6785; and

[0172] -PD-L1 binding variable domain, comprising a VH region, having the amino acid sequence of VH of MF5442 (SEQ ID NO: 92), with up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the specified VH amino acid sequence of MF5542. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0173] Certain aspects provide a multispecific antibody or a functional part, derivative and / or analogue thereof for use or method according to the invention, comprising a binding domain that binds to CD137, the binding domain comprising:

[0174] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:50, a CDR2 having an amino acid sequence as shown in SEQ ID NO:51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:52, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; or

[0175] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; or

[0176] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:21, a CDR2 having an amino acid sequence as shown in SEQ ID NO:22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:23, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; or

[0177] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; and / or a binding domain that binds to PD-L1, comprising:

[0178] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a CDR2 having an amino acid sequence as shown in SEQ ID NO:55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:56, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; or

[0179] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, or

[0180] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102, wherein CDR1, CDR2 and / or CDR3 each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, or

[0181] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 90, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 91, wherein each of CDR1, CDR2 and / or CDR3 has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof. In certain aspects, the amino acid sequence of each CDR has a maximum of 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof.

[0182] In certain aspects, the multispecific antibody or its functional part, derivative and / or analogue according to the use or method of the present invention comprises a variable domain that binds to the extracellular part of CD137, which blocks the binding of CD137 to the CD137 ligand, and a variable domain that binds to the extracellular part of PD-L1, which can block the binding of PD-1 to PD-L1. In certain aspects, the variable domain that binds to the extracellular part of PD-L1 in this antibody or its functional part, derivative and / or analogue comprises a VH region having an amino acid sequence of CDR3, or a VH region having an amino acid sequence of CDR1, CDR2 and CDR3 as follows: MF5554; MF5576; MF5578; MF9375; MF9376; MF7702; MF5424; MF5561; MF5439; MF5553; MF5594; MF5426; MF5442. In certain aspects, the variable domain that binds to the extracellular portion of PD-L1 comprises a VH region having the following VH amino acid sequence: 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: 86). NO:92), having up to 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or such as having 0, 1, 2, 3, 4, 5 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF. In certain aspects, the amino acid sequence of the variable heavy chain region has up to 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or a combination thereof, relative to the VH amino acid sequence of the specified MF.

[0183] In the examples of WO2018 / 056821A1, it is shown that the binding of the CD137-specific VH of MF6797 (SEQ ID NO: 49) is related to the presence of amino acids Arg66, Gly70 and Phe72 comprising the CD137 amino acid sequence.

[0184] Therefore, in certain aspects, the present invention also provides the use or treatment method of an isolated, synthetic or recombinant antibody or its functional part, derivative and / or analogue capable of binding to CD137, wherein the binding of the antibody or functional part, derivative or analogue to the CD137 is related to the presence of amino acids Arg66, Gly70 and Phe72 contained in the CD137 amino acid sequence (SEQ ID NO: 117). In certain aspects, the binding of the antibody or functional part, derivative or analogue to CD137 is also related to the amino acid Val71 contained in the CD137 amino acid sequence.

[0185] The term "Arg66" refers to the arginine residue at position 66 in the CD137 sequence. The term "Gly70" refers to the glycine residue at position 70 in the CD137 sequence of SEQ ID NO 117. The term "Val71" refers to the valine residue at position 71 in the CD137 sequence. The term "Phe72" refers to the phenylalanine residue at position 72 in the CD137 sequence.

[0186] In certain aspects, the multispecific antibodies of the invention comprise a binding site comprising a common light chain as set forth in SEQ ID NO: 109, which has 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain aspects, each binding site comprises a common light chain as set forth in SEQ ID NO: 109, which has 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0187] In certain aspects, the multispecific antibodies of the present invention comprise a binding site comprising a LCDR sequence of a common light chain as shown in SEQ ID NO: 109 according to IMGT, which has 0-5 amino acid insertions, deletions, substitutions, additions or combinations thereof. In certain aspects, each of the binding sites comprises a LCDR sequence of a common light chain as shown in SEQ ID NO: 109 according to IMGT, which has 0-5 amino acid insertions, deletions, substitutions, additions or combinations thereof. In certain aspects, the substitutions are conservative substitutions, whereby the amino acids are substituted with conservative amino acids. The skilled person is fully capable of substituting one or more amino acids in the CDR sequences of the present invention. For example, conservative amino acid substitutions are used. Examples of conservative amino acid substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and substitution of one polar residue for another polar residue, such as substitution of arginine for lysine, substitution of glutamic acid for aspartic acid, or substitution of asparagine for glutamine. In certain aspects, the multispecific antibodies of the invention comprise a binding site comprising a common light chain LCDR sequence as shown in SEQ ID NO: 109 (according to the IMGT database world network imgt.org), which has 1 or 2 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain aspects, according to IMGT, each of the binding sites comprises a common light chain LCDR sequence as shown in SEQ ID NO: 109, which has 1 or 2 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0188] In certain aspects, the light chain variable regions of one, two or more binding domains of the multispecific antibodies of the invention comprise LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT (i.e., CDRs of IGKV1-39 according to IMGT).

[0189] In certain aspects, one, two or more binding domains of the multispecific antibodies of the invention comprise a light chain variable region comprising an amino acid sequence that is at least 90%, in certain aspects at least 95%, in certain aspects at least 97%, in certain aspects at least 98%, in certain aspects at least 99%, or in certain aspects 100% identical to the amino acid sequence of SEQ ID NO: 109.

[0190] In certain aspects, the multispecific antibodies described herein comprise a common light chain variable region, such as the common light chain variable region shown in SEQ ID NO: 110, which has 0-5 amino acid insertions, deletions, substitutions, additions or combinations thereof, such as 0, 1, 2, 3 or 4; or such as 0, 1, 2 or 3, or such as 0, 1 or 2; or such as having 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof. In certain aspects, the multispecific antibodies described herein comprise a common light chain constant region, such as the common light chain variable region shown in SEQ ID NO: 111, which has 0-5 amino acid insertions, deletions, substitutions, additions or combinations thereof. The term "common light chain" according to the present invention refers to a light chain that is equal or has some amino acid sequence differences but does not affect the binding specificity of the full-length antibody. For example, within the scope of the definition of the common light chain used herein, light chains that are not identical but are still functionally equivalent can be prepared or discovered, 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, or similar changes. The terms "common light chain", "common LC", "cLC", "single light chain" with or without the term "rearrangement" are used interchangeably herein. The terms "common light chain variable region", "common VL", "common LCv", "cLCv", "single VL" with or without the term "rearrangement" are used interchangeably herein. In certain aspects of the present invention, the multispecific antibody has a common light chain (variable region) that can be combined with at least two or more heavy chains (variable regions) with different binding specificities to form an antibody with a functional antigen binding domain (WO2004 / 009618, WO2009 / 157771). In certain aspects, the common light chain (variable region) is a human light chain (variable region). In certain aspects, the common light chain (variable region) has a germline sequence. In certain aspects, the germline sequence is a light chain variable region frequently used in the human library, with good thermodynamic stability, yield and solubility. In certain aspects, the common light chain is a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01 (SEQ ID NO 109). In certain aspects, the common light chain variable region is a variable region of a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01. In certain aspects, the common light chain comprises a light chain variable region as shown in SEQ ID NO 110, with 0-5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, such as 0, 1, 2, 3, or 4; such as 0, 1, 2, or 3, such as 0, 1, or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions, or combinations thereof. In certain aspects, the common light chain further comprises a light chain constant region, such as a κ light chain constant region. The nucleic acid encoding the common light chain may be a codon-optimized cell system for expressing the common light chain protein.The encoding nucleic acid may deviate from the germline nucleic acid sequence.

[0191] In certain aspects, the multispecific antibody according to the uses or methods of the invention is a full-length antibody or an antibody fragment, such as a Fab fragment or a single-chain variable fragment (scFv). In certain aspects, the multispecific antibody according to the uses or methods of the invention is a full-length antibody.

[0192] In certain aspects, the multispecific antibody for use or method according to the invention is an IgG. In certain aspects, the multispecific antibody is an IgG1 molecule without Fc effector function.

[0193] The Fc region mediates the effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it may be necessary to reduce or increase the effector function. The present invention is preferably a reduced effector function. In some aspects of the present invention, when the immune response is activated, enhanced or stimulated, a reduced effector function may be required. Antibodies with reduced effector function can be used to target cell surface molecules of immune cells. In certain aspects, the CH2 region of the multispecific antibody of the present invention is modified to reduce the ADCC and / or CDC activity of the antibody. In certain aspects, the CH3 region of the multispecific antibody is modified to promote heterodimerization of the heavy chain.

[0194] The antibody with reduced effector function is preferably an IgG antibody comprising a modified CH2 / lower hinge region, such as to reduce interaction with Fc-receptors or reduce binding to Clq. In certain aspects, the antibody of the present invention is an IgG antibody with a mutated CH2 and / or lower hinge domain, such that the multispecific IgG antibody has reduced interaction with Fc-γ receptors. In certain aspects, the CH2 region of the present invention comprises an amino acid sequence as shown in SEQ ID NO: 114, which has 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain aspects, the hinge region of the present invention comprises an amino acid sequence as shown in SEQ ID NO: 113, which has 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof.

[0195] In some aspects, the CH3 region of multispecific antibodies is transformed to promote the heterodimerization of heavy chain. In some aspects, there are these variations to produce only multispecific full-length IgG molecules substantially, which have amino acid substitutions at positions 351 and 366, such as L351K and T366K (numbered according to EU numbering) in the first CH3 domain ('KK- variant ' heavy chain), and amino acid substitutions at positions 351 and 368, such as L351D and L368E in the second CH3 domain ('DE- variant ' heavy chain), and vice versa. The homodimerization (DE-DE homodimer) of DE- variant heavy chain or KK- variant heavy chain (KK-KK homodimer) hardly occurs, because the strong repulsion between charged residues in the CH3-CH3 interface between the same heavy chain. In certain aspects, the multispecific antibodies of the invention comprise the CH3 domains of SEQ ID NOs 115 and 116 with 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof, provided that the DE / KK variants are not altered.

[0196] In certain aspects, the PD-1 or PD-L1 inhibitor is an antibody or other binding molecule.

[0197] In some aspects, PD-1 or PD-L1 inhibitors are PD-1 inhibitors. In some aspects, PD-1 inhibitors are selected from Nivolumab, Pembrolizumab, Cemiplimab, Penpulimab, Retifanlimab, Sintilimab, Tislelizumab, Toripalimab and Dostarlimab. In some aspects, PD-1 inhibitors are Pembrolizumab. In some aspects, PD-1 or PD-L1 inhibitors are PD-L1 inhibitors. In some aspects, PD-L1 inhibitors are selected from Atezolizumab, Avelumab and Durvalumab. The amino acid sequences of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab and Durvalumab are known in the art.

[0198] In certain aspects, the PD-1 or PD-L1 inhibitor is administered according to FDA-approved recommendations.

[0199] In certain aspects, the PD-1 inhibitor is Nivolumab and is administered at 3 mg / kg as an intravenous infusion (such as over 60 minutes) every 2 weeks until disease progression or unacceptable toxicity.

[0200] In another aspect, Nivolumab is administered at 240 mg every 2 weeks or 480 mg every 4 weeks, or 1 mg / kg, followed by ipilimumab 3 mg / kg on the same day for 4 doses every 3 weeks, followed by 240 mg every 2 weeks or 480 mg every 4 weeks.

[0201] In another aspect, Nivolumab is administered at 3 mg / kg every 2 weeks with ipilimumab 1 mg / kg every 6 weeks, or at 360 mg every 3 weeks with ipilimumab 1 mg / kg every 6 weeks, and 2 cycles of platinum-doublet chemotherapy.

[0202] In another aspect, Nivolumab is administered at 360 mg every 3 weeks with ipilimumab at 1 mg / kg every 6 weeks.

[0203] In another aspect, Nivolumab is administered at 240 mg every 2 weeks or 480 mg every 4 weeks, or at 3 mg / kg every 2 weeks, or at 3 mg / kg, followed by ipilimumab 1 mg / kg on the same day for 4 doses every 3 weeks, followed by 240 mg every 2 weeks or 480 mg every 4 weeks.

[0204] In certain aspects, the PD-1 inhibitor is Cemiplimab and is administered in an amount of 350 mg as an intravenous infusion (such as over 30 minutes) every 3 weeks until disease progression or unacceptable toxicity.

[0205] In certain aspects, the PD-1 inhibitor is Dostarlimab, and the first to fourth doses are administered at 500 mg every 3 weeks, followed by subsequent doses starting 3 weeks after the fourth dose (starting with the fifth dose): 1000 mg administered as an intravenous infusion (such as over 30 minutes) every 6 weeks until disease progression or unacceptable toxicity.

[0206] In certain aspects, the PD-L1 inhibitor is atezolizumab and is administered as an intravenous infusion (e.g., over 60 minutes) at a dose of 1200 mg every 3 weeks until disease progression or unacceptable toxicity occurs. If the first infusion can be tolerated, all subsequent infusions can be completed within 30 minutes.

[0207] In certain aspects, the PD-L1 inhibitor is atezolizumab and is administered at a dose of 10 mg / kg as an intravenous infusion (e.g., over 60 minutes) every 2 weeks until disease progression or unacceptable toxicity.

[0208] In certain aspects, the PD-L1 inhibitor is durvalumab and is administered at a dose of 10 mg / kg as an intravenous infusion (such as over 60 minutes) every 2 weeks until disease progression or unacceptable toxicity occurs.

[0209] In certain aspects, the PD-1 inhibitor is pembrolizumab and is administered at a dose of 400 mg as an intravenous infusion every 3 weeks, or 2 mg / kg as an intravenous infusion. In certain aspects, the administration is a fixed dose of 400 mg once every 6 weeks until disease progression or unacceptable toxicity occurs.

[0210] In some aspects, the PD-1 inhibitor is pembrolizumab, and is administered as an intravenous infusion at a dose of 400 mg every 6 weeks. In some aspects, the PD-1 inhibitor is pembrolizumab, and is administered as an intravenous infusion at a fixed dose of 400 mg every 6 weeks. In some aspects, the PD-1 inhibitor is pembrolizumab, and is administered as an intravenous infusion at a dose of 200 mg every 3 weeks. In some aspects, the PD-1 inhibitor is pembrolizumab, and is administered as an intravenous infusion at a fixed dose of 200 mg every 3 weeks. In some aspects, the administration is a fixed dose of 400 mg and once every 6 weeks until disease progression or unacceptable toxicity occurs. In some aspects, the administration is a fixed dose of 200 mg and once every 3 weeks until disease progression or unacceptable toxicity occurs.

[0211] The term "fixed dose" herein refers to an administration regimen in which a fixed amount of a therapeutic substance is administered to a subject in multiple administrations and is independent of the subject's weight. Fixed doses are often abbreviated as qnw, where n is an integer representing an interval and w is a week. For example, a q6w fixed dose administration regimen of 400 mg of an antibody refers to administration of a fixed amount of 400 mg of the antibody every 6 weeks. In certain aspects, pembrolizumab is administered with a q6w administration regimen of 400 mg. In certain aspects, pembrolizumab is administered with a q3w administration regimen of 200 mg.

[0212] The fixed dose may be pre-administered, meaning that the drug is administered to the subject prior to administration of the antibody of the invention. In certain aspects, the fixed dose of pembrolizumab or a multispecific antibody of the invention is pre-administered with an antihistamine, analgesic, antipyretic, and / or anti-inflammatory drug.

[0213] In some aspects, PD-1 or PD-L1 inhibitors are full-length antibodies or antibody fragments, such as Fab fragments or single-chain variable fragments (scFv). In some aspects, the PD-1 or PD-L1 inhibitors according to the purposes or methods of the present invention are full-length antibodies. In some aspects, PD-1 or PD-L1 inhibitors are or include antibodies selected from Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab and Durvalumab or their functional fragments or variants.

[0214] In certain aspects, the PD-1 or PD-L1 inhibitor comprises or consists of the amino acid sequence of the antigen binding site of one of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab and Durvalumab, in particular Pembrolizumab, or an amino acid sequence having substantial sequence identity thereto.

[0215] In certain aspects, the PD-1 or PD-L1 inhibitor is or comprises at least one, such as at least two, or even such as three CDRs that are consistent or substantially consistent with one of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab, and Durvalumab, especially Pembrolizumab. In certain aspects, the PD-1 or PD-L1 inhibitor comprises at least one, such as at least two, or even such as three CDRs that are consistent with the CDRs in Pembrolizumab.

[0216] In certain aspects, the PD-1 or PD-L1 inhibitor is or comprises a CDR1 that is identical to the CDR1 of one of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab, and Durvalumab, or a CDR1 that is identical to the CDR1 of one of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, ), atezolizumab, avelumab, and durvalumab, or a CDR3 that is identical to a CDR3 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, in certain aspects, is CDR1 and CDR2 identical to the CDR1 and CDR2 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, and CDR1 and CDR2 identical to the CDR1 and CDR2 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, and CDR1 and CDR2 identical to the CDR1 and CDR2 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab the CDR1 and CDR3 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, or the CDR2 and CDR3 of one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab,Even CDR1, CDR2 and CDR3 that are consistent with CDR1, CDR2 and CDR3 of one of Nivolumab, Pembrolizumab, Cemiplimab, Dostarlimab, Atezolizumab, Avelumab and Durvalumab. In certain aspects, the PD-1 or PD-L1 inhibitor comprises CDR1, CDR2 and CDR3 that are consistent with CDR1, CDR2 and CDR3 of Pembrolizumab. ,

[0217] In certain aspects, the multispecific antibody is administered at a dose of between 10-300 mg, such as between 10-150 mg or 25-100 mg, such as between 25-75 mg. In certain aspects, the multispecific antibody is administered at a dose of 25, 30, 35, 40, 45, or 50 mg. In certain aspects, the multispecific antibody is administered at a fixed dose of 25, 30, 35, 40, 45, or 50 mg.

[0218] In certain aspects, the multispecific antibody is administered approximately once every 7, 10, 11, 12, 13, 14, 15, 16, 17 or 18 or 21 days, particularly approximately once every 10, 14 or 18 days, more particularly approximately once every 14 days.

[0219] In certain aspects, the PD-1 or PD-L1 inhibitor is administered at a dose according to a USFDA-approved dose and an approved schedule. In certain aspects, pembrolizumab is administered at a dose of between about 300-500 mg, such as between 350-450 mg, such as between 375-425 mg. In certain aspects, the PD-1 or PD-L1 inhibitor is administered at a dose of 325, 350, 375, 400, 425, 450, or 475 mg.

[0220] In some aspects, 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, 100 mg, 125 mg, or 150 mg.

[0221] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered as a fixed dose.

[0222] In certain aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose or fixed dose of between 50-150 mg, such as between 75-150 mg, such as between 25-125 mg or between 100-150 mg. In certain aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose or fixed dose of between 50-150 mg, or between 75-150 mg, or between 75-125 mg or between 100-150 mg.

[0223] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered at a dose of between 75-125 mg or a fixed dose.

[0224] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered at a dosage or fixed dose of between 50-150 mg, such as between 75-125 mg. In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered at a dosage or fixed dose of between 50-100 mg, or between 75-100 mg, or between 75-125 mg.

[0225] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered at a dose of between 25-75 mg or a fixed dose.

[0226] In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dosage of between 10-50 mg, such as between 25-50 mg, or a fixed dose. In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dosage of between 10-50 mg or between 25-50 mg, or a fixed dose.

[0227] In some aspects, the multispecific antibody according to the uses or methods of the present invention is administered once a week, once every two weeks, or once every three weeks. In some aspects, the multispecific antibody according to the uses or methods of the present invention is administered once every two weeks. In some aspects, if the multispecific antibody according to the uses or methods of the present invention is administered once a week, the multispecific antibody is administered at a dosage of between 10-100 mg, such as between 15-75 mg. In some aspects, if the multispecific antibody according to the uses or methods of the present invention is administered once a week, the multispecific antibody is administered at a dosage of between 10-100 mg, or between 15-75 mg, such as between 15-50 mg, or between 15-40 mg, or between 15-30 mg, or between 15-25 mg.

[0228] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered as a fixed dose once every two weeks.

[0229] In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose of 25, 30, 35, 40, 45 or 50 mg or a fixed dose once every two weeks. In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose of 25 mg or a fixed dose once every two weeks. In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose of 40 mg or a fixed dose once every two weeks. In some aspects, the multispecific antibodies according to the uses or methods of the invention are administered at a dose of 50 mg or a fixed dose once every two weeks.

[0230] In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every 3 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 10 mg, once every two weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every 3 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 15 mg, once every two weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every 3 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 25 mg, once every two weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every 3 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 40 mg, once every 2 weeks.

[0231] In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 10 mg, once every two weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 15 mg, once every three weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 25 mg, once every two weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 40 mg, once every two weeks.

[0232] In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 10 mg once every 3 weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 15 mg once every three weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 25 mg once every three weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 200 mg every three weeks, and the treatment further comprises administering the multispecific antibody of the present invention in an amount of 40 mg once every three weeks.

[0233] In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the invention in an amount of 10 mg once every 3 weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the invention in an amount of 15 mg once every 3 weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the invention in an amount of 25 mg once every 3 weeks. In certain aspects, the PD-1 inhibitor is pembrolizumab, administered as an intravenous infusion in an amount of 400 mg every 6 weeks, and the treatment further comprises administering the multispecific antibody of the invention in an amount of 40 mg once every 3 weeks.

[0234] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered intravenously.

[0235] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered during between 30 minutes and 4 hours, such as between 1 and 3 hours, or such as 2 hours.

[0236] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered during between 30 minutes and 4 hours, such as between 1 and 3 hours, or such as 2 hours.

[0237] In certain aspects, the multispecific antibody according to the uses or methods of the invention is administered intravenously as a fixed dose over a 2-hour period every 14 days in a 28-day cycle.

[0238] In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 10 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 15 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 25 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 30 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 40 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 50 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 60 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 70 mg every 14 days in a 28-day cycle, over a 2-hour period. In certain aspects, the multispecific antibodies according to the uses or methods of the present invention are administered intravenously at a (fixed) dose of 75 mg every 14 days in a 28-day cycle, over a 2-hour period.

[0239] In some aspects, the multispecific antibodies according to the uses or methods of the invention are formulated into a liquid at a concentration of 1 mg / mL to 100 mg / mL, such as at or about 20 mg / mL. In some aspects, the multispecific antibodies according to the uses or methods of the invention are formulated into a liquid at a concentration of 1 mg / mL to 100 mg / mL, or at or about 20 mg / mL.

[0240] In certain aspects, pembrolizumab is administered at a dose of between 100-500 mg, or 100-300 mg or 300-500 mg, or 150-250 mg or 350-450 mg, or 175-225 mg or 375-425 mg, or a fixed dose.

[0241] In certain aspects, the PD-1 or PD-L1 inhibitor is administered at a dose of about 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg or 475 mg, or about 175 mg, 200 mg, 225 mg, 375 mg, 400 mg, or 425 mg, or a fixed dose.

[0242] In certain aspects, a PD-1 or PD-L1 inhibitor, such as pembrolizumab, is administered once every 3 weeks. In certain aspects, a PD-1 or PD-L1 inhibitor, such as pembrolizumab, is administered once every 6 weeks.

[0243] In certain aspects, pembrolizumab is administered at a dose of between 100-300 mg, or 150-250 mg, or 175-225 mg, or a fixed dose, once every 3 weeks. In certain aspects, pembrolizumab is administered at a dose of between 300-500 mg, or 350-450 mg, or 375-425 mg, or a fixed dose, once every 6 weeks.

[0244] In certain aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab and is administered at a dose of about 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or a fixed dose once every 21 days. In certain aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab and is administered at a dose of about 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a fixed dose once every 42 days.

[0245] In certain aspects, the first administration of the PD inhibitor is about 21 days after the first administration of the multispecific antibody. In certain aspects, the first administration of the PD inhibitor is about 42 days after the first administration of the multispecific antibody.

[0246] In some aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or a fixed dose, once every 21 days. In some aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a fixed dose, once every 42 days. In some aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg, or a fixed dose, once every 21 days. In some aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 400 mg, or a fixed dose, once every 42 days.

[0247] In certain aspects, the PD-1 or PD-L1 inhibitor is administered via IV, particularly IV infusion. Such an infusion can be administered, for example, over 15, 30 or 45 minutes.

[0248] In certain aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or a fixed dose via IV infusion once every 21 days. In certain aspects, the PD-1 or PD-L1 inhibitor is pembrolizumab, and administered at a dose of about 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a fixed dose via IV infusion once every 42 days.

[0249] In certain aspects, the multispecific antibody is administered at a dose of 10 mg, 25 mg, 30 mg, 40 mg, or 50 mg, or a fixed dose, once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, or a fixed dose, once every 21 days. In certain aspects, the multispecific antibody is administered at a dose of 10 mg, 25 mg, 30 mg, 40 mg, or 50 mg, or a fixed dose, once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 350 mg, 375 mg, 400 mg, 425 mg, or 450 mg, or a fixed dose, once every 42 days.

[0250] In certain aspects, the multispecific antibody is administered at a fixed dose of 10 mg, 15 mg, 25 mg, or 40 mg once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab at a fixed dose of about 200 mg once every 21 days. In certain aspects, the multispecific antibody is administered at a fixed dose of 10 mg, 15 mg, 25 mg, or 40 mg once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab at a fixed dose of about 400 mg once every 42 days.

[0251] In certain aspects, the multispecific antibody is administered at a fixed dose of 10 mg, 15 mg, 25 mg, or 40 mg once every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab at a fixed dose of about 200 mg once every 21 days. In certain aspects, the multispecific antibody is administered at a fixed dose of 10 mg, 15 mg, 25 mg, or 40 mg once every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab at a fixed dose of about 400 mg once every 42 days.

[0252] In certain aspects, the multispecific antibody is administered at a dose of 40 mg or a fixed dose once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg or a fixed dose once every 21 days, or at a dose of about 400 mg or a fixed dose once every 42 days.

[0253] In certain aspects, the multispecific antibody is administered at a dose of 40 mg or a fixed dose once every 21 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg or a fixed dose once every 21 days, or at a dose of about 400 mg or a fixed dose once every 42 days.

[0254] In certain aspects, the multispecific antibody is administered at a dose or fixed dose of 25 mg once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose or fixed dose of about 200 mg once every 21 days, or at a dose or fixed dose of about 400 mg once every 42 days.

[0255] In certain aspects, the multispecific antibody is administered at a dose of 30 mg or a fixed dose once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg or a fixed dose once every 21 days, or at a dose of about 400 mg or a fixed dose once every 42 days.

[0256] In certain aspects, the multispecific antibody is administered at a dose of 40 mg or a fixed dose once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg or a fixed dose once every 21 days, or at a dose of about 400 mg or a fixed dose once every 42 days.

[0257] In certain aspects, the multispecific antibody is administered at a dose of 50 mg or a fixed dose once every 14 days, and the PD-1 or PD-L1 inhibitor is pembrolizumab, administered at a dose of about 200 mg or a fixed dose once every 21 days, or at a dose of about 400 mg or a fixed dose once every 42 days.

[0258] In certain aspects, the PD-1 or PD-L1 inhibitor is formulated as a liquid at a concentration of 1 mg / mL to 100 mg / mL, or at or about 25 mg / mL.

[0259] In certain aspects, the multispecific antibodies according to the uses or methods of the invention are administered before, simultaneously with, or after administration of antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), anesthetics, intravenous fluids, antipyretics, bronchodilators, oxygen, corticosteroids (IV / oral), vasopressors, or any combination thereof, to reduce infusion-related reactions. If an infusion-related reaction occurs, pre-administration of a specified substance may be selected for the subject to prevent and reduce its incidence and severity.

[0260] In certain aspects, the multispecific antibodies according to the uses or methods of the invention are administered after the subject has been pretreated with standard of care therapy, such as chemotherapy, immunotherapy or targeted therapy, for advanced metastatic disease.

[0261] In certain aspects, the multispecific antibody according to the uses or methods of the invention 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.

[0262] In some aspects, the cancer is an advanced or metastatic solid tumor. Specifically, the cancer can be selected from lung cancer, especially locally advanced or metastatic NSCLC, or melanoma, especially a cancer that has been previously treated with immune checkpoint therapy and / or is PD-L1 positive. In some aspects, the cancer is an advanced or metastatic solid tumor with 1% or higher PD-L1 expression. In some aspects, the cancer or a subject comprising the cancer has received a previous anti-PD-1 / PD-L1 approved immunotherapy and has a cancer with 1% or higher PD-L1 expression, or the cancer or subject has not been treated and has a cancer with 1% or higher PD-L1 expression.

[0263] In certain aspects, the cancer is an advanced or metastatic solid tumor, such as selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma. In certain aspects, the melanoma is selected from cutaneous, acral or mucosal melanoma. In certain aspects, the cancer is Merkel cell carcinoma. Merkel cell carcinoma, also referred to herein as neuroendocrine carcinoma of the skin or trabecular carcinoma, is a very rare skin cancer that forms when Merkel cells grow out of control and form a cancer or tumor.

[0264] The expression of PD-L1 is determined by one of ordinary skill in the art, see de Ruiter et al. (2021) for exemplary methods, which is incorporated herein in its entirety. High expression determined by any of these methods shall constitute high expression for the purposes of the present invention. When assessed by the tumor proportion score (TPS), a TPS score of 1% or greater is a cancer that is PD-L1 positive. When assessed by the combined positive score (CPS), a score of 1% or greater is a cancer that is PD-L1 positive. If the PD-L1 expression score determined using the tumor proportion score (TPS) is 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more, the cancer is high PD-L1. If the PD-L1 expression score determined using the combined positive score (CPS) is 5% or more, 15% or more, or 20% or more, the cancer is high PD-L1. If assessed by multiple such methods, a tumor meets the criteria of the present invention if one or more of the methods identifies it as PD-L1 positive.

[0265] In some aspects, the subject has not been treated with immune checkpoint inhibitors before. In some aspects, the subject has not been treated with immune checkpoint inhibitors before, and their cancer is PD-L1 expression positive. In some aspects, the subject has not been treated with immune checkpoint inhibitors before, and is receiving melanoma treatment, particularly locally advanced or metastatic melanoma. In some aspects, the subject has not been treated with immune checkpoint inhibitors before, and is receiving lung cancer treatment, particularly non-small cell lung cancer (NSCLC).

[0266] NSCLC may be non-squamous or squamous. In some aspects, the subject may have received treatment based on BRAF, ALK, EGFR and / or ROS1 status. In some aspects, the subject may have made progress on such treatment or cannot tolerate such treatment. In some aspects, the NSCLC is unresectable.

[0267] In some aspects, the melanoma is a skin, acral or mucosal melanoma. Melanoma can be confirmed by standard histological or cytological methods. In some aspects, the melanoma is non-ocular or uveal melanoma. In some aspects, the melanoma is a phase III or IV melanoma (according to a standard staging system, such as that determined by the American Joint Staging Committee). It may not be resectable and / or is not suitable for local treatment.

[0268] In certain aspects, the melanoma may be BRAF positive. In these aspects, the subject may have received BRAF±MEF targeted therapy, and in particular may have shown progression.

[0269] In certain aspects, the subject has received previous standard therapy for advanced or recurrent / metastatic disease, particularly systemic therapy, or the subject may be intolerant to such therapy. The standard therapy may include, for example, chemotherapy, immunotherapy, and targeted therapy. The subject may have received, for example, 1, 2, 3, 4 or 5, but particularly 4 or fewer regimens.

[0270] In some aspects, the subject has received a prior immune checkpoint therapy, particularly PD-L1 / PD-1 therapy. In some such aspects, the subject has received no more than one prior PD-1 therapy for advanced or metastatic cancer. In some such aspects, the subject may have relapsed after a prior therapy, e.g., their disease progressed or remained stable at best after at least 6 months of treatment. In some aspects, the subject has not been previously treated with an immune checkpoint inhibitor.

[0271] In some aspects, the subject may have received monotherapy with the multi-specific antibody for 1, 2, 3, 4, 5 or 6 months, in particular 1 or 2 months, prior to starting the combination therapy.

[0272] In certain aspects, the subject is an adult. In certain aspects, the subject is a mammal, particularly a human.

[0273] In some aspects, the subject does not suffer from one or more symptoms selected from Burkitt lymphoma (Burkitt lymphoma), lymphoblastic leukemia / lymphoma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, CNS metastasis / lymphoma, carcinomatous meningitis, autoimmune disease, active HBV, active HCV, HIV. In some aspects, the subject has not received therapy including 4-1BB promoters, CAR T- cell therapy or solid organ or allogeneic stem cell transplantation. In some aspects, the subject does not have a medical history of primary pulmonary fibrosis, interstitial lung disease, organizing pneumonia, drug-induced pneumonia or primary pneumonia or active pneumonia.

[0274] In certain aspects, the multispecific antibodies according to the uses or methods of the invention increase the number of CD8+ T cells, particularly in breast cancer, more particularly in immunodeficient mice bearing human MDA-MB-231 tumors.

[0275] In certain aspects, the multispecific antibodies according to the uses or methods of the invention are significantly less toxic than the combination of urelumab and atezolizumab, e.g. as determined in the same studies, particularly in breast cancer, more particularly in immunodeficient mice bearing human MDA-MB-231 tumors.

[0276] In certain aspects, the multispecific antibodies according to the uses or methods of the invention do not induce graft-versus-host disease.

[0277] The term "antibody" as used herein refers to a protein molecule, such as a protein belonging to the immunoglobulin class, containing one or more variable domains that bind antigenic epitopes, wherein such domains are derived from or share sequence homology with the variable domains of antibodies. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for human subjects). Antibody binding can be represented by specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Affinity is a measure of the binding strength to a specific antigen or epitope. Antibodies, such as the multispecific antibodies of the present invention, typically comprise the constant domains (Fc portions) of natural antibodies, which can be modified as described elsewhere herein, for example to reduce ADCC and / or CDC activity.

[0278] "Multispecific antibody" refers to an antibody comprising at least two binding sites with different antigen or epitope specificities. In some aspects, one or more antigen binding sites comprise an immunoglobulin VH / VL pair. In some aspects, each antigen binding site comprises an immunoglobulin VH / VL pair.

[0279] In some aspects, the multispecific antibodies according to the invention have no more than two antigen binding sites. This means that the antigen binding portion of such multispecific antibodies consists of two antigen binding sites, with no additional antigen binding sites present. In some aspects, each of the two antigen binding sites comprises an immunoglobulin VH / VL pair.

[0280] In some aspects, the VL in each VH / VL pair is similar. In some aspects, the VL in each VH / VL pair is identical. In some aspects, the multispecific antibody is a full-length antibody having a heavy chain / light chain (H / L) combination that binds to the extracellular portion of CD137 and an H / L chain combination that binds to the extracellular portion of a member in the B7 family. In some aspects, the light chain in the first H / L chain combination is similar to the light chain in the first H / L chain combination. In some aspects, the light chains in the first and second H / L chain combinations are identical.

[0281] In certain aspects, the multispecific antibody is a bispecific antibody.

[0282] The term "bispecific antibody" refers to an antibody in which one part binds to an epitope on an antigen, while the second part binds to a different epitope on the same antigen or a different antigen. Different epitopes are usually present on different antigens. However, different epitopes can also be present on the same antigen. Depending on the expression level, (sub-) cellular localization and stoichiometry of the two antigens recognized by the bispecific antibody, the two Fab arms of the antibody may or may not bind their epitopes simultaneously. The arms of a bispecific antibody usually contain the variable domain of one antibody, while the other arm contains the variable domain of another antibody (i.e., one arm of a bispecific antibody is formed by pairing a heavy chain with a light chain, while the other arm is formed by pairing a different heavy chain with a light chain). In certain aspects, the heavy chain variable regions of the bispecific antibodies of the present invention are different from each other, while the light chain variable regions are the same in the bispecific antibodies of the present invention. Bispecific antibodies in which different heavy chain variable regions are combined with the same or common light chain variable region are also referred to as bispecific antibodies with a common light chain variable region (cLcv). In certain aspects, the light chain constant region is also the same. Such bispecific antibodies are referred to as having a common light chain (cLc).

[0283] Certain preferred aspects are immunoglobulins having an IgG format, providing the advantage that the half-life of the bivalent binding molecules / antibodies / variants of the invention is generally longer, compared to multivalent compounds. In addition, the immunogenicity of the bivalent binding molecules of the invention is generally lower than that of multivalent compounds. The molecules / antibodies / variants in these aspects preferably retain the native IgG structure, and therefore retain all the benefits associated with the native IgG structure.

[0284] "Variants" of antibodies or multispecific antibodies as described herein include functional parts, derivatives and / or analogs of antibodies or multispecific antibodies. Variants can be antibody fragments, such as Fab fragments. Variants can be single-chain variable fragments (scFv). The variants retain the binding specificity of the antibody. The functional parts, derivatives and / or analogs retain the binding specificity of the antibody. Binding specificity is defined as the ability to bind to the extracellular part of the first membrane protein and the second membrane protein described herein. Variants may have up to 15 amino acid insertions, deletions, substitutions or combinations thereof relative to a specified amino acid sequence (e.g., any SEQ ID NO. of the present invention), such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as 0, 1, 2, 3, 4 or 5, such as 0, 1, 2, 3 or 4; such as 0, 1, 2 or 3, such as 0, 1 or 2; or such as 0 or 1 amino acid insertions, deletions, substitutions or combinations thereof.

[0285] As used herein, the term "antigen binding site" refers to a site of a binding molecule or antibody that specifically binds to an antigenic epitope. Such antigen binding sites are preferably derived from the variable domains of an antibody, particularly its CDR region, or share sequence homology therewith. In some preferred aspects, the antigen binding site is an immunoglobulin variable domain formed by an immunoglobulin VH / VL pair. In other aspects, the antigen binding site is derived from an antibody mimetic, such as derived from an affibody molecule, human ubiquitin (affilin), affimer, affitin, alphabody, anticalin, avimer, DARPin, fynomer, kunitz domain peptide or monomer, which have been previously described.

[0286] According to the present invention, the term "full length" is defined as comprising a substantially complete antibody without one or more artificially added fragments having a size greater than 20 amino acid residues, such as, for example, additional antigen binding sites or additional activation sites or additional ligands or additional ligand binding moieties. However, a full-length antibody does not necessarily have all the functions of a complete antibody. For the avoidance of doubt, a full-length antibody comprises two heavy chains and two light chains. Each chain comprises a constant (C) and a variable (V) region, which can be divided into CH1, CH2, CH3, VH domains on the heavy chain, and CL, VL domains on the light chain. The domains of the heavy chain preferably exist in the order of natural antibodies (VHCH1-CH2-CH3; refers to the VH domain adjacent to the CH1 domain, followed by the CH2 domain, followed by the CH3 domain). The domains of the light chain also preferably exist in the order of natural antibodies (VL-CL; refers to the VL domain adjacent to the CL domain). The antibody binds to the antigen via the variable domains contained in the Fab fragment portion. The antibody can interact with the molecules and cells of the immune system through the constant domain, mainly through the Fc portion.

[0287] In certain aspects, full-length IgG antibodies are preferred because they generally have a favorable half-life, and for reasons of immunogenicity, it is desirable to be as close to a fully autologous (human) molecule as possible. In certain aspects, the multispecific antibodies of the invention are full-length IgG1, full-length IgG2, full-length IgG3, or full-length IgG4 antibodies.

[0288] Full-length antibodies encompass antibodies in which there may be mutations that provide desired characteristics or that simply replace characteristics in the original chain. Such mutations are generally not deletions of a large portion of any region. However, antibodies in which one or more amino acid residues are inserted, deleted, substituted, or a combination thereof, and do not substantially change the antigen-binding characteristics of the resulting antibody are encompassed within the term "full-length antibody". For example, an IgG antibody may have 1-20 amino acid residues inserted, substituted, deleted, or a combination thereof in the constant region.

[0289] Please note that in this specification, unless otherwise indicated, the amino acid positions of the CDRs and frameworks in the heavy chain variable region of an antibody or antibody fragment are designated according to the Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids in the constant region are indicated according to the EU numbering system.

[0290] In certain aspects, the "percentage of identity" of nucleic acid or amino acid sequences mentioned herein is defined as the percentage of residues in the candidate sequence that are consistent with the residues in the selected sequence after the sequences are aligned to achieve the best comparison purpose. In order to optimize the comparison between the two sequences, a gap can be introduced in either of the two sequences being compared. This comparison can be performed over the full length of the compared sequences. Alternatively, the comparison can be performed over a shorter length, such as about 20, about 50, about 100 or more nucleic acids / bases or amino acids. The comparison can also be performed on a separate CDR sequence. Sequence identity is the percentage of identical matches between two sequences in the reported comparison region.

[0291] In certain aspects, the comparison of sequences and the determination of the percentage of sequence identity between two sequences are completed using a mathematical algorithm. The technician will appreciate that there are several different computer programs that can be used to compare two sequences and determine the consistency between the two sequences (Kruskal, JB (1983) An overview of sequence comparison InD. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44, Addison Wesley). The percentage sequence identity between two amino acid sequences or nucleic acid sequences can be determined using the Needleman and Wunsch algorithm for comparing two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm has been completed in the computer program NEEDLE. In some aspects, the NEEDLE program from the EMBOSS software package is used to determine the percent identity of amino acids and nucleic acid sequences (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6), pp. 276-277, http: / / emboss.bioinformatics.nl / ). In some aspects, for protein sequences, EBLOSUM62 is used for substitution matrices. In some aspects, DNAFULL is used for DNA sequences. The parameters used are blank-open penalty 10 and blank extension penalty 0.5.

[0292] After alignment as described above by the program NEEDLE, the percent sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions showing identical amino acids or identical nucleotides in both sequences divided by the total length of the alignment minus the total number of gaps after alignment.

[0293] In certain aspects, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1-IF20 are applied to the subject being treated. The start of treatment refers to the first administration of the multispecific antibody of the invention. In certain aspects, the subject comprises or meets all IF1-IF:

[0294] IF1. Life expectancy ≥ 12 weeks.

[0295] IF2.ECOG expression status is 0 or 1.

[0296] IF3. Locally advanced tumor recurrence with PD-L1 / PD-1, and tumor and / or tumor-associated immune cells are positive for PD-L1 expression (≥1%) (based on local testing after completion of most recent prior therapy)

[0297] IF4. With measurable disease according to RECIST v1.1 or Lugano criteria.

[0298] IF5. Previously received, was unable to tolerate, or refused standard therapy for advanced or recurrent / metastatic disease appropriate to the tumor type.

[0299] IF6. Has received up to 4 prior systemic treatment regimens (including chemotherapy, immunotherapy, and targeted therapy regimens) for advanced or recurrent / metastatic disease unless approved by the medical monitor.

[0300] IF7. Prior treatment with up to 1 anti-PD-1 therapy in the advanced / metastatic setting, including an immunotherapy regimen, provided that combination of anti-PD-1 therapy with chemotherapy, targeted therapy, or other immunotherapy is permitted.

[0301] Tumor-specific criteria

[0302] IF8. Subjects with histologically or cytologically confirmed melanoma, with histologically or cytologically confirmed cutaneous, acral, or mucosal melanoma.

[0303] IF9. Unresectable stage III or IV melanoma not amenable to local therapy according to the American Joint Committee on Cancer staging system.

[0304] IF10. Have V600-activating BRAF mutation status or agree to BRAF testing during screening (if not previously tested).

[0305] IF11. If BRAF mutation-positive, patients must receive BRAF±MEK targeted therapy and have documented progression.

[0306] IF12. Relapse after PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with at least stable disease as best response)

[0307] IF13. Relapse after PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with at least stable disease as best response)

[0308] IF14. Not received PD-L1 / PD-1 therapy

[0309] IF15. Subjects with documented progression on anti-PD-1 therapy meeting the following criteria:

[0310] IF15a. Primary refractory: Have previously received anti-PD-1 therapy (alone or as part of a combination) for at least 12 weeks in the advanced or metastatic setting and have PD as best response to therapy.

[0311] IF15b. Secondary resistance: Patients who have 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 later developed PD while receiving anti-PD-1 therapy (confirmed PD at least 4 weeks [no less than 28 days] later).

[0312] IF16. Subjects with histologically or cytologically confirmed NSCLC have a histologically or cytologically confirmed diagnosis of NSCLC (either non-squamous or squamous).

[0313] IF16a. If the tumor is of non-squamous histology only, record the test results for EGFR, ALK, BRAF, and ROS1 mutations or gene rearrangements, and if mutations or gene rearrangements are present, the subject has progressed on or is intolerant of targeted therapy.

[0314] IF17. Unresectable advanced or metastatic NSCLC not amenable to local therapy according to the AJCC staging system.

[0315] IF18. Subject has relapsed after PD-L1 / PD-1 therapy (e.g., progressed after more than 6 months of treatment with at least stable disease as best response)

[0316] IF19. Subjects have relapsed following PD-L1 / PD-1 therapy (e.g., progressed after more than 6 months of treatment with at least stable disease as best response), or have not received prior PD-L1 / PD-1 therapy.

[0317] IF20. Subjects have a record of progression on anti-PD-1 therapy, defined as meeting one of the following criteria:

[0318] IF20a. For primary refractory disease, subjects have received at least 12 weeks of anti-PD-1 therapy (alone or as part of a combination) in the advanced or metastatic setting and PD is their best response to treatment.

[0319] IF20b. For secondary resistance, the subject has received prior anti-PD-1 therapy (alone or as part of a combination) in the advanced or metastatic setting and achieved CR, PR, or SD, but was later confirmed to have PD while receiving anti-PD-1 therapy (confirmed PD at least 4 weeks later [no less than 28 days]).

[0320] In certain aspects, the subject receiving treatment meets any one or more factors selected from IF1-IF20.

[0321] In certain aspects, at the start of treatment, at least one, more than one, or all of the following exclusion factors EF1-EF23 will be applied to treat the subject:

[0322] EF1. Prior to administration of the first dose of the multispecific antibody of the present invention, the patient has received treatment with an anticancer drug or an investigational drug within the following intervals:

[0323] EF1a: Chemotherapy (mitomycin C and nitrosourea for 6 weeks), targeted small molecule therapy, or radiotherapy for at least 14 days in which the subject does not develop radiation pneumonitis due to treatment, and a one-week washout period is allowed for palliative radiotherapy for non-CNS disease.

[0324] EF1b: Prior antibody used for anticancer therapy for at least 14 or 28 days,

[0325] EF1c: For other agents with long half-lives (e.g., >5 days), administration of the multispecific antibodies of the invention requires approval by the medical monitor before the fifth half-life,

[0326] EF1d: Radioimmunotherapy for at least 10 weeks, or

[0327] EF1e: Subjects have not undergone allogeneic SCT within the past 6 months or autologous SCT within the past 3 months.

[0328] EF2: The subject has not recovered to ≤ Grade 1 or baseline from toxic effects of prior therapy (including prior immunotherapy) and / or complications of prior surgical intervention prior to the first administration of the multispecific antibody of the invention, provided that alopecia and stable neuropathy (≤ Grade 2) are permitted.

[0329] EF3. Subjects with prior anti-PD-1 therapy (± anti-CTLA-4 therapy) with Grade 1 or higher hepatotoxicity, provided that baseline transaminases and bilirubin are outside normal limits.

[0330] EF4. Subjects have previously experienced Grade ≥ 3 immune-mediated adverse events while receiving anti-PD-1 therapy, with the following Grade 3 or higher adverse events allowed: Grade 3 rash resolved with local treatment; immune-mediated adrenal insufficiency, type 1 diabetes, or other endocrine abnormalities due to prior immunotherapy that is medically stable and adequately controlled on stable doses of alternative therapy; and asymptomatic amylase or lipase elevations that do not require treatment interruption.

[0331] EF5. History of immune-mediated ocular adverse events of any grade.

[0332] EF6. Subjects with laboratory values ​​at Screening as defined in the table below.

[0333] Laboratory values ​​for exclusion

[0334]

[0335]

[0336] EF7. Subjects with clinically significant cardiac disease, including a known history of left ventricular ejection fraction <50%, unstable angina, acute myocardial infarction within 6 months of Cycle 1 Day 1, New York Heart Association Class III or IV congestive heart failure, or arrhythmias requiring treatment, provided that subjects with arrhythmias receive antiarrhythmic medications and are in sinus rhythm on the screening ECG.

[0337] EF7. Subjects with a history or current ECG abnormalities that the investigator deems clinically significant.

[0338] EF8. Subject has experienced immune-related toxicity during prior checkpoint inhibitor therapy for which permanent discontinuation of treatment is recommended (per product labeling or consensus guidelines), or any immune-related toxicity requiring intensive or long-term immunosuppressive management (except endocrine disorders, which are well controlled with hormone replacement).

[0339] EF9. Subjects with a history of primary pulmonary fibrosis, interstitial lung disease, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonitis, or primary pneumonia, or subjects with evidence of active pneumonia on chest CT scan.

[0340] EF10. Subjects with a history of uncontrolled asthma or chronic obstructive pulmonary disease, provided that a history of radiation pneumonitis (fibrosis) in the radiation field is permitted.

[0341] EF11. Subjects with known active CNS metastases / lymphoma and / or carcinomatous meningitis, provided that if the subject has previously treated brain metastases, then either: EF11a: radiographically stable (i.e., no evidence of worsening by repeat imaging for at least 28 days prior to the first dose of the multispecific antibody of the invention, EF11b: no need for steroid therapy for at least 14 days prior to the first dose of the multispecific antibody of the invention, or EF11c: clinically stable with any neurological signs or symptoms returning to baseline, provided that carcinomatous meningitis is excluded regardless of clinical stability.

[0342] EF12: Subjects with evidence of cerebral edema or subjects <28 days after CNS radiation therapy.

[0343] EF13: Subjects with active or inactive autoimmune diseases or syndromes (e.g., rheumatoid arthritis, moderate or severe psoriasis, multiple sclerosis, inflammatory bowel disease) who have required systemic therapy in the past 2 years, or subjects who are receiving systemic therapy for autoimmune or inflammatory diseases (i.e., using disease-modifying agents, corticosteroids, or immunosuppressive drugs), provided that they have vitiligo, resolved childhood asthma / atopic disease, hypothyroidism stable on hormone replacement therapy, controlled asthma, type 1 diabetes, Graves' disease, Hashimoto's disease, or with approval by the medical supervisor, are allowed if all other eligibility criteria are met.

[0344] EF14: Subjects using systemic corticosteroids (≥10 mg / day prednisone or equivalent) within 7 days prior to the first dose of a multispecific antibody of the invention, provided that the use of inhaled or topical corticosteroids or systemic corticosteroids for imaging procedures is permitted, provided that the use of physiologic corticosteroid replacement therapy is approved in consultation with a medical supervisor.

[0345] EF15. Receipt of a live vaccine within 30 days prior to the first dose of the multispecific antibody of the invention or pembrolizumab, including but not limited to the following live vaccines: measles, mumps, German measles, varicella, yellow fever, rabies, Bacillus Calmette–Guérin, and typhoid vaccine.

[0346] EF16. If a live COVID-19 vaccine is available, consultation with a medical monitor is required prior to administration.

[0347] EF17. Injectable seasonal influenza vaccines are generally inactivated vaccines and are allowed to be used; however, the use of intranasal influenza vaccines, which are live attenuated vaccines, is not allowed.

[0348] EF18. Active infection requiring systemic therapy.

[0349] EF19. History of solid organ or allogeneic stem cell transplantation.

[0350] EF20. Subjects with active HBV or HCV infection requiring treatment, and must have undetectable hepatitis B virus DNA and HCV RNA, provided that the subject has previously cleared HBV infection (defined as hepatitis B surface antigen negative, hepatitis B surface antibody positive, hepatitis B core antibody positive) and subjects with no previous history of HBV infection and who have been vaccinated against HBV and have antibodies against hepatitis B surface antigen positive as the only evidence of previous exposure, and hepatitis C antibody-positive subjects who have received and completed hepatitis C treatment aimed at eradication of the virus and have undetectable HCV RNA levels may participate.

[0351] EF21. Known history of HIV infection (HIV 1 / 2 antibodies).

[0352] EF22. Known allergic or severe reaction to any component of the multispecific antibody of the invention, to pembrolizumab, or to a component of the formulation.

[0353] EF23. Pregnancy or lactation or anticipated pregnancy or childbirth during the expected duration of the trial, starting from the Screening Visit until 90 days after the last dose of the multispecific antibody of the invention.

[0354] In certain aspects, the treated subject complies with any one or more factors selected from the group consisting of EF1-EF23. In certain aspects, the treated subject complies with all factors EF1-EF23.

[0355] Terms

[0356] Certain aspects of the invention are defined in the following clauses.

[0357] 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 the treatment also comprises administering a PD-L1 or PD-1 inhibitor.

[0358] 2. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a multispecific antibody and a PD-1 or PD-L1 inhibitor, the 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.

[0359] 3. The multispecific antibody for use as described in item 1 or the method as described in item 2, wherein the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

[0360] 4. The multispecific antibody for use or the method according to any of the preceding clauses, wherein the cancer is an advanced or metastatic solid tumor, such as selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma.

[0361] 5. The multispecific antibody for use or the method as described in any of the preceding clauses, wherein the cancer is NSCLC.

[0362] 6. The multispecific antibody for use or the method of claim 4, wherein the melanoma is selected from cutaneous, acral or mucosal melanoma.

[0363] 7. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the cancer relapses after PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression.

[0364] 8. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is an antibody or a variant or functional fragment thereof.

[0365] 9. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is a PD-1 inhibitor

[0366] 10. The multispecific antibody or method for use as described in Item 9, wherein the PD-1 inhibitor is selected from Nivolumab, Pembrolizumab, Cemiplimab, Penpulimab, Retifanlimab, Sintilimab, Tislelizumab, Toripalimab and Dostarlimab.

[0367] 11. The multispecific antibody or method for use as described in item 10, wherein the PD-1 inhibitor is pembrolizumab.

[0368] 12. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the PD-1 or PD-LI inhibitor is a PD-L1 inhibitor.

[0369] 13. The multispecific antibody for use or method as described in Item 12, wherein the PD-L1 inhibitor is selected from Atezolizumab, Avelumab and Durvalumab.

[0370] 14. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the multispecific antibody is administered at a dose of between 10 mg-1200 mg, 15-1200 mg or 25-1200 mg, such as between 10-600 mg, 15-600 mg, 25-600 mg, 10-300 mg, 15-300 mg, or 25-300 mg, such as between 10-150 mg, 10-100 mg, 10-75 mg, 15-150 mg, 15-100 mg, 15-75 mg, 25-150 mg, 25-100 mg or 25-75 mg, or such as between 10-50 mg, 15-50 mg, 25-50 mg or 50-100 mg.

[0371] 15. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is administered at a dose of about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg or 75 mg.

[0372] 16. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the PD-1 or PD-L1 inhibitor is administered at a dose of about 300-500 mg, such as 325-475 mg, such as 350-450 mg, or such as 375-425 mg.

[0373] 17. The multispecific antibody for use or the method as described in any of the preceding clauses, wherein the multispecific antibody is administered once every 14 days.

[0374] 18. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the PD-1 or PD-L1 inhibitor is administered once every 4, 5, 6, 7 or 8 weeks.

[0375] 19. The multispecific antibody for use or the method as described in Item 18, wherein the PD-1 or PD-L1 inhibitor is administered once every 6 weeks.

[0376] 20. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the PD-1 or PD-L1 inhibitor is administered in the dosage combination shown in the following table:

[0377]

[0378]

[0379]

[0380] 21. The multispecific antibody for use or the method as described in any of the preceding clauses, wherein the multispecific antibody is administered intravenously.

[0381] 22. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is administered once every two weeks.

[0382] 23. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is not a member of the TNF receptor superfamily.

[0383] 24. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is a member of the B7 family.

[0384] 25. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is PD-L1 or PD-L2, such as PD-L1.

[0385] 26. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody comprises an antigen binding site that binds to the PD-1 binding domain of PD-L1.

[0386] 27. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody comprises an antigen binding site that binds to the CD137L binding domain of CD137.

[0387] 28. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody comprises an antigen binding site that blocks ligand binding to CD137 or binds to an extracellular ligand-blocking binding site of CD137, such as a CD137L blocking binding site.

[0388] 29. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the variable domain that binds to the extracellular portion of CD137 is defined as, when in the form of a bivalent monospecific antibody comprising two of said variable domains that bind to CD137, the variable domain does not stimulate CD137 activity on cells, or stimulates CD137 activity on cells at a reduced level compared to one of said variable domains as part of a bispecific antibody having a second variable domain that binds to a tumor-associated antigen (such as a B7 family member, such as PD-L1).

[0389] 30. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein when combined with a multispecific antibody having a second variable domain that binds to PD-L1, and when the multispecific antibody is in the presence of a first cell expressing CD137 and a second cell expressing PD-L1, the variable domain that binds to the extracellular portion of CD137 is capable of stimulating CD137 activity on the cell.

[0390] 31. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is capable of binding to CD137 and PD-L1, such as simultaneously.

[0391] 32. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody only induces or activates CD137 signaling in the presence of cells expressing PD-L1.

[0392] 33. A multispecific antibody for use or method as described in any of the preceding clauses, wherein the antigen binding site of the multispecific antibody consists of an immunoglobulin variable domain that binds to CD137 and an immunoglobulin variable domain that binds to the extracellular portion of a second membrane protein.

[0393] 34. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is a full-length antibody.

[0394] 35. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is an IgG1 molecule without Fc effector function.

[0395] 36. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is not expressed to a significant extent on T cells.

[0396] 37. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins.

[0397] 38. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

[0398] 39. A multispecific antibody for use or method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR3 region having an amino acid sequence as shown in SEQ ID NO:23; SEQ ID NO:27; SEQ ID NO:34 or SEQ ID NO:52, which binds to the extracellular portion of CD137 or a variant thereof.

[0399] 40. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR2 region having an amino acid sequence as shown in SEQ ID NO:22; SEQ ID NO:26; SEQ ID NO:33; or SEQ ID NO:51, which binds to the extracellular portion of CD137 or a variant thereof.

[0400] 41. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO:21; SEQ ID NO:25; SEQ ID NO:32; or SEQ ID NO:50, which binds to the extracellular portion of CD137 or a variant thereof.

[0401] 42. The multispecific antibody for use or method of any of the preceding clauses, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1; SEQ ID NO: 5; SEQ ID NO: 9; SEQ ID NO: 13; SEQ ID NO: 17; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 31; SEQ ID NO: 35; SEQ ID NO: 39; SEQ ID NO: 43; SEQ ID NO: 46; or SEQ ID NO: 49, which binds to an extracellular portion of CD137 (such as SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 31; or SEQ ID NO: 49, or a variant thereof).

[0402] 43. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR3 region having an amino acid sequence as shown 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, which binds to an extracellular portion of PD-L1 such as SEQ ID NO:56; SEQ ID NO:91; SEQ ID NO:95; or SEQ ID NO:102 or a variant thereof.

[0403] 44. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR2 region having an amino acid sequence as shown 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, which binds to the extracellular portion of PD-L1 or a variant thereof.

[0404] 45. A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region comprising a CDR1 region having an amino acid sequence as shown 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, which binds to the extracellular portion of PD-L1 or a variant thereof.

[0405] 46. ​​A multispecific antibody for use or a method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:53; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:73; SEQ ID NO:77; SEQ ID NO:81; SEQ ID NO:85; SEQ ID NO:89; SEQ ID NO:92; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:100; SEQ ID NO:103; SEQ ID NO:104; SEQ ID NO:107, which binds to an extracellular portion of PD-L1 such as SEQ ID NO:67, SEQ ID NO:89, SEQ ID NO:92, or SEQ ID NO:100 or a variant thereof.

[0406] 47. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6797 and CDR1, CDR2 and CDR3 of MF7702, or variants thereof.

[0407] 48. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody comprises SEQ ID NO. 49 and SEQ ID NO. 67, or a variant thereof.

[0408] 49. The multispecific antibody for use or the method as described in any of the preceding clauses, wherein the cancer is Merkel Cell Carcinoma.

[0409] 50. A multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising:

[0410] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0411] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0412] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0413] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0414] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0415] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0416] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0417] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0418] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0419] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0420] The multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

[0421] 51. The multispecific antibody for use or method as described in any of the preceding clauses, wherein the multispecific antibody is administered at a dose of between 10-300 mg, 15-300 mg or 25-300 mg, such as 25-150 mg or 25-100 mg, such as between 50-100 mg.

[0422] 52. The multispecific antibody for use or the method of clause 50 or 51, wherein the cancer is Merkel cell carcinoma.

[0423] 53. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a multispecific antibody comprising a binding domain that binds to CD137, the binding domain comprising:

[0424] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0425] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0426] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0427] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0428] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0429] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0430] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0431] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0432] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0433] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0434] and PD-1 or PD-L1 inhibitors.

[0435] 54. The multispecific antibody for use or the method as described in clause 53 above, wherein the multispecific antibody is administered at a dose of between 10-300 mg, 25-300 mg, such as between 25-150 mg or 25-100 mg, such as 50-100 mg.

[0436] 55. The multispecific antibody for use or the method of any one of items 50 to 54, wherein the cancer is an advanced or metastatic solid tumor.

[0437] 56. The multispecific antibody for use or method of item 55, wherein the advanced or metastatic solid tumor is selected from: advanced or metastatic lung cancer, in particular non-small cell lung cancer (NSCLC); and advanced or metastatic melanoma.

[0438] 57. A multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising:

[0439] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 50, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 52; or

[0440] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or

[0441] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or

[0442] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or

[0443] The antibody comprises a binding domain that binds to PD-L1, comprising:

[0444] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 68, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 56; or

[0445] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or

[0446] A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 102; or

[0447] a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91;

[0448] The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof,

[0449] wherein the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately; and

[0450] Wherein the cancer is selected from advanced or metastatic solid tumors.

[0451] 58. The multispecific antibody for use or the method of any one of items 50 to 57, wherein the multispecific antibody is administered intravenously.

[0452] 59. The multispecific antibody for use or the method of any one of items 50 to 58, wherein the multispecific antibody is administered once every 2 weeks.

[0453] 60. The multispecific antibody for use or method as described in any one of items 50 to 59, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6797 and CDR1, CDR2 and CDR3 of MF7702, or variants thereof.

[0454] 61. The multispecific antibody for use or method as described in item 60, wherein the multispecific antibody comprises SEQ ID NO. 49 and SEQ ID NO. 67, or a variant thereof.

[0455] 62. The multispecific antibody for use or method of any one of items 50 to 59, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6783 and CDR1, CDR2 and CDR3 of MF5542, or variants thereof.

[0456] 63. The multispecific antibody for use or method as described in item 62, wherein the multispecific antibody comprises SEQ ID NO. 1 and SEQ ID NO. 92, or a variant thereof.

[0457] 64. The multispecific antibody for use or method of any one of items 50 to 59, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6754 and CDR1, CDR2 and CDR3 of MF5561, or variants thereof.

[0458] 65. The multispecific antibody for use or method as described in item 64, wherein the multispecific antibody comprises SEQ ID NO. 20 and SEQ ID NO. 100, or a variant thereof.

[0459] 66. The multispecific antibody for use or method as described in any one of items 50 to 59, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6785 and CDR1, CDR2 and CDR3 of MF5439, or variants thereof.

[0460] 67. The multispecific antibody for use or method as described in item 66, wherein the multispecific antibody comprises SEQ ID NO. 31 and SEQ ID NO. 89, or a variant thereof.

[0461] 68. The multispecific antibody for use or method of any one of items 50 to 59, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6795 and CDR1, CDR2 and CDR3 of MF5442, or variants thereof.

[0462] 69. The multispecific antibody for use or method as described in item 68, wherein the multispecific antibody comprises SEQ ID NO. 9 and SEQ ID NO. 92, or a variant thereof.

[0463] 70. A multispecific antibody or method for use as described in any of the preceding clauses, wherein the antibody comprises CDR1, CDR2 and CDR3 sequences of a common light chain variable domain having an amino acid sequence as shown in SEQ ID NO: 110, or a common light chain having an amino acid sequence as shown in SEQ ID NO: 109, or a variant thereof, wherein the CDR naming or numbering is according to IMGT.

[0464] 71. A multispecific antibody for use or method as described in any of the preceding clauses, wherein the antibody comprises a common light chain variable domain having an amino acid sequence as shown in SEQ ID NO: 110, or a common light chain having an amino acid sequence as shown in SEQ ID NO: 109, or a variant thereof.

[0465] 72. A multispecific antibody for use or method as described in any of the preceding clauses, wherein the antibody comprises a heavy chain constant domain 1 (CH1) having an amino acid sequence as shown in SEQ ID NO: 112, a heavy chain constant domain 2 (CH2) having an amino acid sequence as shown in SEQ ID NO: 114, a heavy chain constant domain 3 (CH3) having an amino acid sequence as shown in SEQ ID NO: 115, and a heavy chain constant domain 3 (CH3) having an amino acid sequence as shown in SEQ ID NO: 116, or variants thereof.

[0466] 73. The multispecific antibody or method of any one of items 50 to 72, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor.

[0467] 74. The multispecific antibody or method of item 73, wherein the PD-1 inhibitor is selected from Nivolumab, Pembrolizumab, Cemiplimab, Penpulimab, Retifanlimab, Sintilimab, Tislelizumab, Toripalimab and Dostarlimab.

[0468] 75. The multispecific antibody or method of item 74, wherein the PD-1 inhibitor is pembrolizumab.

[0469] 76. The multispecific antibody or method of any one of items 50 to 72, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor.

[0470] 77. The multispecific antibody or method of item 76, wherein the PD-L1 inhibitor is selected from Atezolizumab, Avelumab and Durvalumab.

[0471] 78. A kit comprising a multispecific antibody as defined in any of the preceding clauses, and instructions for use of the multispecific antibody in combination with a PD-1 or PD-L1 inhibitor.

[0472] 79. The kit as described in item 78, further comprising a PD-1 or PD-L1 inhibitor.

[0473] 80. The kit of item 78 or 79, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor.

[0474] 81. The kit of item 80, wherein the PD-1 inhibitor is selected from Nivolumab, Pembrolizumab, Cemiplimab and Dostarlimab.

[0475] 82. The kit of item 81, wherein the PD-1 inhibitor is pembrolizumab.

[0476] 83. The kit of item 78 or 79, wherein the PD-1 or PD-L1 inhibitor is a PD-1 inhibitor.

[0477] 84. The kit of item 83, wherein the PD-L1 inhibitor is selected from Atezolizumab, Avelumab and Durvalumab.

[0478] 85. The kit of any one of items 78 to 84, further comprising instructions for using the multispecific antibody at a dose of between 10-75 mg or between 25-75 mg or between 25-50 mg or between 25-40 mg or between 25-30 mg, or at a dose of 10 mg or 25 mg or 30 mg or 40 mg or 50 mg or 60 mg or 70 mg or 75 mg or a fixed dose.

[0479] 86. The kit of any one of items 78 to 85, wherein the kit comprises instructions for using the multispecific antibody to treat any advanced or metastatic solid tumor.

[0480] 87. The kit of item 86, wherein the advanced or metastatic solid tumor is selected from: locally advanced or metastatic lung cancer, in particular non-small cell lung cancer (NSCLC); and locally advanced or metastatic melanoma.

[0481] 88. A combination of a multispecific antibody and a PD-1 or PD-L1 inhibitor, the 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 treating cancer in a subject in need thereof, wherein the multispecific antibody is as defined in any of the preceding clauses and the inhibitor is as defined in any of the preceding clauses.

[0482] 89. A PD-1 or PD-L1 inhibitor as defined in any of the preceding clauses for use in treating cancer in a subject in need thereof, wherein the PD-L1 inhibitor is for simultaneous or sequential administration with the 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.

[0483] 90. 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, the subject having been or to be administered PD-L1 or a PD-1 inhibitor, as defined in any of the preceding clauses.

[0484] 91. A PD-1 or PD-L1 inhibitor as defined in any of the preceding clauses for use in treating cancer in a patient in need thereof, wherein the patient has been or is to be administered 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.

[0485] Example

[0486] Example 1: Multispecific Antibodies Binding to PD-L1 and CD137

[0487] Multispecific antibodies comprising heavy chain variable regions as mentioned in Table 1 were obtained as described in WO2018 / 056821.

[0488] Table 1: SEQ ID NOs of heavy chain variable regions of multispecific antibodies that bind to CD137 and PD-L1.

[0489]

[0490]

[0491]

[0492]

[0493] Example 2 - Phase 1, open-label, dose-escalation, safety, tolerability, and preliminary efficacy study of MCLA-145 in combination with pembrolizumab in participants with advanced or metastatic malignancies

[0494] Treatment Group and Period:

[0495] An open-label, nonrandomized, Phase 1 trial has been initiated to determine the safety, tolerability, and preliminary efficacy of a multispecific antibody targeting CD137 and PD-L1 in combination with a PD-1 or PD-L1 inhibitor in adult participants with advanced or metastatic malignancies.

[0496] The following multispecific antibodies are suitable for use in the assays and methods of the invention: MF6797 x MF7702, MF6763 x MF5442, MF6754 x MF5561, MF6785 x MF5439 and MF6785 x MF5442, such as MF6797 x MF7702. Each multispecific antibody comprises two VHs, designated by MF numbers capable of binding to CD137 and PD-L1, respectively, and an Fc region having a KK / DE CH3 heterodimerization domain as shown in SEQ ID NO: 115 and SEQ ID NO: 116, respectively, a CH2 domain as shown in SEQ ID NO: 114, a CH1 domain as shown in SEQ ID NO: 112, and a common light chain as shown in SEQ ID NO: 109. In particular, bispecific antibodies comprising the heavy chain sequence of MF6797 x MF7702 are particularly suitable for use in the assays and methods of the invention.

[0497] A dose escalation study was conducted in participants with advanced or recurrent / metastatic solid tumors to determine the MTD and / or RDE of a combination of a multispecific antibody and a fixed dose of a PD-1 inhibitor. Participants received increasing doses of one of the above exemplary multispecific antibodies, further referred to herein as "test antibodies," every 2 weeks until the MTD or RDE was reached. The duration of each treatment cycle was 28 days with increasing doses. Participants also received a fixed dose of 400 mg of pembrolizumab every 6 weeks.

[0498] Overall experimental design

[0499] This is an open-label, non-randomized Phase 1 trial designed to determine the safety, tolerability, and preliminary efficacy of MCLA-145 in adult participants with advanced or metastatic malignancies and will be conducted in two parts, dose escalation and dose expansion. MCLA-145 will be administered intravenously over 2 hours at a fixed dose every 14 days in a 28-day cycle in combination with pembrolizumab administered at a fixed dose every 6 weeks.

[0500] Part 1: Dose escalation

[0501] Part 1 is dose escalation to determine the maximum tolerated dose (MTD) and / or recommended dose expansion (RDE) of MCLA-145 in combination with pembrolizumab administered every 14 days in participants with advanced or metastatic solid tumors.

[0502] Participants will receive MCLA-145 at a 10mg dose level in combination with a fixed dose of pembrolizumab. Each dose level will include 3-6 evaluable patients; if no dose-limiting toxicity (DLT) occurs within the first 28-day cycle, the dose of MCLA-145 will be escalated to the next level. Up to 6 evaluable patients will be enrolled at each dose level until the MTD or RDE is reached, or the sponsor's decision based on the global safety of the product.

[0503] Participants will initially follow the following process:

[0504]

[0505] Further combination doses may be explored, including 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 100 mg, 125 mg, and 150 mg. Pembrolizumab may also be provided at 200 mg every 3 weeks.

[0506] Part 2: Dose Confirmation / Safety Extension

[0507] Part 2 is a dose expansion to confirm the dose of MCLA-145 in combination with pembrolizumab by further evaluating safety, tolerability, pharmacokinetics, preliminary anti-tumor activity, and binding to functional targets. Participants with advanced or metastatic tumors will be enrolled according to the following:

[0508] Adult men or women with advanced or metastatic solid tumors that have relapsed after PD1 / PD-L1. Participants with NSCLC and melanoma with PD-L1>1%, relapsed after PD1 / PD-L1, who are considered by the site investigator to benefit from combination treatment with pembrolizumab.

[0509] Any other tumor histology in Part 1, if preliminary efficacy is observed with MCLA-145 or emerging data suggest a response, may be treated in Part 2 with the consent of the sponsor’s medical monitor. Initial enrollment in the expansion cohorts will be limited to 10 or 20 participants per indication.

[0510] Inclusion may be limited to PD-L1-positive tumors, defined as tumor expression ≥1%, as determined at the site by any commercially available assay if supported by new data.

[0511] Inclusion criteria

[0512] Only participants who met all of the following criteria were eligible for inclusion in the trial. Participants with melanoma or NSCLC must meet all of the criteria listed in the All Participants section and the applicable tumor-specific criteria section to confirm eligibility.

[0513] All participants

[0514] Be able to understand and be willing to sign a written ICF for the trial.

[0515] Be at least 18 years old when signing the informed consent form.

[0516] Willing and able to comply with all protocol requirements, including all scheduled visits and protocol procedures.

[0517] Life expectancy ≥12 weeks.

[0518] ECOG performance status is 0 or 1.

[0519] Part 1 : Locally advanced or metastatic NSCLC or melanoma recurrent to PD-L1 / PD-1 and positive (≥1%) for PD-L1 expression on tumor and / or tumor-associated immune cells (based on local testing completed after most recent prior line of therapy).

[0520] Part 2: Locally advanced or metastatic NSCLC or melanoma that has relapsed or has not received prior immunotherapy with PD-L1 / PD-1 and is positive (≥1%) for PD-L1 expression on tumor and / or tumor-associated immune cells (based on local testing completed after the most recent prior line of therapy).

[0521] Measurable disease according to RECIST v1.1 or Lugano criteria.

[0522] Note: Tumor lesions located in previously irradiated areas or areas that received other locoregional treatments were considered measurable if they had demonstrated significant progression in lesions.

[0523] Have received, are intolerant to, or refuse prior standard therapy for advanced or recurrent / metastatic disease appropriate for the tumor type.

[0524] Received up to 4 prior systemic treatment regimens (including chemotherapy, immunotherapy, and targeted therapy regimens) for advanced or recurrent / metastatic disease unless approved by the medical monitor.

[0525] Received up to 1 prior anti-PD-1 therapy containing an immunotherapy regimen in the advanced / metastatic setting. Combination of anti-PD-1 therapy with chemotherapy, targeted therapy, or other immunotherapy is allowed.

[0526] Willing to undergo tumor biopsies before and during treatment to obtain tumor tissue.

[0527] NOTE: In participants where tumor tissue cannot be safely obtained, the participant may still be included in the trial after discussion with the medical monitor.

[0528] Part 2 only : Participants with known PD-L1 status or fresh tumor sections for local assessment of PD-L1 expression during screening.

[0529] Willing to avoid pregnancy or childbearing based on the following criteria.

[0530] Men must agree to take appropriate precautions to avoid childbearing for 90 days after the last dose of MCLA-145 (with at least 99% certainty) and must refrain from donating sperm during this period. Approved methods of contraception that are at least 99% effective should be communicated to participants and their understanding confirmed.

[0531] 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 use adequate contraception (with at least 99% certainty) to avoid pregnancy from screening until 90 days after the last dose of MCLA-145. Approved methods of contraception that are at least 99% effective should be communicated to participants and their understanding confirmed. Females of childbearing potential should refrain from donating eggs from 30 days before the first dose of MCLA-145 until 90 days after the last dose of MCLA-145.

[0532] Women of infertile potential (ie, surgically sterilized by hysterectomy and / or bilateral oophorectomy or amenorrhea for ≥12 months and at least 50 years of age) were eligible.

[0533] For combination therapy

[0534] Patients who had received MCLA-145 monotherapy for at least 2 months were eligible for enrollment in this trial. These patients must not have had reports of grade ≤2 hepatotoxicity while receiving MCLA-145 monotherapy.

[0535] Tumor-specific criteria

[0536] Participants with histologically or cytologically confirmed melanoma:

[0537] Histologically or cytologically confirmed cutaneous, acral, or mucosal melanoma.

[0538] Note: Participants with ocular or uveal melanoma were excluded.

[0539] Unresectable stage III or IV melanoma not amenable to local therapy according to the current AJCC staging system.

[0540] Record V600-activating BRAF mutation status or consent for BRAF testing during screening if not previously tested.

[0541] If BRAF mutation-positive, patients must receive BRAF±ME targeted therapy and have progression documented.

[0542] Part 1 participants in combination therapy : Relapse on PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with at least stable disease as best response)

[0543] Part 2 participants in combination therapy :

[0544] Relapsed on PD-L1 / PD-1 therapy (e.g., progressed after more than 6 months of treatment, with at least stable disease as the best response), naive to PD-L1 / PD-1 therapy

[0545] Part 2 participants receiving monotherapy only: There must be documentation of progress on anti-PD-1 therapy meeting one of the following criteria:

[0546] Primary Refractory: Must have received at least 12 weeks of prior treatment with anti-PD-1 therapy (alone or as part of combination therapy with anti-CTLA-4 therapy) in the advanced or metastatic setting and have PD as their best treatment response. Participants who have previously received at least 12 weeks of anti-PD-1 therapy in the adjuvant setting and progressed are also allowed.

[0547] Secondary resistance: Must have previously received anti-PD-1 therapy (alone or as part of a combination therapy) in the advanced or metastatic setting and achieved CR, PR, or SD, but was later confirmed to be PD while receiving anti-PD-1 therapy (confirmed as PD at least 4 weeks later [no less than 28 days]).

[0548] Participants with histologically or cytologically confirmed NSCLC:

[0549] Histologically or cytologically confirmed NSCLC (nonsquamous or squamous).

[0550] If the tumor was of nonsquamous histology only, test results for EGFR, ALK, BRAF, and ROS1 mutations or gene rearrangements were recorded (molecular testing is not currently part of the diagnostic guidelines for predominantly squamous histology). If a mutation or gene rearrangement was present, the participant must have progressed on or been intolerant of targeted therapy.

[0551] Unresectable advanced or metastatic NSCLC not amenable to local therapy according to the current AJCC staging system.

[0552] Part 1 participants in combination therapy : Relapse on PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with at least stable disease being the best response)

[0553] Part 2 participants in combination therapy :

[0554] Relapse on PD-L1 / PD-1 therapy (e.g., progression after more than 6 months of treatment, with at least stable disease being the best response)

[0555] No prior PD-L1 / PD-1 therapy

[0556] Part 2 participants only : There must be documentation of progression on anti-PD-1 therapy meeting one of the following criteria:

[0557] Primary Refractory: Must have received at least 12 weeks of prior treatment with anti-PD-1 therapy (alone or as part of a combination therapy) in the advanced or metastatic setting and have progressive disease (PD) as their best treatment response.

[0558] Secondary resistance: Must have received anti-PD-1 therapy (alone or as part of a combination therapy) in the advanced or metastatic setting and achieved CR, PR, or SD, but later had PD confirmed while receiving anti-PD-1 therapy (PD confirmed at least 4 weeks [no less than 28 days] later).

[0559] Participants with solid tumor indicators with confirmed MSI-H or dMMR status:

[0560] MSI-H or dMMR solid tumors, determined by local laboratories using IHC or polymerase chain reaction, and tissue must also be available for central diagnostic confirmation.

[0561] Participants in Part 2 only were considered primary refractory to anti-PD-1 therapy, defined as having received at least 12 weeks of prior treatment with anti-PD-1 therapy (alone or as part of combination therapy) in the advanced or metastatic setting with documented PD as their best treatment response.

[0562] Exclusion criteria

[0563] Participants will be excluded from the trial if they meet any of the following criteria:

[0564] The following B-cell neoplasms: Burkitt lymphoma, lymphocytic leukemia / lymphoma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia.

[0565] Prior treatment containing a 4-1BB agonist or prior treatment with CAR T-cell therapy.

[0566] Treatment with anticancer drugs or investigational drugs within the following time intervals before the first dose of MCLA-145:

[0567] Chemotherapy (mitomycin C and nitrosourea for 6 weeks), targeted small molecule therapy, or radiation therapy for at least 14 days.

[0568] NOTE: Participants must not develop radiation pneumonitis as a result of treatment. A 1-week washout period is permitted for palliative radiation therapy for non-CNS disease, with sponsor approval.

[0569] mAbs previously used for anticancer therapy for at least 28 days.

[0570] Note: Participants receiving bisphosphonates and / or denosumab were eligible for inclusion.

[0571] For other agents with longer half-lives (e.g., >5 days), inclusion prior to the fifth half-life requires approval by the Medical Supervisor.

[0572] Radioimmunotherapy was continued for at least 10 weeks.

[0573] Patients must not have undergone allogeneic SCT within the past 6 months or autologous SCT within the past 3 months.

[0574] Have not recovered to ≤ Grade 1 or baseline from toxic effects of prior therapy (including prior immunotherapy) and / or complications of prior surgical intervention prior to initiation of MCLA-145.

[0575] Note: Alopecia and stable neuropathy (≤grade 2) were allowed.

[0576] There was no prior anti-PD-1 therapy (± anti-CTLA-4 therapy)-related hepatotoxicity > Grade 1.

[0577] For patients treated with combination therapy only: Baseline transaminases and bilirubin outside normal range

[0578] Prior immune-mediated adverse events of ≥ grade 3 on anti-PD-1 therapy.

[0579] NOTE: The following Grade 3 or higher adverse events were allowed: Grade 3 rash that resolved with topical therapy; immune-mediated adrenal insufficiency, type 1 diabetes mellitus, or other endocrine abnormalities due to prior immunotherapy who were medically stable and adequately controlled on stable doses of alternative therapy; asymptomatic amylase or lipase elevations that did not require interruption of treatment.

[0580] History of immune-mediated ocular adverse events of any grade.

[0581] Participants who had laboratory values ​​at Screening as defined in the table below.

[0582] Laboratory values ​​for exclusion

[0583]

[0584]

[0585] Subjects with clinically significant cardiac disease, including known history of left ventricular ejection fraction <50%, unstable angina, acute myocardial infarction within 6 months of Cycle 1 Day 1, New York Heart Association Class III or IV congestive heart failure, or arrhythmia requiring treatment.

[0586] NOTE: Participants with arrhythmias may be included if they are receiving antiarrhythmic medication and are in sinus rhythm on the screening ECG.

[0587] A history of or current ECG findings that the investigator deems clinically significant.

[0588] Permanent discontinuation of therapy is recommended for immune-related toxicity that occurred during prior checkpoint inhibitor therapy (per product labeling or consensus guidelines), or any immune-related toxicity that requires intensive or prolonged immunosuppressive management (except for endocrine disorders, which are well controlled with hormone replacement).

[0589] Subjects with a history of primary pulmonary fibrosis, interstitial lung disease, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonitis, or primary pneumonia, or evidence of active pneumonia on chest CT scan. Participants with a history of uncontrolled asthma or chronic obstructive pulmonary disease were excluded.

[0590] NOTE: A history of radiation pneumonitis (fibrosis) in the radiation field is allowed.

[0591] Known active CNS metastatic / lymphoma and / or carcinomatous meningitis.

[0592] NOTE: Participants with previously treated brain metastases may participate if they are 1) radiologically stable (i.e., no evidence of worsening by repeat imaging for at least 28 days prior to the first dose of a multispecific antibody of the invention [repeat imaging may be performed during the screening period approved by the medical monitor]), 2) have not required steroid therapy for at least 14 days prior to the first dose of MCLA-145, and 3) are clinically stable with any neurological signs or symptoms having returned to baseline. This exception does not include carcinomatous meningitis, which is excluded regardless of clinical stability.

[0593] NOTE: Participants with evidence of cerebral edema or <28 days post-CNS radiation therapy will be excluded from the trial.

[0594] Subjects with active or inactive autoimmune diseases or syndromes (e.g., rheumatoid arthritis, moderate or severe psoriasis, multiple sclerosis, inflammatory bowel disease) who have required systemic treatment in the past 2 years, or participants who are receiving systemic treatment for autoimmune or inflammatory diseases (i.e., using disease-modifying agents, corticosteroids, or immunosuppressive drugs).

[0595] NOTE: Patients with vitiligo, resolved childhood asthma / atopy, stable hypothyroidism on hormone replacement therapy, controlled asthma, type 1 diabetes, Graves' disease, Hashimoto's disease, or approved by a medical supervisor are eligible if all other eligibility criteria are met.

[0596] Use systemic corticosteroids (≥10 mg / day prednisone or equivalent) within 7 days prior to the first dose of a multispecific antibody of the invention.

[0597] NOTE: Use of inhaled or topical corticosteroids or systemic corticosteroids is permitted for imaging procedures.

[0598] NOTE: Physiologic corticosteroid replacement therapy may be authorized in consultation with the medical supervisor.

[0599] Receive a live vaccine within 30 days prior to administration of the first dose of the multispecific antibody of the invention or pembrolizumab.

[0600] NOTE: Examples of live vaccines include, but are not limited to: measles, mumps, German measles, varicella, yellow fever, rabies, Bacillus Calmette–Guérin, and typhoid vaccines. If a live COVID-19 vaccine is available, a medical monitor will need to be consulted prior to administration. Injectable seasonal influenza vaccines are generally inactivated and are permitted; however, intranasal influenza vaccines, which are live attenuated vaccines, are not permitted.

[0601] Active infection requiring systemic therapy.

[0602] History of solid organ or allogeneic stem cell transplantation.

[0603] Active HBV or HCV infection requiring treatment. HBV DNA and HCV RNA must be undetectable. Participants who had previously cleared HBV infection (defined as negative hepatitis B surface antigen, positive hepatitis B surface antibody, positive hepatitis B core antibody) were eligible for the trial.

[0604] NOTE: For participants who have previously cleared HBV infection, HBV prophylaxis should be considered at the investigator's discretion. HBV reactivation should be monitored every 3 cycles by performing HBV viral load and hepatitis B surface antigen serology testing. Additional viral serology testing may be performed at the investigator's discretion.

[0605] NOTE: Participants with no history of prior HBV infection who have been vaccinated against HBV and have positive antibodies to hepatitis B surface antigen as the only evidence of prior exposure may participate in this trial.

[0606] Note: Participants who have received and completed hepatitis C treatment aimed at eradicating the virus and have undetectable HCV RNA levels may participate.

[0607] Have a known history of HIV (HIV 1 / 2 antibodies). HIV testing is not required unless mandated by local health authorities or regulations.

[0608] Known allergic or severe reaction to any ingredient or formulation component of MCLA-145 or pembrolizumab.

[0609] Be pregnant or breastfeeding or anticipate pregnancy or delivery during the expected duration of the trial, starting from the Screening Visit through 90 days after the last dose of MCLA-145.

[0610] Any condition that, in the investigator's judgment, would interfere with full participation in the study, including administration of MCLA-145 or pembrolizumab; create a significant risk to the participant; or interfere with the interpretation of study data.

[0611] Example 3

[0612] An 80-year-old male patient was diagnosed with Merkel cell carcinoma with a PD-L1 score of 1-10%. The patient had received radiotherapy, surgery, and systemic therapy. Treatment with avelumab resulted in disease progression. Treatment with carboplatin and etoposide produced a partial response.

[0613] Subsequently, the patient was administered a 25 mg dose (25 mg, q3w) of a multispecific antibody having two VHs, designated by MF numbers as MF6797 and MF7702, an Fc region with a KK / DE CH3 heterodimerization domain, as shown in SEQ ID NO:115 and SEQ ID NO:116, respectively, a CH2 domain as shown in SEQ ID NO:114, a CH1 domain as shown in SEQ ID NO:112, and a common light chain as shown in SEQ ID NO:109, and a 200 mg dose of pembrolizumab once every three weeks (200 mg, q3w). The site of onset was a skin nodule on the foot. Tumor assessment involved the sum of the target lesion diameters. After the initial dose, the patient developed a clinically significant reduction in lesion diameter.

[0614] Example 4

[0615] A bispecific antibody having two VHs, designated by MF numbers as MF6797 and MF7702, an Fc region having a KK / DECH3 heterodimerization domain, as shown in SEQ ID NO: 115 and SEQ ID NO: 116, respectively, a CH2 domain as shown in SEQ ID NO: 114, a CH1 domain as shown in SEQ ID NO: 112, and a common light chain as shown in SEQ ID NO: 109, using a dose of 40 mg once every three weeks (40 mg, q3w) or once every two weeks (40 mg, q2w), and 200 mg of pembrolizumab once every three weeks (200 mg, q3w) as in Example 2, followed by tumor assessment involving the sum of target lesion diameters. After the initial dose, patients can develop a clinically significant reduction in lesion diameter.

[0616] sequence

[0617] SEQ ID NO: 1: Heavy chain variable region

[0618] QVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRRAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWGVIGGHYMDVWGKGTTVTVSS

[0619] SEQ ID NO:2: HCDR1 according to Kabat from SEQ ID NO:1

[0620] NFAMN

[0621] SEQ ID NO:3: HCDR2 according to Kabat from SEQ ID NO:1

[0622] WINTNTGNPTYAQGFTG

[0623] SEQ ID NO:4: HCDR3 according to Kabat from SEQ ID NO:1

[0624] DWGVIGGHYMDV

[0625] SEQ ID NO:5: Heavy chain variable region

[0626] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDSDGYGPKAFDYWGQGTLVTVSS

[0627] SEQ ID NO:6: HCDR1 according to Kabat

[0628] SYGIS

[0629] SEQ ID NO:7: HCDR2 according to Kabat

[0630] WISAYNGNTNYAQKLQG

[0631] SEQ ID NO:8: HCDR3 according to Kabat

[0632] DSDGYGPKAFDY

[0633] SEQ ID NO:9: Heavy chain variable region

[0634]

[0635] SEQ ID NO: 10: HCDR1 according to Kabat

[0636] SYWIG

[0637] SEQ ID NO: 11: HCDR2 according to Kabat

[0638] IIYPDDSDTRYSPSFQG

[0639] SEQ ID NO: 12: HCDR3 according to Kabat

[0640] FYTGIVGATGAFDV

[0641] SEQ ID NO: 13: Heavy chain variable region

[0642]

[0643] SEQ ID NO: 14: HCDR1 according to Kabat

[0644] SDAIS

[0645] SEQ ID NO: 15: HCDR2 according to Kabat

[0646] GMIPILGTANYAQKFQG

[0647] SEQ ID NO: 16: HCDR3 according to Kabat

[0648] GATYYYGSGTYYSINWFDP

[0649] SEQ ID NO: 17: Heavy chain variable region

[0650]

[0651] SEQ ID NO: 18: HCDR1 according to Kabat

[0652] NFAMT

[0653] SEQ ID NO: 19: HCDR3 according to Kabat

[0654] DWASVMVRGDLDY

[0655] SEQ ID NO:20: Heavy chain variable region

[0656]

[0657] SEQ ID NO:21: HCDR1 according to Kabat

[0658] ELSIH

[0659] SEQ ID NO:22: HCDR2 according to Kabat

[0660] GFYPEDVEPIYARKFQG

[0661] SEQ ID NO:23: HCDR3 according to Kabat

[0662] EGFDNYGSGIRGNWFDP

[0663] SEQ ID NO:24: Heavy chain variable region

[0664]

[0665] SEQ ID NO:25: HCDR1 according to Kabat

[0666] ELSMH

[0667] SEQ ID NO:26: HCDR2 according to Kabat

[0668] SFYPEDGETIYAQKFQG

[0669] SEQ ID NO:27: HCDR3 according to Kabat

[0670] EGVGVIRGNWFDP

[0671] SEQ ID NO:28: Heavy chain variable region

[0672]

[0673] SEQ ID NO:29: HCDR2 according to Kabat

[0674] IIFPDDSDTRYSPSFQG

[0675] SEQ ID NO:30: HCDR3 according to Kabat

[0676] LGGYSGYAEDFVDF

[0677] SEQ ID NO:31: Heavy chain variable region

[0678]

[0679] SEQ ID NO:32: HCDR1 according to Kabat

[0680] KLSMH

[0681] SEQ ID NO:33: HCDR2 according to Kabat

[0682] GFEPEDGETINAQKFQG

[0683] SEQ ID NO:34: HCDR3 according to Kabat

[0684] DLRLGASYYYSYMDV

[0685] SEQ ID NO:35: Heavy chain variable region

[0686]

[0687] SEQ ID NO:36: HCDR1 according to Kabat

[0688] TSGMSVG

[0689] SEQ ID NO:37: HCDR2 according to Kabat

[0690] LIYWNDDKYFSPSLKS

[0691] SEQ ID NO:38: HCDR3 according to Kabat

[0692] TLWGSDDVFDV

[0693] SEQ ID NO:39: Heavy chain variable region

[0694]

[0695] SEQ ID NO:40: HCDR1 according to Kabat

[0696] NYWIG

[0697] SEQ ID NO:41: HCDR2 according to Kabat

[0698] IIYPGDSDTRYSPSFQG

[0699] SEQ ID NO:42: HCDR3 according to Kabat

[0700] HQGYSFSGSHIDDY

[0701] SEQ ID NO:43: Heavy chain variable region

[0702]

[0703] SEQ ID NO:44: HCDR1 according to Kabat

[0704] TYWIG

[0705] SEQ ID NO:41: HCDR2 according to Kabat

[0706] IIYPGDSDTRYSPSFQG

[0707] SEQ ID NO:45: HCDR3 according to Kabat

[0708] HAGFIITSQNIDDY

[0709] SEQ ID NO:46: Heavy chain variable region

[0710]

[0711] SEQ ID NO:47: HCDR2 according to Kabat

[0712] WINTNTGNPTYAQDFTG

[0713] SEQ ID NO:48: HCDR3 according to Kabat

[0714] DWGLVAIGYFDY

[0715] SEQ ID NO:49: Heavy chain variable region

[0716]

[0717] SEQ ID NO:50: HCDR1 according to Kabat

[0718] TTGVGV

[0719] SEQ ID NO:51: HCDR2 according to Kabat

[0720] LIYWNDDTYYSPSLKS

[0721] SEQ ID NO:52: HCDR3 according to Kabat

[0722] EGIIGFLGGNWFDP

[0723] SEQ ID NO:53: Heavy chain variable region

[0724]

[0725] SEQ ID NO:54: HCDR1 according to Kabat

[0726] SHAMN

[0727] SEQ ID NO:55: HCDR2 according to Kabat

[0728] WINPNTGNPTYAQGFTG

[0729] SEQ ID NO:56: HCDR3 according to Kabat

[0730] DRKYVTNWVFAEDFQH

[0731] SEQ ID NO:57: Heavy chain variable region

[0732]

[0733] SEQ ID NO:58: HCDR3 according to Kabat

[0734] DRGYMSNWVFAEYFPH

[0735] SEQ ID NO:59: Heavy chain variable region

[0736]

[0737] SEQ ID NO:60: HCDR1 according to Kabat

[0738] SYAMN

[0739] SEQ ID NO:61: HCDR3 according to Kabat

[0740] DRGYISSWVFAEDFQH

[0741] SEQ ID NO:62: Heavy chain variable region

[0742]

[0743] SEQ ID NO:63: HCDR2 according to Kabat

[0744] CVNPNTGSPTYAQGSTG

[0745] SEQ ID NO:64: Heavy chain variable region

[0746]

[0747] SEQ ID NO:65: HCDR1 according to Kabat

[0748] NYAMN

[0749] SEQ ID NO:66: HCDR2 according to Kabat

[0750] WMNPNTGNPTYAQGSTG

[0751] SEQ ID NO:67: Heavy chain variable region

[0752]

[0753] SEQ ID NO:68: HCDR1 according to Kabat

[0754] NYAIN

[0755] SEQ ID NO:69: Heavy chain variable region

[0756]

[0757] SEQ ID NO:70: HCDR1 according to Kabat

[0758] TYSIT

[0759] SEQ ID NO:71: HCDR2 according to Kabat

[0760] SIVPIFGTINNAQKFQG

[0761] SEQ ID NO:72: HCDR3 according to Kabat

[0762] DNTMVRGVDYYYMDV

[0763] SEQ ID NO:73: Heavy chain variable region

[0764]

[0765] SEQ ID NO:74: HCDR1 according to Kabat

[0766] TYAIS

[0767] SEQ ID NO:75: HCDR2 according to Kabat

[0768] GIIPIFDTPNYAQKFQG

[0769] SEQ ID NO:76: HCDR3 according to Kabat

[0770] NVRGYSAYDLDY

[0771] SEQ ID NO:77: Heavy chain variable region

[0772]

[0773] SEQ ID NO:78: HCDR1 according to Kabat

[0774] TYGTN

[0775] SEQ ID NO:79: HCDR2 according to Kabat

[0776] GIIPIFGTANYAQKFQG

[0777] SEQ ID NO:80: HCDR3 according to Kabat

[0778] GGADMGTLDY

[0779] SEQ ID NO:81: Heavy chain variable region

[0780]

[0781] SEQ ID NO:82: HCDR1 according to Kabat

[0782] TYTII

[0783] SEQ ID NO:83: HCDR2 according to Kabat

[0784] GIIPIFDTPNFAQKFQG

[0785] SEQ ID NO:84: HCDR3 according to Kabat

[0786] EGCNHGVCYPY

[0787] SEQ ID NO:85: Heavy chain variable region

[0788]

[0789] SEQ ID NO:86: HCDR1 according to Kabat

[0790] SYGIT

[0791] SEQ ID NO:87: HCDR2 according to Kabat

[0792] GIIPIFGTTNYAQKFQG

[0793] SEQ ID NO:88: HCDR3 according to Kabat

[0794] RRGYSNPHWLDP

[0795] SEQ ID NO:89: Heavy chain variable region

[0796]

[0797] SEQ ID NO:90: ​​HCDR1 according to Kabat

[0798] TYGIL

[0799] SEQ ID NO:91: HCDR3GGGNYYEFVY according to Kabat

[0800] SEQ ID NO:92: Heavy chain variable region

[0801]

[0802] SEQ ID NO:93: HCDR1 according to Kabat

[0803] TYAIN

[0804] SEQ ID NO:94: HCDR2 according to Kabat

[0805] RIIPIFDTANYAQKFQG

[0806] SEQ ID NO:95: HCDR3 according to Kabat

[0807] DETGYSSSNFQH

[0808] SEQ ID NO:96: Heavy chain variable region

[0809]

[0810] SEQ ID NO:97: Heavy chain variable region

[0811]

[0812] SEQ ID NO:98: HCDR2 according to Kabat

[0813] GIIPVFGTSKYAQKFQD

[0814] SEQ ID NO:99: HCDR3DPSFSSSSGWFDP according to Kabat

[0815] SEQ ID NO: 100: Heavy chain variable region

[0816]

[0817] SEQ ID NO: 101: HCDR2 according to Kabat

[0818] GIIPIFDTANYAQRFQG

[0819] SEQ ID NO: 102: HCDR3 according to Kabat

[0820] DQTGYSSTLFDY

[0821] SEQ ID NO: 103: Heavy chain variable region

[0822]

[0823] SEQ ID NO: 104: Heavy chain variable region

[0824]

[0825] SEQ ID NO: 105: HCDR2 according to Kabat

[0826] WIIPIFDTGNYAQKIQG

[0827] SEQ ID NO: 106: HCDR3 according to Kabat

[0828] HDYTNTVDAFDI

[0829] SEQ ID NO: 107: Heavy chain variable region

[0830]

[0831] SEQ ID NO: 108: HCDR2 according to Kabat GIIPVFGTTNYAQKFQG

[0832] SEQ ID NO: 109: Amino acid sequence of human common light chain IGKV1-39 / jk1

[0833]

[0834] SEQ ID NO: 110: Amino acid sequence of common light chain variable domain

[0835]

[0836] SEQ ID NO: 111: Amino acid sequence of common light chain constant domain

[0837]

[0838] SEQ ID NO: 112: Amino acid sequence of CH1

[0839]

[0840] SEQ ID NO: 113: Amino acid sequence of hinge

[0841] EPKSCDKTHTCPPCP

[0842] SEQ ID NO: 114: Amino acid sequence of CH2

[0843]

[0844] SEQ ID NO: 115: Amino acid sequence of CH3 with KK mutation

[0845]

[0846] SEQ ID NO: 116: Amino acid sequence of CH3 with DE mutation

[0847]

[0848] SEQ ID NO: 117: Amino acid sequence of CD137 according to HGNC: 11924

[0849]

[0850] SEQ ID NO 118: Amino acid sequence of pembrolizumab heavy chain

[0851]

[0852] SEQ ID NO 119: Amino acid sequence of pembrolizumab light chain

[0853]

Claims

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 the treatment further comprises administering a PD-L1 or PD-1 inhibitor.

2. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a multispecific antibody and a PD-1 or PD-L1 inhibitor, wherein the multispecific antibody comprises 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.

3. The multispecific antibody for use according to claim 1 or the method according to claim 2, wherein the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately.

4. The multispecific antibody for use or the method according to any one of the preceding claims, wherein the cancer is an advanced or metastatic solid tumor, such as selected from locally advanced or metastatic lung cancer and locally advanced or metastatic melanoma.

5. The multispecific antibody for use or the method according to any one of the preceding claims, wherein the cancer is NSCLC.

6. The multispecific antibody for use or method according to claim 4, wherein the melanoma is selected from the group consisting of cutaneous, acral or mucosal melanoma.

7. The multispecific antibody for use or the method according to any one of the preceding claims, wherein the cancer is Merkel cell carcinoma (MCC).

8. The multispecific antibody for use or method according to any one of the preceding claims, wherein the cancer relapses after PD-1 / PD-L1 therapy and / or is positive for PD-L1 expression.

9. The multispecific antibody for use or method according to any one of the preceding claims, wherein the PD-1 or PD-LI inhibitor is an antibody or a variant or functional fragment thereof.

10. The multispecific antibody for use or method according to any one of the preceding claims, wherein the PD-1 or PD-LI inhibitor is a PD-1 inhibitor.

11. The multispecific antibody for use or method according to claim 10, wherein the PD-1 inhibitor is Pembrolizumab.

12. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is administered at a dose of between 10 mg and 1200 mg, 15 mg and 1200 mg, or 25 mg and 1200 mg.

13. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is administered at a dose of about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg or 75 mg.

14. The multispecific antibody for use or method according to any one of the preceding claims, wherein the PD-1 inhibitor is pembrolizumab and is administered at a dose of about 300 mg to 500 mg.

15. The multispecific antibody for use or method according to any one of the preceding claims, wherein the PD-1 inhibitor is pembrolizumab and is administered at a dose of about 100 to 300 mg.

16. The multispecific antibody for use or the method according to any one of the preceding claims, wherein the multispecific antibody is administered once every 14 days.

17. The multispecific antibody for use or the method according to any one of the preceding claims, wherein the multispecific antibody is administered once every 21 days.

18. The multispecific antibody for use or method according to any one of the preceding claims, wherein the PD-1 or PD-L1 inhibitor is administered once every 2, 3, 4, 5, 6, 7 or 8 weeks.

19. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is administered intravenously.

20. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is administered once every two weeks.

21. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is administered once every three weeks.

22. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is not a member of the TNF receptor superfamily.

23. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is a member of the B7 family.

24. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is PD-L1 or PD-L2, such as PD-L1.

25. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody comprises an antigen binding site that binds to the PD-1 binding domain of PD-L1.

26. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody comprises an antigen binding site that binds to the CD137L binding domain of CD137.

27. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody comprises an antigen binding site that blocks ligand binding to CD137 or binds to an extracellular ligand-blocking binding site of CD137, such as a CD137L blocking binding site.

28. The multispecific antibody for use or method according to any of the preceding claims, wherein the variable domains that bind to the extracellular portion of CD137 are defined such that, when in the form of a bivalent monospecific antibody comprising two of said variable domains that bind to CD137, said variable domains do not stimulate CD137 activity on cells, or stimulate CD137 activity on cells at a reduced level compared to one of said variable domains being part of a bispecific antibody having a second variable domain that binds a tumor associated antigen (such as a B7 family member, such as PD-L1).

29. The multispecific antibody for use or method according to any one of the preceding claims, wherein the variable domain that binds the extracellular portion of CD137 is capable of stimulating CD137 activity on a cell when combined with a multispecific antibody having a second variable domain that binds PD-L1, and when the multispecific antibody is in the presence of a first cell expressing CD137 and a second cell expressing PD-L1.

30. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is capable of binding to CD137 and PD-L1, such as simultaneously.

31. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody only induces or activates CD137 signaling in the presence of cells expressing PD-L1.

32. The multispecific antibody for use or method according to any one of the preceding claims, wherein the antigen binding site of the multispecific antibody consists of an immunoglobulin variable domain that binds to CD137 and an immunoglobulin variable domain that binds to the extracellular portion of a second membrane protein.

33. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is a full-length antibody.

34. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody is an IgGl molecule without Fc effector function.

35. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is not expressed to a significant extent on T cells.

36. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is present on the cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins.

37. The multispecific antibody for use or method according to any one of the preceding claims, wherein the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

38. The multispecific antibody for use or method according to any one of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR3 region having an amino acid sequence as shown in SEQ ID NO: 23; SEQ ID NO: 27; SEQ ID NO: 34 or SEQ ID NO: 52, said CDR3 region binding to the extracellular portion of CD137 or a variant thereof.

39. The multispecific antibody for use or method according to any one of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR2 region having an amino acid sequence as shown in SEQ ID NO: 22; SEQ ID NO: 26; SEQ ID NO: 33; or SEQ ID NO: 51, wherein the CDR2 region binds to an extracellular portion of CD137 or a variant thereof.

40. The multispecific antibody for use or method according to any one of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR1 region having an amino acid sequence as shown in SEQ ID NO:21; SEQ ID NO:25; SEQ ID NO:32; or SEQ ID NO:50, wherein the CDR1 region binds to an extracellular portion of CD137 or a variant thereof.

41. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1; SEQ ID NO: 5; SEQ ID NO: 9; SEQ ID NO: 13; SEQ ID NO: 17; SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 28; SEQ ID NO: 31; SEQ ID NO: 35; SEQ ID NO: 39; SEQ ID NO: 43; SEQ ID NO: 46; or SEQ ID NO: 49, which binds to an extracellular portion of CD137 (such as SEQ ID NO: 20; SEQ ID NO: 24; SEQ ID NO: 31; or SEQ ID NO: 49, or a variant thereof).

42. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR3 region having an amino acid sequence as shown 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, such as SEQ ID NO:56; SEQ ID NO:91; SEQ ID NO:95; or SEQ ID NO:102, which CDR3 region binds to the extracellular portion of PD-L1 or a variant thereof.

43. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR2 region having an amino acid sequence as shown 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, wherein the CDR2 region binds to an extracellular portion of PD-L1 or a variant thereof.

44. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising a CDR1 region having an amino acid sequence as shown 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, wherein the CDR1 region binds to an extracellular portion of PD-L1 or a variant thereof.

45. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:53; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:73; SEQ ID NO:77; SEQ ID NO:81; SEQ ID NO:85; SEQ ID NO:89; SEQ ID NO:92; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:100; SEQ ID NO:103; SEQ ID NO:104; SEQ ID NO:107, such as SEQ ID NO:67, SEQ ID NO:89, SEQ ID NO:92, or SEQ ID NO:100, which amino acid sequence binds to the extracellular portion of PD-L1 or a variant thereof.

46. ​​The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6797 and CDR1, CDR2 and CDR3 of MF7702, or variants thereof.

47. The multispecific antibody for use or method according to any one of the preceding claims, wherein the multispecific antibody comprises SEQ ID NO. 49 and SEQ ID NO. 67, or a variant thereof.

48. A multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:50, a CDR2 having an amino acid sequence as shown in SEQ ID NO:51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:52; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or The antibody comprises a binding domain that binds to PD-L1, comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a CDR2 having an amino acid sequence as shown in SEQ ID NO:55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:56; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:102; or a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91; The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof, The multispecific antibody is administered simultaneously, sequentially or separately with the PD-1 or PD-L1 inhibitor.

49. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a multispecific antibody and a PD-1 or PD-L1 inhibitor, the multispecific antibody comprising a binding domain that binds to CD137, the binding domain comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:50, a CDR2 having an amino acid sequence as shown in SEQ ID NO:51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:52; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or The antibody comprises a binding domain that binds to PD-L1, comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a CDR2 having an amino acid sequence as shown in SEQ ID NO:55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:56; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:102; or a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91; The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof.

50. A multispecific antibody for use in a method of treating cancer in a subject in need thereof, wherein the antibody comprises a binding domain that binds to CD137, the binding domain comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:50, a CDR2 having an amino acid sequence as shown in SEQ ID NO:51, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:52; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:40, a CDR2 having an amino acid sequence as shown in SEQ ID NO:41, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:42; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 32, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 34; and / or The antibody comprises a binding domain that binds to PD-L1, comprising: A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:68, a CDR2 having an amino acid sequence as shown in SEQ ID NO:55, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:56; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:94, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:95; or A variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:93, a CDR2 having an amino acid sequence as shown in SEQ ID NO:101, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:102; or a variable domain comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:90, a CDR2 having an amino acid sequence as shown in SEQ ID NO:79, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:91; The individual SEQ ID NOs each have 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions or substitutions, or a combination thereof, wherein the multispecific antibody and the PD-1 or PD-L1 inhibitor are administered simultaneously, sequentially or separately; and Wherein the cancer is selected from advanced or metastatic solid tumors.

51. The multispecific antibody for use or method according to any one of claims 48 to 50, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6797 and CDR1, CDR2 and CDR3 of MF7702, or variants thereof.

52. The multispecific antibody for use or method according to claim 51, wherein the multispecific antibody comprises SEQ ID NO. 49 and SEQ ID NO. 67, or a variant thereof.

53. The multispecific antibody for use or method according to any one of claims 48 to 50, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6783 and CDR1, CDR2 and CDR3 of MF5542, or variants thereof.

54. The multispecific antibody for use or method according to claim 53, wherein the multispecific antibody comprises SEQ ID NO. 1 and SEQ ID NO. 92, or a variant thereof.

55. The multispecific antibody for use or method according to any one of claims 48 to 50, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6754 and CDR1, CDR2 and CDR3 of MF5561, or variants thereof.

56. The multispecific antibody for use or method according to claim 55, wherein the multispecific antibody comprises SEQ ID NO. 20 and SEQ ID NO. 100, or a variant thereof.

57. The multispecific antibody for use or method according to any one of claims 48 to 50, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6785 and CDR1, CDR2 and CDR3 of MF5439, or variants thereof.

58. The multispecific antibody for use or method according to claim 57, wherein the multispecific antibody comprises SEQ ID NO. 31 and SEQ ID NO. 89, or a variant thereof.

59. The multispecific antibody for use or method according to any one of claims 48 to 50, wherein the multispecific antibody comprises CDR1, CDR2 and CDR3 of MF6795 and CDR1, CDR2 and CDR3 of MF5442, or variants thereof.

60. The multispecific antibody for use or method according to claim 59, wherein the multispecific antibody comprises SEQ ID NO. 9 and SEQ ID NO. 92, or a variant thereof.

61. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a common light chain variable domain having an amino acid sequence as shown in SEQ ID NO: 110, or a common light chain having an amino acid sequence as shown in SEQ ID NO: 109, or a variant thereof.

62. The multispecific antibody for use or method according to any of the preceding claims, wherein the antibody comprises a heavy chain constant domain 1 (CH1) having an amino acid sequence as shown in SEQ ID NO: 112, a heavy chain constant domain 2 (CH2) having an amino acid sequence as shown in SEQ ID NO: 114, a heavy chain constant domain 3 (CH3) having an amino acid sequence as shown in SEQ ID NO: 115, and a heavy chain constant domain 3 (CH3) having an amino acid sequence as shown in SEQ ID NO: 116, or variants thereof.

63. A pharmaceutical composition comprising 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, and instructions for use of the multispecific antibody in combination with a PD-1 or PD-L1 inhibitor.

64. The pharmaceutical composition of claim 61, further comprising instructions for use.

65. The pharmaceutical composition of claim 62, wherein the instructions for use include instructions for intravenous administration and / or dosage.

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