Binding molecules that modulate biological activity expressed by cell

Abstract

To provide means and methods of stimulating activity of a member of the TNF receptor superfamily on a cell, and to provide binding molecules such as antibodies that comprises at least two antigen binding sites, where a first antigen binding site can bind an extracellular part of said member and a second antigen binding site can bind an extracellular part of a second (different) membrane protein.SOLUTION: There is provided a method of stimulating activity of a member of the TNF receptor superfamily on a cell comprising providing a first cell and a second cell. The first cell has the member on the cell membrane and the second cell has a second membrane protein on the cell membrane, the method comprising contacting the cells with a bispecific antibody that comprises two variable domains. Here, one variable domain comprises a first antigen binding site that can bind an extracellular part of the member and another variable domain comprises a second antigen binding site that can bind an extracellular part of' the second membrane protein, thereby stimulating activity of the member on the first cell.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Despite significant advances in cancer treatment and increased knowledge of the molecular events that lead to cancer, cancer remains a leading cause of morbidity and mortality worldwide. It is the second leading cause of death worldwide. According to the World Health Organization, cancer was responsible for 8.8 million deaths in 2015. Globally, nearly one in six deaths is attributable to cancer. For example, colorectal cancer (CRC) is the third most common cancer worldwide. In 2008, 1.23 million people were diagnosed with the disease. It is the second most common cancer in Europe, with approximately 447,000 new cases diagnosed in 2012 (13% of the total). Colorectal cancer is the fourth leading cause of cancer death, estimated to be responsible for 608,000 deaths annually (148,000 in the EU). While several new treatments have advanced in CRC, many have failed clinical trials, and metastatic CRC remains largely incurable.

[0003] Traditionally, most cancer drug discovery has focused on agents that block essential cellular functions and kill dividing cells. However, in advanced cancers, no matter how aggressively applied, chemotherapy rarely results in a complete cure, even to the point where patients suffer life-threatening side effects from treatment. In most cases, a patient's tumor only stops growing or shrinks temporarily (called remission), then begins growing again, sometimes more rapidly (called relapse), making treatment increasingly difficult. Recently, the focus of cancer drug development has shifted away from broad-spectrum cytotoxic chemotherapy toward less toxic targeted cytostatic therapies. Treatments for advanced cancers have been clinically validated in leukemia and several other cancers. However, for the majority of carcinomas, targeted approaches have yet to be sufficiently effective to completely eradicate the disease in a large proportion of patients. Melanoma is another example of a highly prevalent cancer. If not detected early enough, the cancer can metastasize, making treatment extremely difficult. Immune interventional therapy has been shown to be effective in at least some patients with metastatic melanoma. Non-small cell lung cancer is a type of cancer that is rarely detected early enough for surgery, and these types of cancer have been successfully treated with immune interventional therapies.

[0004] Targeting cancer has been achieved using a variety of different methods, including, for example, small molecules directed against signaling proteins that cancers depend on for survival and / or growth, vaccines with tumor-specific proteins, cell therapies involving antibodies that target immune cells and cytotoxic molecules to tumors that actively kill tumor cells, and disrupting signal transduction and / or redirecting the host's immune system against tumor cells. Monoclonal antibodies that block the CTLA-4 or PD-1 axis have been shown to induce durable clinical responses in subsets of patients with melanoma, NSCLC, renal cell carcinoma, and urothelial carcinoma. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 157954 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides novel means and methods for redirecting immune system components. The present invention also relates to means and methods for modulating biological activities expressed by cells. [Means for solving the problem]

[0007] The present invention provides methods for stimulating the activity of a member of the TNF receptor superfamily on a cell, the method comprising providing first and second cells, wherein the first cell has the member on its cell membrane and the second cell has a second membrane protein on its cell membrane, and contacting the cells with a binding molecule comprising two antigen-binding sites, wherein the first antigen-binding site is capable of binding to the extracellular portion of the member (first membrane protein) and the second antigen-binding site is capable of binding to the extracellular portion of the second membrane protein, thereby stimulating the activity of the member on the first cell. In some embodiments, the method is an in vitro method. In some embodiments, the member of the TNF receptor superfamily is CD137 or OX40, preferably CD137. In some embodiments, the second membrane protein is not a member of the TNF receptor superfamily. In some embodiments, the second membrane protein is a member of the B7 family. In some embodiments, the second membrane protein is PD-L1.

[0008] In a preferred embodiment, the method further comprises providing a further binding molecule (second binding molecule) comprising an antigen binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and an antigen binding site capable of binding to the extracellular portion of a second membrane protein, wherein the first and second binding molecules are a distinct epitope on the first membrane protein, a different epitope on a second membrane protein, or A different epitope on a first membrane protein and a different epitope on a second membrane protein binds to The method further includes stimulating or enhancing activity of a member of the TNF receptor superfamily on the first cell by incubating the first cell and the second cell with the first and second binding molecules.

[0009] The present invention also provides binding molecules comprising an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily (a first membrane protein) and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein. The TNF receptor superfamily member is preferably CD137 or OX40, preferably CD137. In some embodiments, the second membrane protein is not a member of the TNF receptor superfamily. The second membrane protein is preferably a member of the B7 family. In some embodiments, the second membrane protein is PD-L1.

[0010] The present invention further provides a composition or kit of parts comprising one or more binding molecules comprising an antigen binding site capable of binding to the extracellular part of a member of the TNF receptor superfamily (a first membrane protein) and an antigen binding site capable of binding to the extracellular part of a second membrane protein. In a preferred embodiment, the present invention provides a composition or kit of parts comprising two or more such binding molecules, wherein at least two of the binding molecules are a distinct epitope on the first membrane protein, a different epitope on a second membrane protein, or A different epitope on a first membrane protein and a different epitope on a second membrane protein Preferably, the at least two binding molecules bind to the same epitope on a first membrane protein and to different epitopes on a second membrane protein.

[0011] The present invention also provides a method for stimulating the activity of CD137 or OX40 on a cell, the method comprising: providing a first cell and a second cell, wherein the first cell has CD137 or OX40 on its cell membrane and the second cell has a second membrane protein on its cell membrane; and contacting the first cell and the second cell with a binding molecule (first binding molecule) comprising an antigen-binding site capable of binding to the extracellular portion of CD137 or OX40 (the first membrane protein) and a binding molecule (first binding molecule) comprising an antigen-binding site capable of binding to the extracellular portion of the second membrane protein, the method further comprising incubating the first cell and the second cell with the first binding molecule, thereby stimulating the activity of CD137 or OX40 on the first cell. In some embodiments, the second membrane protein is not a member of the TNF receptor superfamily. In some embodiments, the method is an in vitro method.

[0012] In a preferred embodiment, the method further comprises providing a further binding molecule (second binding molecule) comprising an antigen binding site capable of binding to the extracellular portion of the first membrane protein and an antigen binding site capable of binding to the extracellular portion of the second membrane protein, wherein the first and second binding molecules are a distinct epitope on the first membrane protein, a different epitope on a second membrane protein, or A different epitope on a first membrane protein and a different epitope on a second membrane protein binds to The method further includes stimulating the activity of CD137 or OX40 on the first cell by incubating the first cell and the second cell with the first and second binding molecules.

[0013] In some embodiments, a binding molecule according to the invention comprises an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, the antigen-binding site of a binding molecule according to the invention consists of one antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a member of the B7 family. In some embodiments, a binding molecule according to the invention comprises an antigen-binding site capable of binding to the extracellular portion of CD137 and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, a binding molecule according to the invention comprises an antigen-binding site capable of binding to CD137 and an antigen-binding site capable of binding to PD-L1. In some embodiments, the antigen-binding site of a binding molecule according to the invention consists of one antigen-binding site capable of binding to CD137 and one antigen-binding site capable of binding to PD-L1. In some embodiments, a binding molecule according to the invention has no more than two antigen binding sites.

[0014] The binding molecules described herein are preferably antibodies.

[0015] The present invention further provides a method of stimulating the activity of a member of the TNF receptor superfamily on a cell, the method comprising providing a first cell and a second cell, wherein the first cell has the member (a first membrane protein) on its membrane and the second cell has a second membrane protein on its membrane, the method comprising contacting the cells with an antibody according to the invention comprising at least two variable domains, wherein one variable domain comprises a first antigen-binding site capable of binding to the extracellular portion of the first membrane protein and another variable domain comprises a second antigen-binding site capable of binding to the extracellular portion of the second membrane protein, thereby stimulating the activity of the member on the first cell. In some embodiments, the method is an in vitro method.

[0016] The present invention provides a variable domain capable of binding to the extracellular portion of a member of the TNF receptor superfamily (first membrane protein), and a variable domain capable of binding to the extracellular portion of a second membrane protein Further provided is an antibody or functional portion, derivative and / or analog thereof comprising: The first membrane protein is preferably CD137 or OX40, preferably CD137. In some embodiments, the second membrane protein is not a member of the TNF receptor superfamily.

[0017] The binding molecule is preferably a bispecific antibody. The present invention further provides a method for stimulating the activity of a member of the TNF receptor superfamily on a cell, comprising providing a first cell and a second cell, wherein the first cell has a member (first membrane protein) on its cell membrane and the second cell has a second membrane protein on its cell membrane, and the method comprises contacting the cells with a bispecific antibody comprising two variable domains, one variable domain comprising a first antigen-binding site capable of binding to the extracellular portion of the first membrane protein and another variable domain comprising a second antigen-binding site capable of binding to the extracellular portion of the second membrane protein, thereby stimulating the activity of the member on the first cell. In some embodiments, the method is an in vitro method. Also provided are bispecific antibodies comprising a variable domain having an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily (first membrane protein) and a variable domain having an antigen-binding site capable of binding to the extracellular portion of the second membrane protein. The first membrane protein is preferably CD137 or OX40, preferably CD137. The second membrane protein is preferably not a member of the TNF receptor superfamily.

[0018] In some embodiments, antibodies according to the invention comprise variable domains comprising an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, the antigen-binding site of an antibody according to the invention consists of one antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a member of the B7 family. In some embodiments, antibodies according to the invention comprise an antigen-binding site capable of binding to the extracellular portion of CD137 and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, antibodies according to the invention comprise an antigen-binding site capable of binding to CD137 and an antigen-binding site capable of binding to PD-L1. In some embodiments, the antigen-binding site of an antibody according to the invention consists of one antigen-binding site capable of binding to CD137 and one antigen-binding site capable of binding to PD-L1. In some embodiments, antibodies according to the invention have no more than two antigen-binding sites.

[0019] Further provided are pharmaceutical compositions comprising one or more binding molecules, preferably antibodies or variants thereof, of the invention.

[0020] Also provided is a nucleic acid molecule or collection of nucleic acid molecules encoding the heavy chain(s) or heavy chain variable region(s) of an antibody or variant thereof of the invention.

[0021] Also provided is a nucleic acid molecule or collection of nucleic acid molecules encoding the antibodies of the invention.

[0022] The antibody of the present invention preferably comprises a heavy chain variable region comprising the amino acid sequence of MF as shown in Figure 3. In a preferred embodiment, the antibody further comprises a light chain variable region comprising the amino acid sequence of the light chain variable region as shown in Figure 1. In a preferred embodiment, the light chain comprises the amino acid sequence as shown in Figure 1A. In a preferred embodiment, the heavy chain comprises the constant region of an IgG1 antibody, preferably a human IgG1 antibody. In a preferred embodiment, the CH2 region of the IgG1 constant region has been engineered to reduce ADCC and / or CDC activity of the antibody. In a preferred embodiment, the CH2 region comprises the sequence as shown in Figure 2E. In a preferred embodiment, the CH3 region of the antibody has been engineered to promote heterodimerization of the heavy chains. In a preferred embodiment, one heavy chain comprises the sequence as shown in Figure 2F, and another heavy chain comprises the sequence as shown in Figure 2G.

[0023] Also provided are cells comprising one or more nucleic acid molecules encoding, alone or together, an antibody or variant thereof of the invention. Also provided are methods for producing an antibody or variant thereof of the invention using the cells as described, preferably together with harvesting the antibody or variant thereof from culture of the cells.

[0024] Further provided is a cell line comprising an antibody of the invention or a variant thereof.

[0025] Also provided is a method for the treatment of an individual having a disease involving abnormal cells, such as cancer, or having a chronic infection by a virus or parasite, comprising administering to an individual in need thereof a binding molecule of the invention, preferably an antibody or variant thereof.

[0026] The present invention further provides a binding molecule of the invention, preferably an antibody or variant thereof, preferably a bispecific antibody of the invention or variant thereof, for use in treating an individual having a disease involving abnormal cells, such as cancer, or a chronic infection by a virus or parasite.

[0027] In a preferred embodiment, the parasite is an intracellular parasite.

[0028] Further provided is a method for stimulating an immune response in an individual against abnormal cells of the individual, comprising administering to the individual a binding molecule of the invention, preferably an antibody or variant thereof, preferably a bispecific antibody or variant thereof. The abnormal cells are preferably cancer cells, virus-infected cells, parasites or parasite-infected cells. In a preferred embodiment, the cells are cancer cells or neoplastic cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] The tumor necrosis factor receptor superfamily (TNFRSF) is a group of receptors typically characterized by their ability to bind tumor necrosis factor (TNF) via an extracellular cysteine-rich domain. With the exception of nerve growth factor (NGF), all TNFs are homologous to the prototypic TNF-α. In their active form, the majority of TNF receptors form trimeric complexes in the plasma membrane. Therefore, most TNF receptors contain a transmembrane domain (TMD) and are located on the plasma membrane. However, some can be cleaved into soluble forms (e.g., TNFR1) and lack the TMD entirely (e.g., DcR3). Antibodies of the present invention that bind to members of the TNF receptor superfamily bind to membrane-bound members of the superfamily. Members that exist only in membrane-independent forms are not within the scope of the present invention.

[0030] TNF receptors are involved in signal transduction into the cell upon binding of their ligands. Some receptors require specific adaptor proteins, such as TRADD, TRAF, RIP, and FADD, for downstream signal transduction. In the context of the present invention, various members of the TNF superfamily are preferred. These include tumor necrosis factor receptor 1, tumor necrosis factor receptor 2, lymphotoxin β receptor, OX40, CD40, Fas receptor, CD27, CD30, CD137, death receptor 3, death receptor 4, death receptor 5, death receptor 6, RANK, TROY, BAFF receptor, B-cell maturation antigen (BCMA), and transmembrane activator and calcium-modulating cyclophilin ligand-interacting protein (TACI).

[0031] Tumor necrosis factor receptor 1 is one of the primary receptors for tumor necrosis factor-α. This receptor has many alternative names, some of which are tumor necrosis factor receptor superfamily member 1A, TNFRSF1A, TNF-R1, TNF-RI, TNFR-I, TNFR1, TNFAR, P60, P55, tumor necrosis factor receptor 1A isoform beta, tumor necrosis factor binding protein 1, tumor necrosis factor receptor type 1, tumor necrosis factor receptor type I, tumor necrosis factor-α receptor, tumor necrosis factor receptor 1, CD120a antigen, TNFR1-D2, TNF-RI, TNF-R55, CD120a, TNFR55, TNFR60, TNF-R, P55-R, Tbp1, FPF, and MS5. The external Ids for tumor necrosis factor receptor 1 are HGNC:11916, Entrez Gene:7132, Ensembl:ENSG00000067182, OMIM:191190, and UniProtKB:P19438.

[0032] Tumor necrosis factor receptor 2 is a membrane receptor that binds tumor necrosis factor-alpha (TNFα). This receptor has many alternative names, some of which are tumor necrosis factor receptor superfamily member 1B, TNFRSF1B, tumor necrosis factor receptor type II, tumor necrosis factor receptor 2, p80 TNF-α receptor, TNF-RII, TNF-R2, TNFR2, TNFBR, p75, tumor necrosis factor binding protein 2, tumor necrosis factor beta receptor, p75 TNF receptor, CD120b antigen, etanercept, TNF-R-II, TNF-R75, p75TNFR, TNFR-II, CD120b, TNFR1B, TNFR80, and TBPII. The external Ids for tumor necrosis factor receptor 2 are HGNC:11917, Entrez Gene:7133, Ensembl:ENSG00000028137, OMIM:191191, and UniProtKB:P20333.

[0033] The lymphotoxin beta receptor is expressed on the surface of most cell types, including epithelial and myeloid cells, but is not normally expressed on normal T and B lymphocytes. This protein binds to the membrane form of lymphotoxin (a complex of lymphotoxin-α and lymphotoxin-β). The encoded protein and its ligand play a role in the development and organization of lymphoid tissues and transformed cells. Activation of this protein can potentially induce apoptosis. This protein is known by many names, including LTBR, ​​tumor necrosis factor receptor 2-related protein, tumor necrosis factor receptor type III, tumor necrosis factor C receptor, D12S370, TNFRSF3, TNFCR, TNFR3, lymphotoxin beta receptor (TNFR superfamily, member 3), lymphotoxin B receptor, LT-β-R, TNF-R-III, TNFR2-RP, TNF-RIII, TNFR-III, TNFR-RP, and CD18. The exoIds for lymphotoxin beta receptor are HGNC:6718, Entrez Gene:4055, Ensembl:ENSG00000111321, OMIM:600979, and UniProtKB:P36941.

[0034] Unlike CD28, OX40 is not constitutively expressed on resting naive T cells. OX40 is a secondary costimulatory immune checkpoint molecule that is expressed 24 to 72 hours after activation. Its ligand, OX40L, is also not expressed on resting antigen-presenting cells (APCs), but is expressed after their activation. OX40 expression is dependent on T cell activation. In the absence of CD28, OX40 expression is typically delayed and present at lower levels. This protein is known by numerous aliases, including TNFRSF4, tumor necrosis factor receptor superfamily member 4, TAX transcriptional activator glycoprotein 1 receptor, OX40L receptor, ACT35 antigen, CD134 antigen, TXGP1L; Tax transcriptional activator glycoprotein 1 receptor, lymphoid activation antigen ACT35, OX40 cell surface antigen, ATC35 antigen, OX40 antigen, ACT35, CD134, and IMD16. The external Ids for OX40 are HGNC:11918, Entrez Gene:7293, Ensembl:ENSG00000186827, OMIM:600315, and UniProtKB:P43489.

[0035] CD40 is a costimulatory protein found on antigen-presenting cells and is involved in their activation. Binding of CD154 (CD40L) to CD40 on T helper cells activates the antigen-presenting cell, inducing intracellular signaling via CD40 and inducing various downstream effects. This protein is known by several different names, including CD40 molecule, CD40 molecule, TNF receptor superfamily member 5, CD40L receptor, TNFRSF5, CDW40, Bp50, tumor necrosis factor receptor superfamily, member 5, B cell surface antigen CD40, B cell surface antigen CD40, B cell-associated molecule, CD40 antigen, and P50. The external Ids for CD40 are HGNC:11919, Entrez Gene:958, Ensembl:ENSG00000101017, OMIM:109535, and UniProtKB:P25942.

[0036] The Fas receptor is a cell death receptor on the surface of cells that leads to programmed cell death (apoptosis). It forms part of one of the more prominent apoptotic pathways. It is known by several alternative names, such as Fas cell surface death receptor, tumor necrosis factor receptor superfamily, member 6, apoptosis-mediating surface antigen FAS, TNF receptor superfamily member 6, FASLG receptor, CD95 antigen, TNFRSF6, APT1, FAS1, APO-1 cell surface antigen, apoptosis antigen 1, Apo-1 antigen, Fas AMA, ALPS1A, APO-1, FASTM, and CD95. The external IDs for FAS are HGNC:11920, Entrez Gene:355, Ensembl:ENSG00000026103, OMIM:134637, and UniProtKB:P25445.

[0037] CD27 is thought to be important for the development and long-term maintenance of T cell immunity. It binds to the ligand CD70 and plays a role in regulating B cell activation and immunoglobulin synthesis. CD27 transduces signals that lead to the activation of NF-κB and MAPK8 / JNK. CD27-associated protein (SIVA), an apoptosis protein, can bind to this receptor and is thought to play a role in apoptosis induced by this receptor. Alternative names for this protein include, among others, CD27 molecule, tumor necrosis factor receptor superfamily, member 7, T cell activation antigen CD27, CD27L receptor, TNFRSF7, T14, T cell activation antigen S152, CD27 antigen, s152, LPFS2, S152, and Tp55. The external Ids for CD27 are HGNC:11922, Entrez Gene:939, Ensembl:ENSG00000139193, OMIM:186711, and UniProtKB:P26842.

[0038] CD30 is expressed by activated T and B cells. TRAF2 and TRAF5 are thought to interact with this receptor and mediate signaling that leads to NF-κB activation. It is a positive regulator of apoptosis and has also been shown to limit the proliferative potential of autoreactive CD8 effector T cells, protecting the body from autoimmunity. It is known by several names, including TNFRSF8, tumor necrosis factor receptor superfamily member 8, lymphocyte activation antigen CD30, CD30L receptor, Ki-1 antigen, D1S166E, CD30, cytokine receptor CD30, CD30 antigen, and Ki-1. The external IDs for CD30 are HGNC:11923, Entrez Gene:943, Ensembl:ENSG00000120949, OMIM:153243, and UniProtKB:P28908.

[0039] CD137 can be expressed by activated T cells. It is also found on other cells, such as dendritic cells, natural killer cells, granulocytes, and cells of the vascular wall at sites of inflammation. This protein is known for its costimulatory activity for T cell activation. CD137 has been known by several names, including TNFRSF9, TNF receptor superfamily member 9, tumor necrosis factor receptor superfamily member 9, T cell antigen 4-1BB homolog, 4-1BB ligand receptor, T cell antigen ILA, CD137 antigen, CDw137, ILA, interleukin-activating receptor, homolog of mouse Ly63, induced lymphocyte activation (ILA), homolog of mouse 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, and UniProtKB:Q07011. CD137 is an inducible receptor that is most commonly upregulated on activated CD8+ T cells. CD137 signaling enhances T cell function by activating NF-κB [Arch et al., 1998]. Other immune cell types, including CD4+ T cells, monocytes, B cells, dendritic cell (DC) subpopulations, and granulocytes and NK cells, can express CD137 at various levels [Shao et al., 2011]. In monocytes, CD137 is inducible upon activation by lipopolysaccharide (LPS) and IL-1β. On B lymphocytes, CD137 expression is induced by antibodies against cell surface immunoglobulins and by transformation with EBV. On DCs, CD137 ligation induces their maturation through upregulation of B7 costimulatory molecules (CD80 and CD86), in addition to increasing the production of inflammatory cytokines (IL-6 and IL-12) and their survival [Makkouk et al., 2015]. The natural function of CD137 ligation on neutrophils is increased phagocytosis of bacterial and parasitic infections.Additionally, CD137 ligation blocks anti-apoptotic signals mediated by the IL-3 / IL-5 / GM-CSF receptor in neutrophils and eosinophils in vitro, thereby preventing granulocyte accumulation [Simon, 2001; Vinay et al., 2011]. In non-lymphoid cells, such as chondrocytes, endothelial cells, and tumor cells, CD137 expression is upregulated by cytokine stimulation, such as IL-1β for chondrocytes, the inflammatory cytokines TNFα / IFNγ / IL-1β for endothelial cells, and IFNγ for tumor cells. The stimulatory ligand for CD137 (CD137L) is expressed on activated antigen-presenting cells. CD137 exists in the membrane as both a monomer and a dimer [Pollok et al., 1993].

[0040] Death receptor 3 is expressed by activated and antigen-experienced T lymphocytes. This receptor is also expressed by FoxP3+ regulatory T lymphocytes. The receptor's ligand is TL1A (TNFSF15), which is upregulated in antigen-presenting cells and some endothelial cells after Toll-like receptor or Fc receptor activation. Various alternatively spliced ​​transcript variants encoding different isoforms have been reported, most of which are potentially secreted molecules. This receptor is thought to be involved in the regulation of lymphocyte proliferation induced by T cell activation. Activation is thought to depend on prior engagement of the T cell receptor. Ligand engagement increases T cell sensitivity to endogenous IL-2 via the IL-2 receptor, enhancing T cell proliferation. In vivo activation is likely specific to T cells encountering cognate antigen. At rest and in individuals without autoimmunity, the majority of T cells that regularly encounter cognate antigen are FoxP3+ regulatory T cells. Stimulation of death receptor 3 in the absence of any other exogenous signals stimulates the significant and specific proliferation of FoxP3+ regulatory (CD4+) T cells. Therapeutic agonists of death receptor 3 can be used to stimulate the proliferation of Treg cells, which can reduce inflammation in experimental models of asthma, allogeneic solid organ transplantation, and ocular keratitis. On the other hand, costimulation of the receptor with self- or vaccine-antigens can exacerbate immunopathology or enhance vaccine-stimulated immunity, respectively. Receptor stimulation is specific to T cell-mediated immunity, and it can be used to enhance or attenuate inflammation depending on the temporal context and quality of foreign versus self-antigen availability. Stimulation of TNFRSF25 in humans can produce effects similar to but more controllable from costimulatory blockade targeting molecules such as CTLA-4 and PD-1.Death receptor 3 is known by several other names, such as TNFRSF25, tumor necrosis factor receptor superfamily member 25, tumor necrosis factor receptor superfamily, member 12 (translocating chain-associated membrane protein), lymphocyte-associated receptor for cell death, apoptosis-mediating receptor TRAMP, apoptosis-mediating receptor DR3, apoptosis-inducing receptor AIR, protein WSL-1, TNFRSF12, APO-3, DDR3, LARD, DR3, apoptosis-inducing receptor, death receptor β, protein WSL, WSL-LR, TRAMP, WSL-1, APO3, WSL1, TR3, and WSL. The external IDs for death receptor 3 are HGNC:11910, Entrez Gene:8718, Ensembl:ENSG00000215788, OMIM:603366, and UniProtKB:Q93038.

[0041] Death receptor 4 is a cell surface receptor of the TNF receptor superfamily that is thought to bind TRAIL, transduce a cell death signal, and induce cell apoptosis. It is known by several names, including TNFRSF10A, tumor necrosis factor receptor superfamily member 10a, TNF-related apoptosis-inducing ligand receptor 1, death receptor 4, TRAIL receptor 1, TRAIL-R1, TRAILR1, APO2, DR4, tumor necrosis factor receptor superfamily member 10a variant 2, cytotoxic TRAIL receptor, CD261 antigen, TRAILR-1, and CD261. The external Ids for death receptor 4 are HGNC:11904, Entrez Gene:8797, Ensembl:ENSG00000104689, OMIM:603611, and UniProtKB:O00220.

[0042] Death receptor 5 (DR5) is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis. This receptor can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10 / TRAIL / APO-2L) and transduce the apoptotic signal. This receptor is known by several different names, including TNFRSF10B, tumor necrosis factor receptor superfamily member 10b, TNF-related apoptosis-inducing ligand receptor 2, death receptor 5, TRAIL-R2, TRAILR2, KILLER, TRICK2, ZTNFR9, DR5, p53-regulated DNA damage-inducible death receptor (killer), tumor necrosis factor receptor-like protein ZTNFR9, death domain containing receptor for TRAIL / Apo-2L, apoptosis-inducing protein TRICK2A / 2B, apoptosis-inducing receptor TRAIL-R2, TNF receptor superfamily member 10b, cytotoxic TRAIL receptor-2, Fas-like protein, TRAIL receptor 2, CD262 antigen, KILLER / DR5, TRICK2A, TRICK2B, TRICKB, and CD262. The external Ids for death receptor 5 are HGNC:11905, Entrez Gene:8795, Ensembl:ENSG00000120889, OMIM:603612, and UniProtKB:O14763.

[0043] Death receptor 6 can induce cell apoptosis upon activation. Knockout studies in mice suggest that this receptor plays a role in T helper cell activation and may be involved in inflammation and immune regulation. This receptor is also thought to be involved in Alzheimer's disease and neurodegeneration in the brain, triggering signaling in stress responses and cell survival. Overexpression induces apoptosis in expressing cells. APP (amyloid precursor protein) is the natural ligand for this receptor and is initially cleaved into Aβ and N-APP. N-APP is the fragment that interacts with DR6, causing axonal degradation in Alzheimer's patients. Death receptor 6 is also known by several names, including TNFRSF21, tumor necrosis factor receptor superfamily member 21, DR6, TNFR-related death receptor 6, CD358 antigen, BM-018, and CD358. The external Ids for death receptor 6 are HGNC:13469, Entrez Gene:27242, Ensembl:ENSG00000146072, OMIM:605732, UniProtKB:O75509.

[0044] RANK is a receptor for RANK-ligand (RANKL) and is part of the RANK / RANKL / OPG signaling pathway, which regulates osteoclast differentiation and activation. This pathway is involved in bone remodeling and repair, immune cell function, lymph node development, thermoregulation, and mammary gland development. Osteoprotegerin (OPG) is a decoy receptor for RANK and regulates stimulation of the RANK signaling pathway by competing with RANKL. The cytoplasmic domain of RANK transduces signals to downstream targets, such as NF-κB and JNK. RANK is expressed in skeletal muscle, thymus, liver, colon, small intestine, adrenal gland, osteoclasts, mammary epithelial cells, prostate, vascular cells, and pancreas. NF-κB activation is often mediated by RANKL, but overexpression of RANK alone can also activate the NF-κB pathway. RANK is known by several different names, such as TNFRSF11A, tumor necrosis factor receptor superfamily member 11a activator of NFKB, loss of heterozygosity 18, chromosome region 1, osteoclast differentiation factor receptor, receptor activator of NF-KB, Paget's disease of bone 2, ODFR, tumor necrosis factor receptor superfamily member 11a, activator of NFKB, tumor necrosis factor receptor superfamily member 11a, activator of NFKB, receptor activator of nuclear factor-kappa B, CD265 antigen, LOH18CR1, TRANCER, CD265, OPTB7, OSTS, PDB2, FEO, and OFE. External Ids for RANK are HGNC:11908, Entrez Gene:8792, Ensembl:ENSG00000141655, OMIM:603499, and UniProtKB:Q9Y6Q6.

[0045] The BAFF receptor is a membrane protein of the TNF receptor superfamily that recognizes BAFF. B-cell activating factor (BAFF) enhances B cell survival in vitro and is a regulator of peripheral B cell populations. The BAFF receptor is a type III transmembrane protein containing a single extracellular phenylalanine-rich domain. This receptor is thought to be the primary receptor required for BAFF-mediated mature B cell survival. BAFF is also bound by the TNF receptor B-cell maturation antigen (BCMA) and transmembrane activator, as well as calcium-regulated cyclophilin ligand-interacting protein (TACI). The BAFF receptor is known by several names, including TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, B-cell activating factor receptor, BLyS receptor 3, BAFF-R, BAFFR, B-cell activating factor receptor, CD268 antigen, prolixin, BROMIX, CD268, CVID4, and BR3. The external Ids for the BAFF receptor are HGNC:17755, Entrez Gene:115650, Ensembl:ENSG00000159958, OMIM:606269, and UniProtKB:Q96RJ3.

[0046] B-cell maturation antigen (BCMA) is a cell surface receptor of the TNF receptor superfamily that recognizes B-cell activating factor (BAFF). This receptor is preferentially expressed on mature B lymphocytes and is thought to be important for B-cell development and autoimmune responses. This receptor has been shown to specifically bind to tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B / TALL-1 / BAFF), leading to activation of NF-kappa B and MAPK8 / JNK. B-cell maturation antigen is also known by several names, including TNFRSF17, tumor necrosis factor receptor superfamily member 17, B-cell maturation protein, BCM, B-cell maturation factor, CD269 antigen, TNFRSF13A, and CD269. External Ids for BCMA are HGNC:11913, Entrez Gene:608, Ensembl:ENSG00000048462, OMIM:109545, and UniProtKB:Q02223.

[0047] Transmembrane activator and calcium-modulating cyclophilin ligand-interacting protein (TACI). The protein encoded by this gene is a lymphocyte-specific member of the tumor necrosis factor (TNF) receptor superfamily. It interacts with calcium-modulator and cyclophilin ligand (CAML). This protein can also bind to BAFF and APRIL (TNSF13 or CD256). TACI plays a role in humoral immunity by inducing activation of the transcription factors NFAT, AP1, and NF-κB and interacting with TNF ligands. TACI-deficient mice had increased splenic B cells and serum Ig, suggesting a potential negative regulatory role for TACI in B cell survival. However, a simpler explanation may be that the lack of TACI results in the availability of more circulating BAFF, which can bind to BR3 and increase the number of B cells. TACI-associated diseases include common variable immunodeficiency 2 and immunoglobulin a deficiency 2. The gene encoding TACI is located within the Smith-Maginnis syndrome region on chromosome 17. TACI is also known by many other names, including TNFRSF13B, tumor necrosis factor receptor superfamily member 13B, transmembrane activator and CAML interactor, tumor necrosis factor receptor 13B, CD267 antigen, TNFRSF14B, CD267, CVID2, IGAD2, CVID, and RYZN3. The external Ids for TACI are HGNC:18153, Entrez Gene:23495, Ensembl:ENSG00000240505, OMIM:604907, and UniProtKB:O14836.

[0048] TROY is expressed during embryonic development. It has been shown to activate the JNK signaling pathway when overexpressed in cells. Activation of this receptor can induce apoptosis. This receptor is thought to play a role in embryonic development. Alternatively, spliced ​​transcript variants encoding different isoforms have been described. TROY is known by several names, such as TNFRSF19, tumor necrosis factor receptor superfamily member 19, virulence and JNK inducer, TRADE, TAJ, and TAJ-alpha. The external IDs for TROY are HGNC:11915, Entrez Gene:55504, Ensembl:ENSG00000127863, OMIM:606122, and UniProtKB:Q9NS68.

[0049] The B7 family includes many structurally related cell surface proteins that bind to receptors on lymphocytes that regulate immune responses. Lymphocyte activation is initiated by cell surface, antigen-specific T cell receptor, or B cell receptor binding. Additional signals simultaneously delivered by B7 ligands further determine the immune response of these cells. These so-called "costimulatory" or "coinhibitory" signals are delivered by B7 family members via receptors of the CD28 family on lymphocytes. Binding of B7 family members to costimulatory receptors enhances immune responses, while binding to costimulatory receptors attenuates them. Currently, the following members are considered to be part of this family: B7.1 (CD80), B7.2 (CD86), inducible costimulator 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 effects of members on regulating immune responses have been demonstrated in mice with mutations in B7 family genes, resulting in the development of immunodeficiency and autoimmune diseases. Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmune, inflammatory, and cancer diseases.

[0050] Binding molecules or antibodies or variants thereof according to the invention that bind to the extracellular portion of a member of the TNF receptor superfamily and the extracellular portion of a member of the B7 family offer the advantage that the desired immune response may be particularly well promoted because B7 family members deliver "costimulatory" or "coinhibitory" signals to lymphocytes, thereby enhancing or attenuating the immune response. Thus, by targeting members of the B7 family, it is possible to enhance stimulatory signals and / or counteract inhibitory signals, thereby inducing or enhancing the desired immune response, for example against abnormal cells.

[0051] CD80 is a protein found on activated B cells and monocytes that provides costimulatory signals necessary for T cell activation and survival. It is a ligand for two distinct proteins on the surface of T cells: CD28 and CTLA-4. When bound to CD28, it is associated with costimulation, whereas binding to CTLA4 is associated with attenuation of the immune response. CD80 functions in conjunction with CD86 to activate T cells. CD80 has also been reported to bind to PD-L1. CD80 is known by several different names, including CD80 molecule, CD80 antigen, CD28 antigen ligand 1, B7-1 antigen, B lymphocyte activation antigen B7, CTLA-4 counter-receptor B7.1, activating B7-1 antigen, CD28LG1, CD28LG, LAB7, BB1, B7, costimulator CD80, CD80 antigen, and B7-1. The external Ids for CD80 are HGNC:1700, Entrez Gene:941, Ensembl:ENSG00000121594, OMIM:112203, and UniProtKB:P33681.

[0052] CD86 is a protein expressed on antigen-presenting cells. It can provide costimulatory signals for T cell activation and survival. It is a ligand for two different proteins on the surface of T cells: CD28 and CTLA-4. When bound to CD28, it is associated with costimulation, whereas binding to CTLA4 is associated with attenuation of the immune response. CD86 works in conjunction with CD80 to activate T cells. It is known by several different names, including CD86 molecule, CD86 antigen, CD28 antigen ligand 2, B7-2 antigen, CTLA-4 counter-receptor B7.2, CD28LG2, FUN-1, BU63, B70, B lymphocyte activation antigen B7-2, B lymphocyte antigen B7-2, activating B7-2 antigen, CD86 antigen, LAB72, and B7-2. The external Ids for CD86 are HGNC:1705, Entrez Gene:942, Ensembl:ENSG00000114013, OMIM:601020, and UniProtKB:P42081.

[0053] Inducible T cell costimulatory ligand (ICOSL or CD275) is constitutively expressed by APCs and several non-hematopoietic tissues. Expression can be downregulated during ongoing inflammation. ICOSL is now known to interact with ICOS, CD28, and CTLA-4 in humans. The ICOSL / CD28 interaction appears to costimulate primary human T cell responses to alloantigens and memory recall responses. ICOSL / CTLA-4 is thought to provide a co-inhibitory signal. ICOSL is also known as ICOSLG, B7-related protein 1, B7 homolog 2, B7-like protein GI50, B7 homolog 2, B7RP-1, B7-H2, B7RP1, B7H2, transmembrane protein B7-H2, ICOS ligand, CD275 antigen, KIAA0653, ICOS-L, LICOS, and GL50. The external Ids for ICOSL are HGNC:17087, Entrez Gene:23308, Ensembl:ENSG00000160223, OMIM:605717, and UniProtKB:O75144.

[0054] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during certain events, such as pregnancy, tissue allotransplantation, autoimmune diseases, and other disease states, such as hepatitis. PD-L1 is expressed in various types of cancer, particularly NSCLC (Boland et al., 2013; Velcheti et al., 2014), melanoma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, and various leukemias and multiple myeloma (Bernstein et al., 2014; Thompson et al., 2005). PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, although not all cancers or cells within tumors express PD-L1 (Dong et al., 2002). Several tumor microenvironment cells contribute to immune suppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because tumors protect themselves by inducing PD-L1 in response to IFN-γ produced by activated T cells (Sharma et al., 2017). PD-L1 can also be regulated by oncogenes, a mechanism known as intrinsic immune resistance (Akbay et al., 2013). Within the tumor microenvironment, PD-L1 is also expressed on myeloid cells and activated T cells (Tumeh et al., 2014). PD-L1 expression is induced by multiple proinflammatory molecules, including type I and type II IFN-γ, TNF-α, LPS, GM-CSF, and VEGF, as well as the cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer (Kondo et al., 2010; Sznol and Chen, 2013).

[0055] Binding of PD-L1 to PD-1 or B7.1 (CD80) transmits an inhibitory signal, which reduces the proliferation of PD-1 expressing T cells. PD-1 is thought to be able to control the accumulation of foreign antigen-specific T cells through apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in dampening the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins and is 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 cell 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.

[0056] PD-L2 is a second ligand for PD-1. PD-L2 binding 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 is upregulated on antigen-presenting cells by interferon gamma treatment. It is expressed in several normal tissues and various tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. This protein is known by several other names, including programmed cell death 1 ligand 2, B7 dendritic cell molecule, programmed cell 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.

[0057] Expression of B7-H3 (CD276) is increased in various malignancies and can distinguish between normal and tumor-derived circulating endothelial cells (Kraan et al., British Journal of Cancer (2014) 111, 149-156). Stimulation of this receptor induces the differentiation of human bone marrow stromal cells into osteoblasts (Xu et al., 2011; Immunobiology 216 (2011) 1311-1317). This protein contains four Ig-like domains in humans, while the mouse protein appears to have two such domains. This protein is thought to be the first identified ligand of triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2 or TREMML2). The latter protein binds to B7-H3 (4Ig-B7-H3) and costimulates CD8 T cell activation (Hofmeyer et al. 2009 PNAS 105;10277-10278). CD276 is widely expressed. It acts as a T cell costimulatory molecule. CD276 is also known by several other names, such as CD276 molecule, costimulatory molecule, CD276 antigen, B7 homolog 3, 4Ig-B7-H3, B7-H3, B7H3, and B7RP-2. The external IDs for CD276 are HGNC:19137, Entrez Gene:80381, Ensembl:ENSG00000103855, OMIM:605715, and UniProtKB:Q5ZPR3.

[0058] Although B7-H4 (VTCN1) mRNA appears to be widely expressed, only a small number of cells actively express the protein on their membranes. B7-H4 expression and binding to activated T cells inhibits T cell effector function through cell cycle arrest, reduced proliferation, and decreased IL-2 production. B7-H4 is upregulated on the surface of cancer cells and immunosuppressive tumor-associated macrophages (TAMs) in various human cancers. Signaling through the B7-H4 pathway results in the inhibition of TCR-mediated CD4+ and CD8+ T cell proliferation, cell cycle progression, and IL-2 production. B7-H4 is also known by several other names, such as V-Set domain-containing inhibitor of T-cell activation 1, immune costimulatory protein B7-H4, T-cell costimulatory molecule B7x, B7 superfamily member 1, B7 homolog 4, B7h.5, B7H4, T-cell costimulatory molecule B7x, B7 family member, H4, protein B7S1, PRO1291, VCTN1, B7S1, B7X, and H4 2. The external Ids for B7-H4 are HGNC:28873, Entrez Gene:79679, Ensembl:ENSG00000134258, OMIM:608162, and UniProtKB:Q7Z7D3.

[0059] B7-H5 (VISTA) is a 55-65 kDa member of the B7 family. It is a transmembrane molecule expressed in bone, on embryonic stem cells (ESCs), and on the surface of tumor cells. In tumor cells, this protein promotes MT1-MMP expression and activity and serves as a substrate for MT1-MMP, thereby increasing the potential for cell motility. This protein is also known by several names, including chromosome 10 open reading frame 54, V-Set domain-containing immunomodulatory receptor, V-domain Ig inhibitor of T cell activation, stress-induced secreted protein-1, Sisp-1, SISP1, stress-induced secreted protein 1, platelet receptor GI24, platelet receptor Gi24, cell death domain 1 alpha, DD1 alpha, B7H5, and GI24. The external Ids for this protein are HGNC:30085, Entrez Gene:64115, Ensembl:ENSG00000107738, OMIM:615608, and UniProtKB:Q9H7M9.

[0060] B7-H6 belongs to the B7 family (see MIM 605402) and is selectively expressed on tumor cells. Binding of B7-H6 to NKp30 (NCR3; MIM611550) leads to the activation and cytotoxicity of natural killer (NK) cells (Brandt et al. 2009 J Exp Med. 2009 Jul 6:206(7):1495-503). Natural killer (NK) cells are lymphocytes of the innate immune system involved in tumor elimination. B7-H6 is a tumor cell surface molecule that binds to NKp30, a human receptor that triggers the cytotoxicity and cytokine secretion of antitumor NK cells. Other names for B7-H6 are NCR3LG1, natural killer cell cytotoxicity receptor 3 ligand 1, B7 homolog 6, B7H6, putative Ig-like domain-containing protein DKFZp686O24166 / DKFZp686I21167, and DKFZp686O24166. External Ids for B7-H6 are HGNC:42400, Entrez Gene:374383, Ensembl:ENSG00000188211, OMIM:613714, and UniProtKB:Q68D85.

[0061] B7-H7 (HHLA2) protein has been detected in placental trophoblast cells and in the epithelium of the intestine, kidney, gallbladder, and breast, but not in most other organs. HHLA2 protein is widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, and esophagus. High HHLA2 expression is associated with regional lymph node metastasis and stage (Janakiram et al. Clin Cancer Res; 21(10):2359-66; May 15, 2015). TMIGD2 has been identified as one of the receptors for HHLA2. B7-H7 is known by several names, including HERV-H LTR-associated 2, human endogenous retrovirus-H long terminal repeat-associating protein 2, B7H7, and B7y. The external Ids for B7-H7 are HGNC:4905, Entrez Gene:11148, Ensembl:ENSG00000114455, OMIM:604371, and UniProtKB:Q9UM44.

[0062] Programmed Cell Death 1 (PD-1) is a cell surface receptor that belongs to the CD28 family of receptors and is expressed on T cells and pro-B cells. PD-1 is currently known to bind two ligands, PD-L1 and PD-L2. PD-1, functioning as an immune checkpoint, plays an important role in downregulating the immune system by inhibiting T cell activation, which further reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is thought to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is also known by several different names, including PDCD1, programmed cell death 1, systemic lupus erythematosus susceptibility 2, protein PD-1, HPD-1, PD1, programmed cell death 1 protein, CD279 antigen, CD279, HPD-L, HSLE1, SLEB2, and PD-1. The external Ids for PD-1 are HGNC:8760, Entrez Gene:5133, Ensembl:ENSG00000188389, OMIM:600244, and UniProtKB:Q15116. PD-1 inhibitors, a new class of drugs that block the activity of PD-1, activate the immune system to attack tumors and thus have been used successfully to treat several types of cancer.

[0063] CLEC12A also associates with C-type lectin domain family 12, member A, C-type lectin protein CLL-1, MICL, dendritic cell-associated lectin 2, C-type lectin superfamily, myeloid inhibitory C-type lectin-like receptor, C-type lectin-like molecule-1, CLL-1, DCAL2, CLL1, C-type lectin-like molecule 1, DCAL-2, killer cell lectin-like receptor subfamily L, member 1 (KLRL1), CD371 (Bakker A. et al. Cancer Res. 2004, 64, p8843 50; GenBank™ Accession Number: AY547296; Zhang W. et al. GenBank™ Accession Number: AF247788; AS Marshall, et al. J Biol Chem 2004, 279, p14792-802; GenBank™ Accession Number: AY498550; Y. Han et al. Blood 2004, 104, p2858 66; H. Floyd, et al. GenBank™ Accession Number: AY426759; C. H. Chen, et al. Blood 2006, 107, p1459 67). Ids; HGNC: 31713; Entrez Gene: 160364, Ensembl: ENSG00000172322, OMIM: 612088, UniProtKB: Q5QGZ9. CLEC12A is an antigen expressed on leukemic blast cells and leukemic stem cells in acute myeloid leukemia (AML), as well as on CD34-negative or CD34-low expressing leukemic stem cells (side populations) (AB Bakker et al. Cancer Res 2004, 64, p8443 50; Van Rhenen et al. 2007 Blood 110:2659; Moshaver et al. 2008 Stem Cells 26:3059). CLEC12A expression is otherwise thought to be restricted to the hematopoietic system, particularly to myeloid cells in peripheral blood and bone marrow, namely granulocytes, monocytes, and dendritic cell precursors. More importantly, CLEC12A is absent from hematopoietic stem cells.This expression profile makes CLEC12A a particularly preferred target in AML. The full-length form of CLEC12A contains 275 amino acid residues, including an additional intracellular stretch of 10 amino acids rarely present in other isoforms, and exhibits a strict myeloid expression profile (surface expression and mRNA level). The term "CLEC12A or its functional equivalent" refers to all of the above-mentioned variants (such as splices and mutations) and their isoforms that retain the strict myeloid expression profile (both surface expression and mRNA level) as described in Bakker et al. Cancer Res 2004, 64, pp. 8443-50 and Marshall 2004-J Biol Chem 279(15), pp. 14792-802. The CLEC12A-binding antibody of the present invention binds to human CLEC12A. In this specification, when CLEC12A is referred to, it refers to human CLEC12A unless otherwise specified.

[0064] "ErbB1" or "EGFR" is a member of a family of four receptor tyrosine kinases (RTKs), designated HerbB-1, -2, -3, and -4. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and one of which is involved in homodimerization and heterodimerization. Reference numbers used in this section refer to the numbering in the list entitled "References Cited herein." EGFR integrates extracellular signals from various ligands to produce diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to activation of Ras via the Grb2-binding Ras guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is much stronger in the presence of coexpression of Her3. EGFR is involved in several human epithelial malignancies, particularly cancers of the breast, bladder, non-small cell lung cancer, lung, colon, ovary, head and neck, and brain. Activating mutations in this gene, as well as overexpression of the receptor and its ligands, have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target for cancer therapy. Both small molecule inhibitors directed against the extracellular ligand-binding domain of RTKs and monoclonal antibodies (mAbs) have been developed, which have shown some clinical success, although so far, mostly in select patient populations. The database accession number for the human EGFR protein and its encoding gene is (GenBank NM_005228.3).This accession number was provided primarily to provide further methods for identifying EGFR proteins as targets, and the actual sequence of the EGFR protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. References to EGFR herein refer to human EGFR unless otherwise specified. The antigen-binding site that binds to EGFR binds to various mutants of EGFR, including those expressed in some EGFR-positive tumors.

[0065] As used herein, "ErbB-2" or "HER2" refers to the protein encoded by the ERBB-2 gene in humans. Alternative names for this gene or protein include CD340, HER-2, HER-2 / neu, MLN 19, NEU, NGL, and TKR1. The ERBB-2 gene is often referred to as HER2 (human epidermal growth factor receptor 2). When ErbB-2 is referred to herein, this reference refers to human ErbB-2. Antibodies comprising an antigen-binding site that binds to ErbB-2 bind to human ErbB-2. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, ErbB-2 antigen-binding sites may also bind to such orthologs, although this is not necessarily the case. The database accession numbers for the human ErbB-2 protein and the gene encoding it are (NP_001005862.1, NP_004439.2, NC_000017.10, NT_010783.15, NC_018928.2). These accession numbers were provided primarily to provide additional methods for identifying ErbB-2 as a target; the actual sequence of the ErbB-2 protein bound by an antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. The ErbB-2 antigen-binding site binds to ErbB-2 and various variants thereof, such as those expressed by some ErbB-2-positive tumor cells.

[0066] As used herein, "ErbB-3" or "HER3" refers to the protein encoded by the ERBB-3 gene in humans. Alternative names for the gene or protein are HER3, LCCS2, MDA-BF-1, c-ErbB-3, c-erbb-3, erbb-3-S, p180-Erbb-3, p45-sErbb-3, and p85-sErbb-3. When ErbB-3 is referred to herein, this reference refers to human ErbB-3. An antibody comprising an antigen-binding site that binds to ErbB-3 binds to human ErbB-3. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, the ErbB-3 antigen-binding site may, but need not, also bind to such orthologs. The database accession numbers for the human ErbB-3 protein and the gene encoding it are (NP_001005915.1, NP_001973.2, NC_000012.11, NC_0118923.2, NT_0029419.12). These accession numbers were provided primarily to provide further methods for identifying ErbB-3 as a target; the actual sequence of the ErbB-3 protein bound by an antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. The ErbB-3 antigen-binding site binds to ErbB-3 and various variants thereof, such as those expressed by some ErbB-2-positive tumor cells.

[0067] LGR4 is leucine-rich repeat-containing G protein-coupled receptor 4. Alternative names for this gene or protein are GPR48, G protein-coupled receptor 48, BNMD17, leucine-rich repeat-containing G protein-coupled receptor 4, leucine-rich repeat-containing G protein-coupled receptor 4, G protein-coupled receptor 48.

[0068] The proteins or antibodies of the present invention that bind to LGR4 bind to human LGR4. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, the LGR4-binding proteins or antibodies of the present invention also bind to such orthologs, although this is not necessarily the case. Database accession numbers for the human LGR4 protein and its encoding gene are (NC_00000011.10, NC_018922.2, NT_009237.19, NP_060960.2). These accession numbers are provided primarily to provide additional methods for identifying LGR4 as a target; the actual sequence of the bound LGR4 protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The LGR4 antigen-binding site binds to LGR4 and various variants thereof, such as those expressed by some LGR4-positive tumor cells.

[0069] LGR5 is leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for this gene or protein are leucine-rich repeat-containing G protein-coupled receptor 5, leucine-rich repeat-containing G protein-coupled receptor 5, G protein-coupled receptor HG38, G protein-coupled receptor 49, G protein-coupled receptor 67, GPR67, GPR49, orphan G protein-coupled receptor HG38, G protein-coupled receptor 49, GPR49, Hg38, and FEX.

[0070] The proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, the LGR5-binding proteins or antibodies of the present invention also bind to such orthologs, although this is not necessarily the case. Database accession numbers for the human LGR5 protein and its encoding gene are (NC_000012.12, NT_029419.13, NC_018923.2, NP_001264155.1, NP_001264156.1, NP_003658.1). These accession numbers are provided primarily to provide additional methods for identifying LGR5 as a target; the actual sequence of the bound LGR5 protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The LGR5 antigen-binding site binds to LGR5 and various variants thereof, such as those expressed by some LGR5-positive tumor cells.

[0071] ZNRF3 is zinc and ring finger 3. Alternative names for this gene or protein are zinc and ring finger 3, zinc / ring finger protein 3, ring finger protein 203, KIAA1133, RNF203, novel C3HC4-type zinc finger (ring finger), E3 ubiquitin-protein ligase ZNRF3, CTA-292E10.6, EC 6.3.2., and BK747E2.3 3.

[0072] The protein or antibody of the present invention that binds to ZNRF3 binds to human ZNRF3. Due to sequence and tertiary structure similarity between human and other mammalian orthologs, the ZNRF3-binding protein or antibody of the present invention also binds to such orthologs, but this is not necessarily the case. Database accession numbers for the human ZNRF3 protein and its encoding gene are (NC_000022.11, NT_0011520.13, NC_018933.2, NP_001193927.1, NP_115549.2). These accession numbers are provided primarily to provide additional methods for identifying ZNRF3 as a target; the actual sequence of the bound ZNRF3 protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The ZNRF3 antigen-binding site binds to ZNRF3 and various of its variants, such as those expressed by some ZNRF3-positive tumor cells.

[0073] RNF43 is ring finger protein 43. Alternative names for this gene or protein are ring finger protein 43, RNF124, E3 ubiquitin-protein ligase RNF43, ring finger protein 43, EC 6.3.2., URCC.

[0074] The protein or antibody of the present invention that binds to RNF43 binds to human RNF43. Due to sequence and tertiary structure similarity between human and other mammalian orthologs, the RNF43-binding protein or antibody of the present invention also binds to such orthologs, although this is not necessarily the case. Database accession numbers for the human RNF43 protein and its encoding gene are (NC_000017.11, NT_010783.16, NC_018928.2, NP_001292473.1, NP_001292474.1, NP_060233.3). These accession numbers are provided primarily to provide additional methods for identifying RNF43 as a target; the actual sequence of the bound RNF43 protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The RNF43 antigen-binding site binds to RNF43 and various variants thereof, such as those expressed by some RNF43-positive tumor cells.

[0075] Reference to the sequence identifier identifies which protein is targeted. Binding molecules, such as antibodies of the present invention, also recognize at least some variants, such as allelic variants, splice variants, and mutants thereof, as long as the epitope recognized by each variable domain of the antibody is unaffected. Some alternative names may or may not be used to refer to other proteins. Names are provided for reference purposes only. Binding molecules, such as antibodies of the present invention, bind to proteins when expressed on cells. They can also bind to variants of the protein as long as the epitope to which the binding molecule binds is available. Thus, splice variants or mutant proteins (if present) will also be bound as long as the epitope is available. The fact that a binding molecule binds to a designated protein means that it can bind to the protein as a property, but does not mean that the binding molecule actually binds to the target, even if the binding molecule does. Nor does it mean that the antibody does not bind to other proteins. Such cross-reactivity is not currently known for binding molecules, such as antibodies of the present invention. However, it is not expressly excluded that such cross-reactivity may exist.

[0076] The present invention discloses binding molecules capable of binding to the extracellular portion of a member of the TNF receptor superfamily (first membrane protein) and to the extracellular portion of a second membrane protein. The second membrane protein is preferably not a member of the TNF receptor superfamily. Such binding molecules are also referred to as "binding molecules of the invention." The binding molecule is preferably a binding protein. In a preferred embodiment, the binding molecule is an antibody or variant thereof, preferably a bispecific antibody or variant thereof. Compositions and kits-of-parts comprising two or more of the binding molecules described herein are also provided.

[0077] Binding molecules of the present invention are preferably antibodies (or variants thereof as described elsewhere in this application), antibody mimetics, polypeptides, aptamers, or combinations thereof. These proteins or aptamers typically bind to one target. Binding molecules of the present invention bind to two or more targets. Binding molecules preferably bind to two targets. Variants of antibodies or bispecific antibodies maintain this aspect. A binding protein or aptamer has a binding site (antigen-binding site) to which a target binds. A binding protein or aptamer preferably contains two or more domains with target (antigen) binding sites, preferably one binding site per domain. Such domains are preferably antibody variable domains or variants thereof. Antibody variable domains have been the subject of much research. Many variants have been created that resemble variable domains or portions thereof that retain the binding specificity of the normal variable domain. Non-limiting examples of such variants are described elsewhere herein.

[0078] It should be understood that any combination of these antibodies, antibody mimics, polypeptides, and aptamers can be linked together by methods known in the art. For example, in some embodiments, the binding molecules of the present invention are conjugates or fusion proteins. Regarding antibodies, the technology for producing multispecific antibodies has progressed to include bispecific antibodies, which have the same overall structure as normal monospecific antibodies, but each of the two arms of the antibody binds to a different target.

[0079] Antibody mimetics are polypeptides that, like antibodies, can specifically bind to antigens but are structurally unrelated to antibodies. Antibody mimetics are typically artificial peptides or proteins with molar masses of approximately 3-20 kDa. Their common advantages over antibodies include better solubility, tissue penetration, heat and enzymatic stability, and relatively low production costs. Non-limiting examples of antibody mimetics are affibody molecules (typically based on the Z domain of protein A), affilins (typically based on gamma-B crystalloid or ubiquitin), affimers (typically based on cystatins), affitins (typically based on Sac7d from Sulfolobus acidocaldarius), alphabodies (typically based on triple-helical coiled-coils), anticalins (typically based on lipocalins), avimers (typically based on the A domains of various membrane receptors), DARPins (typically based on ankyrin repeat motifs), fynomers (typically based on the SH3 domain of Fyn7), kunitz domain peptides (typically based on the Kunitz domains of various protease inhibitors), and monobodies (typically based on the type III domain of fibronectin).

[0080] Monobodies are synthetic binding proteins constructed using the fibronectin type III domain (FN3) as a molecular scaffold. Monobodies are a simple and robust alternative to antibodies for generating target-binding proteins. The term "monobody" was coined in 1998 by Koide's group, who published the first paper demonstrating the monobody concept using the tenth Fn3 domain of human fibronectin.

[0081] Monobodies and other antibody mimetics are typically generated from combinatorial libraries in which portions of the scaffold are diversified using directed evolution techniques such as molecular display and phage display, mRNA display, and yeast surface display. Many antibody mimetics have high affinity and specificity for their respective targets.

[0082] Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are usually generated by selection from a large pool of random sequences, although natural aptamers also exist in riboswitches. As macromolecules, aptamers can be used for both basic research and clinical purposes.

[0083] As used herein, the term "conjugate" refers to two or more molecules covalently linked, optionally by a linking region. For example, in some embodiments, a conjugate is a first protein or non-protein moiety linked to a second protein or non-protein moiety by a linking region. For example, in some embodiments of a binding molecule of the invention, it comprises or consists of two or more covalently linked antibodies. A conjugate is not limited to a first and second moiety, but in some embodiments may have a third, fourth, or more moieties linked by additional linking regions. As described elsewhere in this application, examples of protein moieties include, but are not limited to, polypeptides, peptidomimetics, or antibodies (or antibody moieties, derivatives, or analogs, as described elsewhere in this application). Examples of non-protein moieties include, but are not limited to, aptamers. Many types of linkers can be used, and the linker is selected as appropriate depending on the type of molecule in the conjugate and the desired properties of the linker (length, flexibility, resistance to protease activity, and other similar properties). Such linkers may comprise nucleotides, polypeptides, or suitable synthetic materials. For example, the linker may be a flexible peptide linker. In certain embodiments, the linker may be a cleavable linker, allowing the moieties of the conjugate to separate from each other. In other embodiments, the peptide linker may be a helical linker. Various examples and kits for linking proteins and other molecules are known in the art. As used herein, the term "fusion protein" refers to a protein comprising two or more polypeptides or proteins recombinantly linked at the DNA level and expressed together as a single polypeptide. The fusion protein may also include a peptide linking region encoded by DNA and co-expressed with the fusion protein. The peptide linker that is part of the fusion protein may be designed to have specific properties, such as flexibility, hydrophilicity, protease resistance, cleavability, etc.All of these characteristics can be designed into the DNA sequence, and methods for designing linkers are well known in the art. For example, antibodies can be linked together by methods well known in the art to form bispecific or multitargeting antibodies, as described herein. Furthermore, bispecific antibodies can be constructed by various methods known in the art, for example, by using technologies such as Biclonics® (see, for example, WO 2013 / 157954). Bispecific monoclonal antibodies (BsMAbs, BsAbs) typically contain the binding domains of two different monoclonal antibodies and, as a result, bind to two different epitopes. Biclonics® molecules, as well as other full-length IgG bispecific antibodies, have two different antigen-binding specificities encoded by two different variable regions of the Fab of a full-length IgG molecule (scFv). Biclonics® can be generated by co-transfection of individual cells with genetic constructs encoding two different common light chain (cLC) antibodies, as detailed elsewhere herein. CH3 engineering ensures efficient heterodimerization and formation of essentially pure bispecific antibodies.

[0084] The binding molecule of the present invention is preferably an antibody or a variant thereof. The binding molecule of the present invention is preferably a bispecific antibody or a variant thereof.

[0085] Antibodies typically bind to their targets via so-called antigen-binding sites. The unmodified antigen-binding site is typically formed by and present in the variable domain of an antibody. The variable domain contains the antigen-binding site. The variable domain that binds to the antigen is the variable domain that contains the antigen-binding site that binds to the antigen.

[0086] In one embodiment, an antibody variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). The antigen-binding site may be present in the combined VH / VL variable domains, or in the VH region alone, or in the VL region alone. When the antigen-binding site is present in one of the two regions of the variable domain, the corresponding variable region may contribute to folding and / or stability of the combined variable region, but does not contribute significantly to binding of the antigen itself.

[0087] As used herein, antigen binding refers to the typical binding ability of antibody to its antigen.The binding of antibody to antigen can be evaluated by various methods.One method is to incubate antibody with antigen (preferably the cell that expresses antigen), remove unbound antibody (preferably by washing step), and detect bound antibody by labeled antibody that binds to bound antibody.

[0088] Antigen binding by an antibody is typically mediated through the antibody's complementarity-determining regions (CDRs) and the specific three-dimensional structure of both the antigen and variable domains, which allows these two structures to bind together precisely (an interaction similar to a lock and key) as opposed to the random, nonspecific adhesion of proteins. Antibodies typically recognize a part of an antigen called its epitope; such epitopes may be present in other compounds as well, and antibodies according to the invention may recognize other proteins as well, if such other compounds contain the same epitope. Thus, the term "binding" does not exclude binding of the antibody to another protein or proteins containing the same epitope. Such other protein(s) are preferably not human proteins.

[0089] A protein of the invention, such as an antibody, typically does not bind to other proteins other than the specific target protein on cell membranes after birth, preferably in adult humans.

[0090] As used herein, the term "antibody" preferably refers to a protein molecule belonging to the immunoglobulin class of proteins containing one or more variable domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable domain of an antibody. Therapeutic antibodies are preferably as close as possible to the natural antibody of the subject to be treated (e.g., a human antibody for a human subject). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. Preferably, the affinity of the separate arms of the antibody according to the invention is in the nanomolar range. Antibodies, such as bispecific antibodies of the present invention, typically comprise the constant domain (Fc portion) of a natural antibody, which may be genetically engineered as described elsewhere herein, e.g., to reduce ADCC and / or CDC activity. Antibodies of the present invention are typically bispecific full-length antibodies, preferably of the human IgG subclass.

[0091] The terms "variable domain", "VH / VL pair", and "VH / VL" are used interchangeably herein. A variable domain is composed of a heavy chain variable region and a light chain variable region. The heavy chain variable region is typically formed by a rearranged VDJ region. The light chain variable region is typically formed by a rearranged VJ region. VDJ / VJ regions here can be artificially generated, for example, using the available sequence information of numerous functional antibodies.

[0092] In some embodiments, a binding molecule or antibody or variant according to the invention comprises an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, a binding molecule or antibody or variant according to the invention comprises an antigen-binding site capable of binding to the extracellular portion of CD137 and an antigen-binding site capable of binding to a member of the B7 family. In some embodiments, a binding molecule or antibody or variant according to the invention comprises an antigen-binding site capable of binding to CD137 and an antigen-binding site capable of binding to PD-L1.

[0093] In some embodiments, a binding molecule, antibody, or variant according to the invention has no more than two antigen-binding sites. This means that the antigen-binding portion of such a binding molecule, antibody, or variant consists of two antigen-binding sites without the presence of an additional antigen-binding site. Each of the two antigen-binding sites preferably contains an immunoglobulin VH / VL pair. Preferably, the antigen-binding portion of a binding molecule, antibody, or variant according to the invention consists of one immunoglobulin variable domain capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to a second membrane protein. A particularly preferred embodiment is an immunoglobulin having an IgG format, which offers the advantage that the half-life of bivalent binding molecules / antibodies / variants according to the invention is typically longer compared to multivalent compounds. Furthermore, the immunogenicity of bivalent binding molecules according to the invention is typically lower compared to multivalent compounds. Molecules / antibodies / variants according to these embodiments preferably maintain the structure of native IgG and thus all the benefits associated with the structure of native IgG.

[0094] As used herein, the term "multivalent" encompasses three or more specificities, which are present, for example, in trivalent and tetravalent binding molecules.

[0095] Some embodiments provide a binding molecule or antibody or variant according to the invention, wherein the antigen-binding site of the binding molecule or antibody or variant consists of one antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a member of the B7 family. In some embodiments, the antigen-binding site of a binding molecule or antibody or variant according to the invention consists of one antigen-binding site capable of binding to the extracellular portion of CD137 and one antigen-binding site capable of binding to a member of the B7 family. In some embodiments, the antigen-binding site of a binding molecule or antibody or variant according to the invention consists of one antigen-binding site capable of binding to CD137 and one antigen-binding site capable of binding to PD-L1.

[0096] As used herein, the term "antigen-binding site" refers to the site of a binding molecule or antibody that specifically binds to an epitope of an antigen. Such antigen-binding sites are preferably derived from or share sequence homology with the variable domains of antibodies, particularly the CDR regions thereof. In some preferred embodiments, the antigen-binding site is an immunoglobulin variable domain formed by an immunoglobulin VH / VL pair. In other embodiments, the antigen-binding site is derived from an antibody mimetic, such as an affibody molecule, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, fynomer, kunitz domain peptide, or monobody, e.g., as previously described herein.

[0097] The antibodies of the present invention are preferably "full-length" antibodies. The term "full-length" according to the present invention is defined to include essentially complete antibodies, free of one or more artificially added moieties having a size greater than 20 amino acid residues, such as additional antigen-binding sites, additional activation sites, additional ligands, or additional ligand-binding moieties. However, full-length antibodies do not necessarily possess all the functions of intact antibodies. For the avoidance of doubt, full-length antibodies comprise two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be broken down into domains designated CH1, CH2, CH3, VH for the heavy chain, and CL and VL for the light chain. The heavy chain domains are preferably present in the order of natural antibodies (VH-CH1-CH2-CH3; i.e., the VH domain is adjacent to the CH1 domain, followed by the CH2 domain, which is then followed by the CH3 domain). The light chain domains are also preferably present in the order of natural antibodies (VL-CL; i.e., the VL domain is adjacent to the CL domain). Antibodies bind to antigens via the variable domains contained in the Fab fragment. Antibodies can interact with molecules and cells of the immune system via the constant domains, mostly through the Fc portion. In some embodiments, the antibodies of the present invention are IgG, preferably full-length IgG. Full-length IgG antibodies are preferred due to their typically favorable half-life and the desire to remain close to the intact autologous (human) molecule for immunogenicity reasons. In some embodiments, the antibodies of the present invention are full-length IgG1, full-length IgG2, full-length IgG3, or full-length IgG4 antibodies.

[0098] Full-length antibodies according to the present invention encompass antibodies in which mutations may be present that provide desired properties or that merely substitute for those in the original chain. Such mutations should not result in the deletion of a substantial portion of any of the regions. However, antibodies in which one or several amino acid residues have been inserted, deleted, substituted, or a combination thereof without essentially altering the antigen-binding properties of the resulting antibody are encompassed within the term "full-length antibody." For example, an IgG antibody can have 1 to 20 amino acid residue insertions, substitutions, deletions, or a combination thereof in the constant region.

[0099] The antibody of the present invention, or a functional part, derivative, and / or analog thereof, is preferably a bispecific antibody, or a functional part, derivative, and / or analog thereof. In a preferred embodiment, it is a bispecific IgG antibody with reduced effector function. In a preferred embodiment of the present invention, the antibody of the present invention is a bispecific full-length antibody. The antibody of the present invention is preferably a bispecific full-length IgG antibody mutated, preferably in the CH2 / lower hinge region, to reduce effector function. IgG1 mutated in the CH2 / lower hinge region to reduce effector function is advantageous due to its long circulating half-life in humans. To prevent any immunogenicity in humans, the bispecific antibody according to the present invention is preferably a human antibody.

[0100] The term "bispecific" (bs) means that one portion of an antibody (as defined above) binds to one epitope on an antigen, while the second portion binds to a different epitope, either on the same antigen or on a different antigen. The different epitopes typically reside on different antigens. However, the different epitopes may also reside on the same antigen. According to the present invention, the first and second antigens are actually two different proteins. A preferred bispecific antibody is an antibody comprising portions of two different monoclonal antibodies, and is thus capable of binding to two different epitopes, preferably two different antigens. Depending on the expression levels, (sub)cellular localization, and stoichiometry of the two antigens recognized by the bispecific antibody, both Fab arms of the antibody may or may not simultaneously bind to their epitopes. One arm of a bispecific antibody typically contains the variable domain of one antibody, and the other arm contains the variable domain of another antibody (i.e., one arm of the bispecific antibody is formed by one heavy chain paired with one light chain, while the other arm is formed by a different heavy chain paired with a light chain). The heavy chain variable regions of bispecific antibodies of the invention are typically different from each other, whereas the light chain variable regions are preferably the same in bispecific antibodies of the invention. Bispecific antibodies in which different heavy chain variable regions are associated with the same or common light chain variable region are also referred to as bispecific antibodies with a common light chain variable region (cLcv). The light chain constant region is preferably also the same. Such bispecific antibodies are referred to as having a common light chain (cLc). Thus, there is further provided a bispecific antibody according to the invention, in which both arms comprise a common light chain.

[0101] The bispecific antibodies described herein preferably comprise a common light chain variable domain, preferably a common light chain. The term "common light chain" according to the present invention refers to a light chain that may be identical or may have some amino acid sequence differences, while the binding specificity of the full-length antibody is not affected. For example, 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, it is possible to prepare or find a non-identical but still functionally equivalent light chain within the definition of a common light chain used herein. The terms "common light chain," "common LC," "cLC," and "single light chain" are all used interchangeably herein, with or without the addition of the term "rearrangement." The terms "common light chain variable region," "common VL," "common LCv," "cLCv," and "single VL" are all used interchangeably herein, with or without the addition of the term "rearrangement." A preferred embodiment of the present invention is that the bispecific antibody has a common light chain (variable region) that can bind to at least two, preferably multiple, heavy chains (variable regions) of different binding specificities to form an antibody having a functional antigen-binding domain (WO 2004 / 009618, WO 2009 / 157771). The common light chain (variable region) is preferably a human light chain (variable region). The common light chain (variable region) preferably has a germline sequence. Preferred germline sequences are light chain variable regions that are frequently used in the human repertoire and have good thermodynamic stability, yield, and solubility. A preferred germline light chain is O12. The common light chain is preferably a rearranged germline human kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 (FIG. 1A). The common light chain variable region is preferably a rearranged germline human kappa light chain IgVκ1-39. * 01 / IGJκ1 *The common light chain preferably comprises a light chain variable region as shown in Figure 1B or 1D, with 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. The common light chain preferably further comprises a light chain constant region, preferably a kappa light chain constant region. The nucleic acid encoding the common light chain may be codon-optimized for the cell system used to express the common light chain protein. The encoding nucleic acid may deviate from the germline nucleic acid sequence.

[0102] In a preferred embodiment, the light chain comprises an O12 / IgVκ1-39 antibody as shown in FIG. 1A, having 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. * The phrase "O12 light chain" refers to an O12 / IgVκ1-39 light chain having 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or a combination thereof, as shown in FIG. 1A. * IgVκ1-39 will be used throughout this specification as shorthand for "a light chain comprising a light chain variable region comprising the amino acid sequence of the O1 gene segment." IgVκ1-39 is shorthand for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39, O12a, or O12. The external Ids for this genetic element are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. A preferred amino acid sequence of IgVκ1-39 is shown in Figure 1E, which lists the sequence of the V region. The V region can be combined with one of five J regions. Figures 1B and 1D describe two preferred sequences for IgVκ1-39 combined with a J region. The combined sequences are designated as IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 *01 (Worldwide Web nomenclature of the IMGT database at imgt.org).

[0103] O12 / IgVκ1-39 containing the light chain variable region * Preferably, O1 is a germline sequence. * 01 or / IGJκ5 * It is further preferred that 01 is a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0104] In a preferred embodiment, the light chain variable region is germline O12 / IgVκ1-39 * 01. In a preferred embodiment, the light chain variable region comprises the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. In a preferred embodiment, IgVκ1-39 * 01 / IGJκ1 * 01. The light chain variable region is the germline kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or germline kappa light chain IgVκ1-39 * 01 / IGJκ5 * 01, preferably germline IgVκ1-39 * 01 / IGJκ1 * Includes 01.

[0105] Mature B cells producing antibodies with O12 light chains often produce light chains that have undergone one or more mutations relative to the germline sequence, i.e., the normal sequence in nonlymphoid cells of an organism. The process involved in these mutations is often referred to as somatic (hyper)mutation. The resulting light chain is referred to as an affinity-matured light chain. When such a light chain is derived from the O12 germline sequence, it is an O12-derived light chain. As used herein, the term "O12 light chain" includes O12-derived light chains. Mutations introduced by somatic hypermutation can, of course, also be artificially introduced in the laboratory. Other mutations can also be introduced in the laboratory in a homogeneous manner, regardless of amount, without affecting the properties of the light chain. A light chain is at least an O12 light chain if it comprises the sequence shown in Figure 1A, Figure 1B; Figure 1D, or Figure 1E, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising the sequence shown in Figure 1A; Figure 1B; Figure 1D or Figure 1E with 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising the sequence shown in Figure 1A; Figure 1B; Figure 1D or Figure 1E with 0 to 5, preferably 0 to 4, and more preferably 0 to 3 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising the sequence shown in Figure 1A; Figure 1B; Figure 1D or Figure 1E with 0 to 2, more preferably 0 to 1, and most preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising the sequence shown in Figure 1A or Figure 1B with the aforementioned amino acid insertions, deletions, substitutions, additions, or combinations thereof. In a preferred embodiment, the light chain comprises the sequence of Figure 1A. In a preferred embodiment, the light chain variable region comprises the sequence of Figure 1B.

[0106] The common light chain (variable region) may be a lambda light chain, which is therefore also provided in the context of the present invention, although a kappa light chain is preferred. The constant portion of the common light chain of the present invention may be the constant region of a kappa or lambda light chain. This is preferably the constant region of a kappa light chain, and preferably the common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising the IgVKI-39 gene fragment, and most preferably a rearranged germline human kappa light chain IgVKI-39. * 01 / IGJKI * 01 (Figure 1). Rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * The terms huVκ1-39 / IGKJ1, IGKV1-39 / IGKJ1, huVκ1-39 light chain, or the shortened form huVκ1-39, or simply 1-39, are used interchangeably throughout this application. Obviously, one of skill in the art will recognize that "common" refers to functional equivalents of light chains that are not identical in amino acid sequence. Many variants of light chains exist, and mutations (deletions, substitutions, additions) exist that do not affect the formation of a functional binding region.

[0107] IgVκ1-39 is short for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV1-39, IGKV1-39, O12a, or O12. The external Ids for this genetic element are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. A preferred amino acid sequence of IgVκ1-39 is shown in Figure 1, which lists the sequence of the V region. The V region can be combined with one of five J regions. Figure 1 lists two preferred sequences for IgVκ1-39 combined with a J region. The combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, and alternative names are IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 *01 (Worldwide Web nomenclature of the IMGT database at imgt.org).

[0108] Preferably, the common light chain variable region is linked to a kappa light chain constant region. In a preferred embodiment, the light chain is the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. In a preferred embodiment, IgVκ1-39 * 01 / IGJκ1 * The number is 01.

[0109] Cells producing a common light chain include, for example, rearranged germline human kappa light chain IgVκ1-39. * 01 / IGJκ1 * Light chains can be produced that comprise the variable regions of the aforementioned light chains fused to O1 and lambda constant regions.

[0110] The bispecific antibodies or variants thereof described herein preferably have one heavy chain variable region / light chain variable region (VH / VL) combination that binds to the extracellular portion of a member of the TNF receptor superfamily, and a second VH / VL combination that binds to the extracellular portion of a second membrane protein, which second membrane protein is not a member of the TNF receptor superfamily.

[0111] The bispecific antibodies or variants thereof described herein preferably have one heavy chain variable region / light chain variable region (VH / VL) combination that binds to the extracellular portion of a member of the TNF receptor superfamily and a second VH / VL combination that binds to the extracellular portion of a member of the B7 family. As described herein, this provides the advantage that a desired immune response may be particularly well promoted, since B7 family members deliver "costimulatory" or "coinhibitory" signals to lymphocytes, thereby enhancing or attenuating the immune response. Thus, by targeting B7 family members, it is possible to enhance stimulatory signals and / or counteract inhibitory signals, thereby inducing or enhancing a desired immune response, for example, against abnormal cells.

[0112] In a preferred embodiment, the VL in the first VH / VL combination is similar to the VL in the second VH / VL combination. In a more preferred embodiment, the VLs in the first and second VH / VL combinations are identical. In a preferred embodiment, the bispecific antibody is a full-length antibody having one heavy / light (H / L) chain combination that binds to the extracellular portion of a member of the TNF receptor superfamily and one H / L chain combination that binds to the extracellular portion of a member of the B7 family. In a preferred embodiment, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In a more preferred embodiment, the light chains in the first and second H / L chain combinations are identical.

[0113] Several methods have been published that favor the production of bispecific antibodies, or conversely, the production of monospecific antibodies. In the present invention, it is preferred that cells prefer the production of bispecific antibodies over the production of the corresponding monospecific antibodies. This is typically achieved by modifying the heavy chain constant regions so that they prefer heterodimerization (i.e., dimerization with the heavy chain of another heavy chain / light chain combination) over homodimerization. In a preferred embodiment, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. This compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. When wild-type CH3 domains are used, coexpression of two different heavy chains (A and B) with a common light chain will result in three different antibody species: AA, AB, and BB. AA and BB are designations for two monospecific bivalent antibodies, and AB is a designation for a bispecific antibody. To increase the proportion of the desired bispecific product (AB), CH3 genetic engineering can be used, or in other words, heavy chains with compatible heterodimerization domains, as defined below, can be used. The art describes various ways in which such heterodimerization of heavy chains can be achieved. One method is to generate a "knob into a hole" in the bispecific antibody. See U.S. Patent Application Publication No. 20030078385 (Arathoon et al.).

[0114] As used herein, the term "compatible heterodimerization domain" refers to a protein domain that has been engineered such that engineered domain A' preferentially forms heterodimers with engineered domain B', and vice versa, with reduced homodimerization between A'-A' and B'-B'.

[0115] U.S. Patent Application No. 13 / 866,747 (now issued as U.S. Patent No. 9,248,181), U.S. Patent Application No. 14 / 081,848 (now issued as U.S. Patent No. 9,358,286), and PCT / NL2013 / 050294 (published as WO 2013 / 157954; incorporated herein by reference) disclose methods and means for producing bispecific antibodies using compatible heterodimerization domains. These means and methods can be suitably employed in the present invention. Specifically, the bispecific antibodies of the present invention preferably contain mutations to produce essentially only bispecific full-length IgG molecules. Preferred mutations are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK-mutant" heavy chain) and amino acid substitutions L351D and L368E in the second domain ("DE-mutant" heavy chain), or vice versa. It has previously been demonstrated in our U.S. Patents Nos. 9,248,181 and 9,358,286 and WO 2013 / 157954 that DE-mutant and KK-mutant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-mutant heavy chains (DEDE homodimers) rarely occurs due to repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0116] Bispecific antibodies can be produced by (transient) transfection of one or more plasmids encoding light chains with two different heavy chains, with CH3 genetic engineering to ensure efficient heterodimerization and formation of the bispecific antibody. Production of these chains in a single cell favors the formation of bispecific antibodies over monospecific antibodies. Preferred mutations for generating essentially only bispecific full-length IgG1 molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbered according to EU numbering) ("KK-mutant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE-mutant" heavy chain), or vice versa.

[0117] In one embodiment, the heavy / light chain combination comprising a variable domain that binds CD137 comprises a DE mutant of the heavy chain. In this embodiment, the heavy / light chain combination comprising a variable domain that can bind to an antigen other than CD137 comprises a KK mutant of the heavy chain. It will be recognized that one embodiment of the present invention may also comprise a variable domain that binds CD137 and includes KK mutants of the heavy chain, as well as other variations known to those skilled in the art that are used to promote heterodimerization with a variable domain that can bind to an antigen other than CD137.

[0118] The Fc region mediates antibody effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). Depending on the application of a therapeutic antibody or Fc fusion protein, it may be desirable to either reduce or increase effector function. In the present invention, reduced effector function is preferred. Reduced effector function may be desired when an immune response is to be activated, enhanced, or stimulated, as in some embodiments of the present invention. Antibodies with reduced effector function can be used to target cell surface molecules of immune cells, among others.

[0119] Binding of IgG to FcγRs or C1q has been found to require residues located within the hinge region and CH2 domain. Two regions of the CH2 domain (Figure 2D) are involved in FcγRs and C1q binding. Substitution of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 into human IgG1 has been shown to significantly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol. 29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604). Furthermore, Idusogie et al. showed that alanine substitutions at different positions, including K322, significantly reduced complement activation (Idusogie et al., 2000, J Immunol. 164(8):4178-84).

[0120] Due to their reduced effector functions, IgG4 antibodies represent an IgG subclass for receptor blocking without cell depletion. IgG4 molecules can exchange half molecules in a dynamic process called Fab arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation is an example of a mutation that ensures a reduced ability for Fab arm exchange. (Labrijn et al., 2009. Nat Biotechnol. 27(8):767-71).

[0121] Antibodies with reduced effector function are preferably IgG antibodies comprising modified CH2 / lower hinge regions, e.g., to reduce Fc receptor interaction or reduce C1q binding. In some embodiments, antibodies of the invention are IgG antibodies with mutant CH2 and / or lower hinge domains such that the interaction of the bispecific IgG antibody with Fc gamma receptors is reduced. Antibodies comprising mutant CH2 regions are preferably IgG1 antibodies. Such mutant IgG1 CH2 and / or lower hinge domains preferably comprise amino acid substitutions at positions 235 and / or 236 (EU numbering), preferably L235G and / or G236R substitutions (Figure 2E).

[0122] Variants of the antibodies or bispecific antibodies described herein include functional parts, derivatives and / or analogs of the antibodies or bispecific antibodies. The variants maintain the binding specificity of the (bispecific) antibody. The functional parts, derivatives and / or analogs maintain the binding specificity of the (bispecific) antibody. The binding specificity is defined by the ability to bind to the extracellular portions of the first membrane protein and the second membrane protein as described herein.

[0123] A functional portion of an antibody or preferably a functional portion of a bispecific antibody described herein is a portion comprising a variable domain that binds to the extracellular portion of a member of the TNF receptor superfamily and a variable domain that binds to the extracellular portion of a second membrane protein. A suitable portion is, for example, an F(ab')2 fragment generated by digestion of the bispecific antibody with pepsin. Other portions comprising variable domains are encompassed by the present invention.

[0124] A functional derivative of an antibody, or preferably a functional derivative of a bispecific antibody, described herein is a protein comprising a variable domain that binds to the extracellular portion of a member of the TNF receptor superfamily and the extracellular portion of a second membrane protein linked by a linker. The variable domain may be such a variable domain or a variable domain-like molecule such as a Fab fragment or a single-chain Fv fragment comprising a VH and a VL linked together via a linker. Another example of a variable domain-like molecule is a so-called single-domain antibody fragment. A single-domain antibody fragment (sdAb) is an antibody fragment that contains a single monomeric variable antibody region. Like a whole antibody, it can selectively bind to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibody fragments are much smaller than common antibodies (150–160 kDa), which are composed of two heavy chain proteins and two light chains, and even smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable regions, one from the light chain and one from the heavy chain). Single-domain antibodies themselves are not much smaller than normal antibodies (typically 90–100 kDa). Most single-domain antibody fragments are genetically engineered from heavy-chain antibodies found in camels; these are called VHH fragments (nanobodies®). Some fish also have heavy-chain-only antibodies (IgNAR, "immunoglobulin new antigen receptor") from which single-domain antibody fragments called VNAR fragments can be derived. Another approach is to split the dimeric variable domain from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Other non-limiting examples of variable domain-like molecules are VHHs, human domain antibodies (dAbs), and unibodies. Preferred functional moieties are those comprising variable domains comprising heavy and light chain variable regions. Non-limiting examples of such variable domains are F(ab)-fragments and single-chain Fv fragments.Bispecific formats for variable domain(-like) linkages include, for example, human serum albumin (HSA) linked to two different scFvs; two different scFvs linked together via a dimerization motif; or bispecific miniantibodies containing self-associating secondary structures such as helix bundles or coiled coils to induce dimerization of the scFv fragments (Morrison (2007) Nat. Biotechnol 25:1233-34). Examples of suitable HSA linkers and methods for linking scFvs to the linker are described in WO 2009 / 126920.

[0125] The antibodies of the present invention, or functional portions, derivatives, and / or analogs thereof, or preferably bispecific antibodies, or functional portions, derivatives, and / or analogs thereof, are preferably used in humans. To this end, the antibodies of the present invention, or functional portions, derivatives, and / or analogs thereof, are preferably human or humanized. Human tolerance to polypeptides is governed by many different mechanisms. Immunity, whether T cell-mediated, B cell-mediated, or otherwise, is one of the variables involved in human tolerance to polypeptides. The constant region of the bispecific antibody of the present invention preferably comprises a human heavy chain constant region, preferably comprising the sequence shown in FIG. 2, and a human light chain constant region, preferably comprising the sequence shown in FIG. 1C. This constant region can contain one or more, preferably no more than 10, and preferably no more than 5, amino acids that differ from the constant region of a naturally occurring human antibody. The constant portion is preferably derived entirely from a naturally occurring human antibody. The various antibodies produced herein are derived from common light chain mice immunized with the respective targets, as described in WO 2009 / 15771. The various antibodies produced herein are derived from a human antibody variable domain library. These variable domains are therefore human. The unique CDR regions may be of human origin, synthetic, or derived from another organism. A variable region is at least human if it has an amino acid sequence identical to that of a variable region of a naturally occurring human antibody, excluding the CDR regions. In such embodiments, the VH of the variable domain of an antibody that binds to a member of the TNF receptor superfamily or a membrane-bound member of the B7 family, or the light chain of an antibody of the present invention, may contain one or more, preferably no more than 10, and preferably no more than 5, amino acids that differ from the variable region of a naturally occurring human antibody (not counting possible differences in the amino acid sequence of the CDR regions). Such mutations also occur inherently in the context of somatic hypermutation.

[0126] Antibodies, at least with respect to the heavy chain variable region, can be derived from various animal species. For example, it is common practice to humanize murine heavy chain variable regions. There are various ways in which this can be achieved, including CDR-grafting onto a human heavy chain variable region having a 3D structure that matches that of the murine heavy chain variable region; preferably, deimmunization of the murine heavy chain variable region, which is carried out by removing known or suspected T-cell or B-cell epitopes from the murine heavy chain variable region. Removal is typically by substituting one or more amino acids in the epitope with other (typically conservative) amino acids, such that the sequence of the epitope is modified so that it is no longer a T-cell or B-cell epitope.

[0127] Deimmunized murine heavy chain variable regions are less immunogenic in humans than the original murine heavy chain variable region. Preferably, the variable regions or domains of the invention are further humanized, e.g., veneering. Using veneering techniques, exterior residues that are readily encountered by the immune system are selectively replaced with human residues to provide hybrid molecules that contain either a weakly immunogenic or a substantially non-immunogenic veneered surface. The animals used in the present invention are preferably mammals, more preferably primates, and most preferably humans.

[0128] Preferably, the antibody or bispecific antibody according to the present invention, or a functional part, derivative, and / or analog thereof, comprises a human antibody constant region. According to differences in their heavy chain constant domains, antibodies are classified into five classes or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes comprise at least one heavy chain designated by the corresponding Greek letter. In a preferred embodiment, the present invention provides an antibody according to the present invention, whose constant region is selected from the group of IgG constant regions, i.e., selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Preferably, the constant region is an IgG4 or IgG1 constant region (FIG. 2), more preferably a mutated IgG1 constant region. Some changes in the IgG1 constant region occur naturally and / or are tolerated without altering the immunological properties of the resulting antibody. Typically, about 1 to 10 amino acid insertions, deletions, substitutions, or a combination thereof are tolerated in the constant region. The constant region may be mutated to allow efficient heterodimerization as demonstrated herein, to reduce effector function, or for other reasons including half-life, stability, etc.

[0129] Rational methods have evolved to minimize the content of non-human residues in the human context. Various methods are available for successfully grafting the antigen-binding properties of an antibody onto another antibody. The binding properties of an antibody may primarily depend on the correct sequence of the CDR3 region, which is often supported by the sequences of the CDR1 and CDR2 regions within the variable domain combined with the appropriate structure of the variable domain as a whole. Various methods are currently available for grafting CDR regions onto a suitable variable domain of another antibody. Some of these methods are reviewed in J.C. Almagro1 and J. Fransson (2008) Frontiers in Bioscience 13, 1619-1633 (incorporated herein by reference).

[0130] The light chain variable region of the variable domain comprising the variable heavy chain sequence shown in FIG. 3 is preferably an O12 germline light chain or is based on O12, preferably a rearranged germline human kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 or a fragment or functional derivative thereof (according to the worldwide web nomenclature of the IMGT database at imgt.org). Rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * The terms IGKV1-39 / IGKJ1, huVκ1-39 light chain, or the shortened form huVκ1-39, are used. The light chain can have 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof. The 1, 2, 3, 4, or 5 amino acid substitutions referred to are preferably conservative amino acid substitutions, and the insertions, deletions, substitutions, or a combination thereof are preferably not in the CDR3 region of the VL chain, and preferably not in the CDR1, CDR2, or CDR3 regions or the FR4 region of the VL chain. A preferred sequence of the common light chain is shown in Figure 1.

[0131] Various methods are available for producing bispecific antibodies. One method involves expressing two different heavy chains and two different light chains in cells and collecting the antibodies produced by the cells. The antibodies produced in this manner typically contain a collection of antibodies with different combinations of heavy and light chains, some of which are the desired bispecific antibodies. The bispecific antibodies can then be purified from the collection. The ratio of bispecific antibodies to other antibodies produced by the cells can be increased in various ways. In a preferred embodiment of the present invention, this ratio is increased by not expressing two different light chains, but by expressing two essentially identical light chains in the cells. The two essentially identical light chains can be light chains with essentially the same light chain variable region and different light chain constant regions, or preferably two essentially identical light chain constant regions. This concept is also known in the art as the "common light chain" method. When essentially identical light chains work together with two different heavy chains to form variable domains with different antigen-binding sites and associated different binding properties, the ratio of bispecific antibodies to other antibodies produced by a cell is significantly improved over the expression of two essentially different light chains. The ratio of bispecific antibodies produced by a cell can be further improved by encouraging the pairing of two different heavy chains with each other versus the pairing of two identical heavy chains. Various methods by which such heterodimerization of heavy chains can be achieved have been described in the art. A preferred method is described in U.S. Provisional Patent Application No. 61 / 635,935, which was followed up by U.S. Regular Patent Application No. 13 / 866,747 and PCT / NL2013 / 050294 (WO 2013 / 157954(A1)), both of which are incorporated herein by reference. Methods and means for producing bispecific antibodies (from a single cell) are disclosed, thereby providing a means for favoring the formation of bispecific antibodies over the formation of monospecific antibodies. In the present invention, these methods can be suitably employed.Thus, the present invention provides a method for producing a bispecific antibody according to the present invention (from a single cell), wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising providing a) a first nucleic acid molecule encoding a first CH3 domain comprising a heavy chain and b) a second nucleic acid molecule encoding a second CH3 domain comprising a heavy chain in the cell, the nucleic acid molecule comprising a means for preferential pairing of the first and second CH3 domains comprising the heavy chain, and the method further comprising culturing the host cell, allowing expression of the two nucleic acid molecules, and harvesting the bispecific antibody from the culture. The first and second nucleic acid molecules may be part of the same nucleic acid molecule, vector, or gene delivery vehicle, or may be integrated into the same site in the genome of the host cell. Alternatively, the first and second nucleic acid molecules are provided separately to the cell. The host cell comprises at least one light chain, preferably a common light chain.

[0132] A preferred embodiment is a method for producing a bispecific antibody according to the invention from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising: providing a cell having: a) a first nucleic acid molecule encoding a heavy chain capable of binding to the extracellular portion of a membrane-bound member of the TNF receptor superfamily, the heavy chain comprising an antigen-binding site comprising a first CH3 domain; and b) a second nucleic acid molecule encoding a heavy chain capable of binding to the extracellular portion of a membrane-bound second protein, the heavy chain comprising an antigen-binding site comprising a second CH3 domain, the nucleic acid molecule comprising means for preferential pairing of the first and second CH3 domains; The method further comprises culturing the cells, allowing expression of the proteins encoded by the two nucleic acid molecules, and harvesting the bispecific IgG antibody from the culture. In a particularly preferred embodiment, the cells also contain a third nucleic acid molecule encoding a common light chain. The first, second, and third nucleic acid molecules may be part of the same nucleic acid molecule, vector, or gene delivery vehicle, and may be integrated into the same site in the genome of the host cell. Alternatively, the first, second, and third nucleic acid molecules are provided to the cells separately. A preferred common light chain is based on O12, preferably a rearranged germline human kappa light chain IgVκ1 39 as described above. * 01 / IGJκ1 *01. Means for preferential pairing of the first and second CH3 domains are preferably corresponding mutations in the CH3 domains of the heavy chain coding region. Preferred mutations for producing essentially only bispecific antibodies are the amino acid substitutions L351K and T366K (numbering according to EU numbering) in the first CH3 domain and the amino acid substitutions L351D and L368E in the second CH3 domain, or vice versa (Figure 2). Thus, further provided is a method according to the invention for producing a bispecific antibody, wherein the first CH3 domain comprises the amino acid substitutions L351K and T366K (numbering according to EU numbering) and the second CH3 domain comprises the amino acid substitutions L351D and L368E, the method further comprising the steps of culturing the cells, allowing expression of the proteins encoded by the nucleic acid molecules, and recovering the bispecific antibody from the culture. Also provided is a method according to the present invention for producing a bispecific antibody, wherein the first CH3 domain comprises the amino acid substitutions L351D and L368E (numbering according to EU numbering) and the second CH3 domain comprises the amino acid substitutions L351K and T366K, and the method further comprises culturing cells, allowing expression of the nucleic acid molecule, and recovering the bispecific antibody from the culture. Antibodies that can be produced by these methods are also part of the present invention. The CH3 heterodimerization domain is preferably an IgG1 heterodimerization domain. The heavy chain constant region comprising the CH3 heterodimerization domain is preferably an IgG1 constant region.

[0133] The member of the TNF receptor superfamily (first membrane protein) is preferably CD137, OX40, CD40, or CD30. In preferred embodiments, the first membrane protein is CD137 or OX40, preferably CD137. A binding molecule of the invention preferably comprises one (antigen) binding site for the first membrane protein. In some embodiments, a binding molecule of the invention is monovalent for the first membrane protein. A binding molecule preferably comprises one (antigen) binding site for the second membrane protein. In some embodiments, a binding molecule of the invention is monovalent for the second membrane protein. In some embodiments, a binding molecule of the invention is monovalent for the first membrane protein and monovalent for the second membrane protein. In some embodiments, a binding molecule of the invention is monovalent for a member of the TNF receptor superfamily and monovalent for a member of the B7 family. In some embodiments, a binding molecule of the invention is monovalent for CD137 and monovalent for a member of the B7 family. In some embodiments, a binding molecule of the invention is monovalent for CD137 and monovalent for PD-L1. Bivalent monoclonal anti-CD137 antibodies are known in the art to activate CD137, but prior art monovalent CD137 binding molecules typically do not.

[0134] The first membrane protein described herein is a member of the TNF receptor superfamily, which is a cell membrane protein. A protein is said to be a cell membrane protein if it has a transmembrane region present in the cell membrane of the cell it is on. This is typically the first cell described herein. The protein may have additional transmembrane regions. In such cases, all transmembrane regions present in the cell membrane are present in the cell membrane of the same cell. When the first membrane protein is present on the cell membrane, it is a cell membrane protein with an extracellular portion. The cell membrane is the membrane of a cell that separates the inside of the cell from the outside of the cell. The first membrane protein is typically present on the cell membrane of the first cell described herein. In terms of the binding molecule of the present invention or in the method or use of the present invention, the first membrane protein is typically present on the cell membrane of the first cell described herein. The first membrane protein may be present on a second cell, but it is preferable that the expression of the first membrane protein is negligible on the second cell. Typically, the level of the first membrane protein on the second cell is at most 10% compared to the expression of the first membrane protein on the first cell.The second cell preferably does not significantly express the first membrane protein.Expression is at least not significant if the first membrane protein cannot be detected (above background) by immunofluorescence in a FACS assay using an antibody specific for the first membrane protein.

[0135] The second membrane protein is also a cell membrane protein. The second membrane protein has a transmembrane region present in the cell membrane of the cell on which it is located. This is typically the second cell described herein. The second protein may have an additional transmembrane region. In such cases, all of the transmembrane regions present in the cell membrane are present in the cell membrane of the same cell. When the second membrane protein is present on the cell membrane, it is a cell membrane protein with an extracellular portion. In terms of the binding molecule of the present invention or the method or use of the present invention, the second membrane protein is typically on the cell membrane of the second cell described herein. Although the second membrane protein may be present on the first cell, it is preferable that the expression of the second membrane protein on the first cell is negligible. Typically, the level of the second membrane protein on the first cell is at most 10% compared to the expression of the second membrane protein on the second cell. The first cell preferably does not significantly express the second membrane protein. Expression is at least not significant if the second membrane protein cannot be detected (above background) by immunofluorescence in a FACS assay using an antibody specific for the second membrane protein.

[0136] According to some embodiments, a binding molecule or (bispecific) antibody or variant according to the invention has one antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and a second antigen-binding site capable of binding to a second membrane protein that is not a member of the TNF receptor superfamily. This offers the advantage of at least partially avoiding 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. These embodiments of the invention contrast with prior art approaches related to binding agents that bind to receptors of the TNF superfamily, particularly binding agents that bind to at least two different receptors of the TNF superfamily. Such prior art approaches may lead to T cell activation in cis, meaning that there is no secondary target, and may entail the risk of excessive T cell responses resulting, for example, in a cytokine storm. Consequently, such prior art approaches have a potential increased risk of side effects compared to binding molecules according to the invention that have a first antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and a second antigen-binding site capable of binding to a membrane protein that is not a member of the TNF receptor superfamily.

[0137] Also provided is an antibody, or functional portion, derivative, and / or analog thereof, comprising an antigen-binding site capable of binding to the extracellular portion of CD137 or OX40 and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein, where the second membrane protein is not a member of the TNF receptor superfamily. Also provided is a method of stimulating the activity of a member of the TNF receptor superfamily on a cell, comprising providing a first cell and a second cell, where the first cell has a member of the TNF receptor superfamily on its membrane and the second cell has a second membrane protein on its membrane, the method comprising contacting the cells with an antibody, or functional portion, derivative, and / or analog thereof, comprising two variable domains, where one variable domain comprises a first antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and another variable domain comprises a second antigen-binding site capable of binding to the extracellular portion of the second membrane protein, thereby stimulating the activity of the member on the first cell, where the second membrane protein is not a member of the TNF receptor superfamily. In some embodiments, the method is an in vitro method.

[0138] In some embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises one antigen-binding site capable of binding to a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a second membrane protein that is not a member of the TNF receptor superfamily. In some embodiments, the antibody of the invention, or functional part, derivative, and / or analog thereof, consists of one immunoglobulin variable domain capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to a second membrane protein that is not a member of the TNF receptor superfamily. The bispecific antibody is preferably a full-length antibody. In some embodiments, the bispecific antibody is a full-length IgG, i.e., full-length IgG1, IgG2, IgG3, or IgG4, preferably full-length IgG1 or full-length IgG4.

[0139] Further provided is an antibody, or functional part, derivative and / or analogue thereof, comprising an antigen-binding site capable of binding to the extracellular part of CD137 or OX40 and an antigen-binding site capable of binding to the extracellular part of a second membrane protein, wherein the second membrane protein is a member of the B7 family, preferably PD-L1. Also provided is a method for stimulating the activity of a member of the TNF receptor superfamily on a cell, the method comprising providing a first cell and a second cell, wherein the first cell has a member of the TNF receptor superfamily on its cell membrane and the second cell has a second membrane protein on its cell membrane, the method comprising contacting the cell with an antibody or functional part, derivative, and / or analogue thereof comprising two variable domains, wherein one variable domain comprises a first antigen-binding site capable of binding to an extracellular portion of the member of the TNF receptor superfamily and another variable domain comprises a second antigen-binding site capable of binding to an extracellular portion of a second membrane protein, thereby stimulating the activity of the member on the first cell, wherein the second membrane protein is a member of the B7 family, preferably PD-L1. In some embodiments, the method is an in vitro method.

[0140] In some embodiments, the antibody, or functional portion, derivative, and / or analog thereof, comprises one antigen-binding site capable of binding to a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a second membrane protein, which is a member of the B7 family, preferably PD-L1. In some preferred embodiments, the antibody, or functional portion, derivative, and / or analog thereof, of the invention consists of one immunoglobulin variable domain capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to a second membrane protein, which is a member of the B7 family, preferably PD-L1. The bispecific antibody is preferably a full-length antibody. In some embodiments, the bispecific antibody is a full-length IgG, i.e., IgG1, IgG2, IgG3, or IgG4, preferably a full-length IgG1 or full-length IgG4.

[0141] A variable domain that "blocks" binding of a first membrane protein to its binding partner interferes with binding of the first membrane protein to its binding partner. Such a variable domain can bind to the first membrane protein. Such a blocking variable domain can bind to an epitope on the first membrane protein and compete with the first membrane protein's binding partner for binding to the epitope. Such a blocking variable domain and the first membrane protein's binding partner can also bind to different epitopes on the first membrane protein. In such cases, the blocking activity can be due, for example, to reduced binding of the binding partner and / or displacement of the binding partner when it is already bound to the first membrane protein, and / or the blocking variable domain can prevent binding of the binding partner to the first membrane protein due to steric hindrance. All of these and other mechanisms can, at least in part, prevent binding of the binding partner to the first membrane protein.

[0142] In one embodiment, a domain comprising an antigen-binding site that binds to a TNF receptor superfamily member blocks the member from binding to its ligand. TNF receptor superfamily member-ligand interactions have been widely studied in the art. In general, members of the TNF receptor superfamily are typically known to have at least one ligand. Examples of known receptor-ligand pairs are the TNF receptor tumor necrosis factor receptors 1 and 2 and the ligand TNF-α, the receptor OX40 and the ligand OX40L, the receptor CD40 and the ligand CD154, the Fas receptor and the ligand FasL, the CD30 receptor and the ligand CD153, and the receptor CD137 and the ligand CD137L. In some embodiments, a variable domain comprising an antigen-binding site that binds to a TNF receptor superfamily member does not block the member from binding to its ligand.

[0143] In some embodiments, this domain comprises an antigen-binding site that binds to a TNF receptor superfamily member and blocks the binding of that TNF receptor superfamily target membrane protein to its binding partner. The variable domain may be further characterized in that, when provided as a monospecific bivalent antibody comprising two variable domains, the variable domain does not stimulate the activity of the TNF receptor superfamily member on cells without cross-linking. In some embodiments, the domain comprising the antigen-binding site that binds to a TNF receptor superfamily member comprises a variable domain that blocks the binding of CD137 to CD137L, and the variable domain is further characterized by the fact that, when provided as a monospecific bivalent antibody comprising two variable domains, the variable domain does not stimulate the activity of CD137 on cells.

[0144] The term "binding partner," binding pair, receptor-ligand pair, etc., refers to proteins that can bind to each other and exert an activity as a result of the binding. At least one of the partners or pairs is a membrane protein on the cell membrane of a cell. The activity is typically exerted by the cell having this membrane protein on it.

[0145] A variable domain that blocks binding of a specific membrane protein binding pair as described herein typically reduces binding of the pair compared to binding in the absence of the variable domain. This is preferably measured in an in vitro assay. Typically, this is done by incubating the variable domain with the membrane protein, followed by binding to the other member of the pair and subsequent incubation. Binding of the pair is then compared to binding of the pair in the absence of the variable domain. The variable domain can completely prevent binding of the first membrane protein to its binding partner. It can also partially prevent binding of the binding pair. A variable domain that blocks binding of a specific membrane protein binding pair preferably reduces binding of the pair by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, and more preferably at least 90% compared to binding in the absence of the variable domain. Blocking of binding by a variable domain is defined herein as blocking achieved using a bivalent monoclonal antibody containing two identical variable domains. Of course, the variable domain will also block binding when present in an antibody comprising a variable domain and a variable domain that binds to a second membrane protein.

[0146] Particular variable domains that can bind to the extracellular portion of CD137 and at least partially block binding of a CD137 ligand to CD137 are variable domains comprising the amino acid sequence of the VH of MF6783, MF6861, MF6795, MF6808, MF6798, MF6754, MF6763, MF6744, MF6785, MF6825, MF6737, MF6749, MF6788, or MF6797.

[0147] Particular variable domains that can bind to the extracellular portion of PD-L1 and block PD1 binding to PD-L1 are variable domains comprising the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5359, MF5377, MF5382, MF5424, MF5426, MF5439, MF5442, MF5553, MF5557, MF5561, MF5576, MF5594, or MF5708. The amino acid sequences are shown in Figure 3.

[0148] The variable domains that bind to a member of the TNF receptor superfamily are preferably variable domains that, when provided in a monospecific, bivalent antibody comprising two of the domains, do not stimulate the activity of the TNF receptor superfamily member on a cell. A variable domain in the context of a bivalent, monospecific antibody comprises two of the same variable domains and does not stimulate the activity of a cell that contains a TNF receptor superfamily member.

[0149] The stimulatory activity of a member of the TNF receptor superfamily on a cell is typically measured by measuring the biological activity of the cell. The type of activity depends on which member of the TNF receptor superfamily is analyzed. For example, for OX40 and CD137, the activation status of OX40- and / or CD137-positive T cells can be measured. OX40 and CD137 are so-called costimulatory proteins that stimulate the activity of activated T cells. Suitable methods for measuring T cell activation are provided in the Examples section. One method is to measure the production of IL-2, IFNγ, and / or TNFα by activated T cells or compositions containing T cells. Other TNF receptors have different biological activities. For example, CD30 is expressed on activated T cells and B cells and is a positive regulator of apoptosis. CD30 stimulation can be measured by measuring the apoptosis of activated B cells or T cells in response to a binding molecule of the invention. CD40 is a costimulatory protein found on antigen-presenting cells, such as macrophages, and stimulation further stimulates the activation of antigen-presenting cells. The activity of a member of the TNF receptor superfamily is stimulated when the activity is measured in the presence of a binding molecule discussed herein, preferably an antibody according to the invention or a functional part, derivative and / or analogue thereof, and is higher than the activity measured under otherwise identical conditions in the absence of the binding molecule, preferably an antibody according to the invention or a functional part, derivative and / or analogue thereof. Stimulating activity includes inducing activity and enhancing already existing activity.

[0150] Stimulation of CD137 or OX40 activity is preferably measured by measuring the biological activity of CD137 or OX40 containing cells. The biological activity is preferably the activation state of CD137- or OX40-expressing cells. CD137 and OX40 are costimulatory molecules expressed on immune cells, including activated T cells. Stimulation of CD137 or OX40 activity is preferably measured by determining the level of activation of immune cells, such as activated T cells. In an individual, stimulation of CD137 or OX40 activity is measured by measuring the activation of the individual's immune cells and / or T cells. Alternatively, it can also be determined by measuring tumor response in an individual, the individual's viral load, or the individual's parasite load, if applicable.

[0151] The present invention also provides a method for engaging and / or activating T cells, comprising providing a system containing T cells and a cell (second cell) to which the T cells are to be engaged or activated; providing at least one bispecific antibody to the system, preferably comprising a variable domain capable of binding to a member of the TNF receptor superfamily and at least one antibody, preferably a variable domain capable of binding to the extracellular portion of a second protein; and incubating the system under conditions that allow the T cells to be engaged and / or activated. In some embodiments, the method is an in vitro method. The TNF receptor superfamily member is preferably CD137 or OX40, most preferably CD137, and the second membrane protein is preferably not a member of the TNF receptor superfamily. The second membrane protein is preferably a member of the B7 family, most preferably PD-L1. The cell to engage or activate the T cells is preferably an immune cell (e.g., an antigen-presenting cell or macrophage), a neoplastic cell, a virus-infected cell, or an intracellular parasite-infected cell. Engaging and / or activating T cells directs T cells to specific targets. Activating T cells activates the T cell receptor on the T cell. Engaging T cells typically activates T cells. Engagement can also direct already activated T cells to targets specified by antibodies. Conditions that allow T cells to be engaged and / or activated are typically culture conditions, but can also be incubated in non-human animals. These conditions are such that T cells will not engage in the absence of antibodies. When a population of T cells is measured, some of them may already be engaged or activated, provided that the population contains sufficient T cells that are not engaged or activated.

[0152] The antibodies of the invention can bring two cells into close proximity to allow an interaction between the cell and a second membrane protein bound by the antibody of the invention that is mediated by a protein other than a member of the TNF receptor superfamily, one such interaction being the interaction of a T cell receptor on one cell with an MHC on another cell.

[0153] The first and second cells are preferably different cells. The different cells can express both the first and second membrane proteins. However, typically, the first membrane protein is expressed only on the first cell, and the second membrane protein is expressed only on the second cell. Biological activity is typically more stimulated when the first cell does not express the second membrane protein, when the second cell does not express the first membrane protein, or more preferably, when a combination of these is used. When the TNF receptor superfamily member is OX40, CD137, CD30, or CD40, the first cell is preferably an immune cell, preferably a T cell. In these cases, the second cell is preferably an abnormal cell, a tumor cell, or an immune cell (e.g., a macrophage or an antigen-presenting cell). An abnormal cell is a cell that is not normally present in a healthy individual. Non-limiting preferred examples of such cells include cancer cells, virus-infected cells, parasite-infected cells, or cells induced to express the second membrane protein. A suitable second cell is also an immune cell. Preferred examples of such cells are dendritic cells, macrophages, other cells of the myeloid lineage, or B cells. In some cases, the cells may express a second membrane protein as a result of inhibitory factors released by neighboring cells, such as immune cells, fibroblasts, or cancer cells. In some embodiments, the second cell is an antigen-presenting cell that presents a tumor antigen or a pathogenic antigen, such as a viral antigen or a parasite antigen, in the context of a major histocompatibility complex (MHC). The MHC complex is preferably a human leukocyte antigen (HLA) complex. In this regard, the antibodies of the present invention can enhance both the proliferation and differentiation of antigen-naive T cells in vitro. Induction and enhancement of novel T cell responses against tumor antigens typically results in more effective tumor immunity and eradication of cancer cells.

[0154] CD137 can be expressed by activated T cells. It is also found on other cells, such as dendritic cells, natural killer cells, granulocytes, and cells of the vascular wall at sites of inflammation. This protein is known for its costimulatory activity for T cell activation. The ligand CD137L is expressed on monocytes, macrophages, and other cells. Binding of CD137 to the CD137 ligand exerts effects on both the receptor and the ligand-bearing cell. Activation of the ligand or receptor can be achieved in various ways. Common methods are coating on tissue culture plates or cross-linking with antibodies (for a review, see Schwartz, 2004). CD137 expression on both innate and adaptive immune cells, combined with its ability to enhance antitumor responses, has established it as a therapeutic target for enhancing tumor immunity. Various CD137-targeted immunotherapies have reached clinical development (for a review, see Makkouk et al., 2016). Activation of receptors or ligands appears to require homomeric association in the cell membrane to exert their effects. Antibodies with two binding sites for CD137 can activate receptors or ligands. Therefore, such antibodies are bivalent for CD137. Molecules with only one binding site for CD137 have also been produced. Such monovalent binding molecules of the prior art were unable to activate the receptor (McNamara 2008). In contrast, the binding molecules or antibodies or variants according to the present invention can activate CD137 even when they are monovalent for CD137.

[0155] OX40 is a secondary costimulatory immune checkpoint molecule expressed on T cells. OX40 expression is not constitutive; it is typically expressed 24–72 hours after activation. Its ligand, OX40L, is also not expressed on resting antigen-presenting cells (APCs), but is expressed after their activation.

[0156] In the present invention, it has been found that a binding molecule having only one binding site for a TNF receptor superfamily member (first membrane protein) can be activated if the binding molecule also has a binding site for a second membrane protein on another cell (second cell), including when the second membrane protein is not a member of the TNF receptor superfamily. The second membrane protein may preferably be a ligand for a receptor of the TNF receptor superfamily. However, typically, it is not a ligand for the TNF receptor superfamily member that is the first membrane protein. The second membrane protein may be a ligand for a TNF receptor superfamily member that is not a ligand for the first membrane protein.

[0157] Stimulation of TNF receptor superfamily member activity on cells typically requires the clustering of two or more receptor complexes. Homotrimeric ligands arrayed on cell surface membranes typically achieve this by binding multiple TNF receptor superfamily member complexes on neighboring cells. Clustering of TNF receptor superfamily complexes is thought to facilitate stimulation of cellular activity. This is evident, for example, from artificial receptors containing only the cytoplasmic portion of the CD137 receptor. The proximity of the cytoplasmic portions of various artificial receptors also stimulates cells containing the artificial receptor. Bivalent, monospecific antibodies specific for TNF receptor superfamily members can also mimic the ligand effect and stimulate activity. The antibody arms are thought to cluster or cluster the receptors together. This activity is also referred to as receptor cross-linking. Activity can sometimes be further stimulated by providing an anti-antibody antibody, which results in further receptor cross-linking. Prior art binding molecules with only one binding site for a TNF receptor superfamily member typically fail to stimulate activity. Such molecules are unable to cluster or cross-link TNF receptor superfamily members. However, the binding molecules or antibodies or variants according to the invention can stimulate the activity of TNF receptor superfamily members, including when they are monovalent for the receptor superfamily member. Without being bound by any theory, it is believed that the bispecific antibodies according to the invention can also cluster TNF receptor superfamily receptor complexes, thereby promoting the activation of TNF receptor superfamily members. This is particularly true when the second membrane protein bound by the antibody according to the invention is a member of the B7 family, such as PD-L1.

[0158] In one aspect of the present invention, the second membrane protein is present on the cell membrane as part of a multimeric protein containing two or more instances of the second membrane protein. Such a multimeric protein can provide two or more epitopes for the antigen-binding site of a binding molecule of the present invention. In such cases, bound TNF receptor superfamily members on other cells will cluster and stimulate the activity of TNF receptor-containing cells. The binding molecules of the present invention can promote the proximity of TNF receptor superfamily members through binding to other proteins on different cells. This is a characteristic known as trans-crosslinking, in contrast to cis-crosslinking, where a binding molecule binds to two or more instances of TNF receptor superfamily members on the same cell. In some embodiments, the second membrane protein is a homodimer or homotrimer. A homodimer is a protein composed of two identical polypeptide units. A homotrimer is a protein composed of three identical polypeptide units. Without being bound by any theory, it is believed that the epitopes of the antigen-binding site of the second membrane protein collectively bind to several binding molecules. In this way, they act as anchors that promote the proximity of two or more TNF receptor superfamily members close enough to stimulate the activity of the TNF receptor superfamily members on the cell.

[0159] In another embodiment, the second membrane protein is a protein present in one or more distinct regions on the cell membrane. The cell membrane does not provide a homogeneous distribution of all of its components. It is now known that the cell membrane has regions where one or more components of the cell membrane are more frequently present than in other parts of the membrane (for a review, see Vereb, G., et al., 2003 Proc. Natl. Acad. Sci. 100.14:8053-8058). The region is preferably a cluster, domain, microdomain, or compartment of proteins on the cell membrane, preferably an immune synapse. Without being bound by any theory, it is believed that non-random distribution promotes close proximity of TNF receptor superfamily members.

[0160] In other embodiments, stimulation of the activity of a TNF receptor superfamily member on a cell is achieved by providing two or more binding molecules that bind to the same member of the TNF receptor superfamily (first membrane protein) and the same second membrane protein. Embodiments involving two or more binding molecules are also referred to as Oligoclonics® embodiments. For example, as shown in Figures 15 and 16, Oligoclonics® embodiments can result in T cell activation. General methods for producing such Oligoclonics® products are disclosed in WO 2013 / 157953 and WO 2004 / 009618, which are incorporated herein by reference. The term "Oligoclonics" is a registered trademark and is designated by (registered trademark). In Oligoclonics® embodiments, at least two of the binding molecules bind to different epitopes on the first membrane protein, different epitopes on the second membrane protein, or different epitopes on the first membrane protein and different epitopes on the second membrane protein. Oligoclonics® embodiments allow two or more binding molecules to bind to the same molecule of a first and / or second membrane protein, thereby stimulating the activity of a TNF receptor superfamily member on a cell. Preferably, at least two of the binding molecules bind to different epitopes on the second membrane protein, or to different epitopes on the first membrane protein and different epitopes on the second membrane protein. In particularly preferred embodiments, at least two of the binding molecules bind to the same epitope on the first membrane protein and different epitopes on the second membrane protein. In some Oligoclonics® embodiments, the two binding molecules block TNF receptor superfamily member-ligand interactions. In other Oligoclonics® embodiments, the two binding molecules do not block TNF receptor superfamily member-ligand interactions.Preferably, the different epitopes on the first and second membrane proteins allow simultaneous binding of a binding molecule that binds to one of the epitopes with a binding molecule that binds to a different epitope. In a preferred embodiment, the different epitopes are non-competing epitopes. In a preferred embodiment, the first and second binding molecules can bind to the same domain of CD137 or OX40. In a preferred embodiment, the first and second binding molecules bind to the same epitope of CD137 or OX40.

[0161] The second membrane protein is preferably a multimeric cytokine receptor, a member of the B7 family, a member of the CD28 family, a member of an ATP-binding cassette transporter (ABC transporter), an aquaporin, a member of the serine / threonine kinase receptor family, or a member of the receptor tyrosine kinase family. In a preferred embodiment, the second membrane protein is a member of the B7 family. In a preferred embodiment, the B7 family member is CD80, CD86, PD-L1, PD-L2, ICOSL, B7-H3, B7-H4, B7-H5, B7-H6, or B7-H7. The second membrane protein is preferably a co-inhibitory protein of the B7 family. In this preferred embodiment, the variable domain binding to the second membrane protein preferably blocks binding of the B7 family member to its binding partner, the CD28 family. In this way, potential co-inhibitory signals provided to the first cell by the second membrane protein are reduced. In a particularly preferred embodiment, the second membrane protein is PD-L1 or PD-L2, preferably PD-L1. In another preferred embodiment, the second membrane protein is a member of the EGF receptor family (ErbB), insulin receptor family, IGF receptor family, FGF receptor family, VEGF receptor family, HGF receptor family, or AXL receptor family. The second membrane protein is preferably a member of the EGF receptor family (ErbB), preferably EGFR; ErbB-2 or ErbB-3, preferably ErbB-2. The variable domain that binds to EGFR, ErbB-3, or ErbB-4 members of the EGF receptor family preferably blocks growth factor binding to the member. In this embodiment, the activity of the EGF receptor family member on the second cell is reduced.

[0162] In an embodiment of the present invention, the second membrane protein is a member of a binding pair. For example, EGF receptor (EGFR) and EGF form a binding pair. Other non-limiting examples of suitable binding pairs are HER3 and heregulin, LGR5-R spondin, LGR4-R spondin, or a B7 family member ligand and its receptor in the CD28 family. In a preferred embodiment of the present invention, the binding molecules described herein block the binding of the second membrane protein to the complementary member of the binding pair. Such binding molecules typically stimulate the activity of a member of the TNF receptor superfamily on a cell and block the activity of the second membrane protein. Such binding molecules are particularly well suited for treating situations in which the second cell is a tumor cell or an individual with cancer. In a preferred embodiment, the second membrane protein is a member of the B7 family, preferably PD-L1 or PD-L2, preferably PD-L1, and at least one binding molecule preferably blocks the binding of the B7 family member to its normal receptor in the CD28 family. In a preferred embodiment, the second membrane protein is PD-L1, and the at least one binding molecule preferably blocks binding of PD-L1 to PD-1.

[0163] The present invention provides a method for enhancing a biological effect in a CD137-expressing cell, comprising providing a system having a first cell and a second cell, wherein the first cell comprises CD137 on its cell membrane and the second cell comprises a protein on its cell membrane with two or more identical epitopes on the extracellular portion of the protein (i.e., two or more copies of the same epitope on the protein are present); providing the system with a binding site for the extracellular portion of CD137 and a binding molecule comprising a binding site for the epitope; and further comprising incubating the system under conditions capable of enhancing the biological activity. In some embodiments, the method is an in vitro method. In some embodiments, the CD137-expressing cell is an immune cell, preferably a T cell, and the second cell is a tumor cell. In some embodiments, the CD137-expressing cell is an immune cell, preferably a T cell, and the second cell is another immune cell. In some embodiments, the CD137-expressing cell is an immune cell, preferably a T cell, and the second cell is a cell of the myeloid lineage. In some embodiments, the CD137-expressing cell is an immune cell, preferably a T cell, and the second cell is an antigen-presenting cell that preferably presents a tumor antigen or a pathogenic antigen in an MHC context, preferably in an HLA context.

[0164] Cells typically have a member of the TNF receptor superfamily on their membrane if the member is expressed by the cell. Expression can be measured in a variety of ways. Quantitative RNA-specific PCR is often used. Immunohistochemistry using immunofluorescence or FACS analysis is also often used.

[0165] A suitable system for providing the first cell and the second cell is a cell culture. Another suitable system is a non-human animal containing the first cell and the second cell. Another suitable system is an ex vivo system in which the cells are maintained in an active form but do not necessarily promote cell proliferation. For example, the first and second cells can be incubated together under assay conditions that do not necessarily promote proliferation but allow biological activity to be measured.

[0166] Incubating a system under conditions that are permissive for cells that express a biological activity mediated by binding between a first membrane protein and a second membrane protein means that the system is maintained under conditions in which the first and second cells can exhibit the biological activity as a result of the binding partners. In vivo or in vitro incubation need not involve the passage of more than a period of time sufficient for the biological activity to become apparent.

[0167] A variable domain that does not block binding of a specific binding pair of membrane proteins as described herein typically does not reduce binding of the pair compared to binding in the absence of the variable domain. This is preferably measured in an in vitro assay. Typically, this is done by incubating the variable domain with the membrane protein, followed by binding to the other member of the pair and subsequent incubation. Binding of the pair is then compared to binding of the pair in the absence of the variable domain. A variable domain is considered not to block binding of a specific binding pair of membrane proteins if it reduces binding of the pair by 50% or less, preferably 40% or less, preferably 30% or less, preferably 20% or less, and more preferably 10% or less compared to binding in the absence of the variable domain. Binding by a variable domain and blocking or non-blocking binding to the other member of the binding pair is defined herein as blocking obtained using a bivalent monoclonal antibody containing two identical variable domains. Blocking or non-blocking is defined as obtained with a bivalent monospecific antibody containing two identical variable domains.

[0168] A particular variable domain that can bind to the extracellular domain of CD137 and does not block binding of CD137 to CD137L is a variable domain comprising the amino acid sequence of the VH of MF6860, MF6848, MF6805, MF6832, MF6870, MF6862, MF6875, or MF6873.

[0169] A particular variable domain that can bind to the extracellular domain of PD-L1 and does not block the binding of PD1 to PD-L1 is a variable domain comprising the amino acid sequence of the VH of MF5361.

[0170] The essential functional aspects of the variable domain, not necessarily the quantity, such as, for example, antigen binding, ability to block receptor-ligand interaction, or biological activity of the variable domain, can be determined in various ways. A preferred format is a Fab fragment or an antibody. A preferred antibody format is a monospecific bivalent antibody comprising two variable domains. Another preferred format is, for example, a bispecific antibody comprising the variable domain to be tested and another variable domain. The other variable domain is preferably a variable domain with a neutral specificity for the assay being performed. A preferred neutral variable domain is a variable domain capable of binding to tetanus toxin.

[0171] The antibodies of the present invention, or functional parts, derivatives, and / or analogs thereof, preferably comprise a variable domain that blocks the binding of its TNF receptor superfamily target membrane protein to its binding partner. In some embodiments, the antibodies of the present invention, or functional parts, derivatives, and / or analogs thereof, comprise a variable domain that blocks the binding of its TNF receptor superfamily target membrane protein to its binding partner, and when provided as a monospecific bivalent antibody comprising two variable domains, do not stimulate the activity of TNF receptor superfamily members on cells. In some embodiments, the antibodies of the present invention, or functional parts, derivatives, and / or analogs thereof, comprise a variable domain that blocks the binding of CD137 to CD137L, and when provided as a monospecific bivalent antibody comprising two variable domains, do not stimulate the activity of CD137 on cells.

[0172] The present invention also provides methods for treating an individual with cancer, comprising administering to an individual in need thereof a binding molecule of the invention, preferably an antibody of the invention or a functional part, derivative, and / or analog thereof, or a bispecific antibody of the invention. The individual preferably has cancer. In some embodiments, the cancer is a cancer comprising cancer cells that express a second membrane protein. In some embodiments, the cancer is a cancer comprising cancer cells that express a member of the B7 family. In some embodiments, immune cells and / or cells of the individual's myeloid lineage express the second membrane protein, preferably a member of the B7 family. In some embodiments, antigen presenting cell (APC) cells of the individual express the second membrane protein, preferably a member of the B7 family. According to these embodiments, the cancer cells may or may not express the second membrane protein. When APCs express the second membrane protein, cancer antigens are presented by such APCs of an individual, and transactivation of immune cells (preferably T cells) can be induced by antibodies, or functional portions, derivatives, and / or analogs, of the present invention, which are capable of binding to immune cells and to the individual's immune cells, APCs, or tumor cells. In some embodiments, antibodies, or functional portions, derivatives, and / or analogs of the present invention, which bind to CD137 and members of the B7 family, preferably PD-L1, are used. Such antibodies, or functional portions, derivatives, and / or analogs of the present invention, can transactivate immune cells by binding to either CD137-expressing immune cells (preferably T cells) and tumor cells and / or immune cells and / or cells of the myeloid lineage and / or APCs expressing a member of the B7 family.

[0173] The cancer is preferably an adenocarcinoma. Preferred cancers are colorectal cancer, pancreatic cancer, lung cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, melanoma, testicular cancer, urothelial cancer, kidney cancer, gastric cancer, or carotenoid cancer. In a preferred embodiment, the cancer is colorectal cancer, pancreatic cancer, lung cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, or melanoma. In a particularly preferred embodiment, the cancer is colorectal cancer, pancreatic cancer, lung cancer, breast cancer, or liver cancer. In a particularly preferred embodiment, the cancer is gastrointestinal cancer. In a preferred embodiment, the cancer is colorectal cancer. In this embodiment, the binding molecule, preferably an antibody or functional part, derivative, and / or analog thereof, is an antibody having a variable domain capable of binding to CD137 or OX40 and a variable domain capable of binding to PD-L1. A variable domain that binds to CD137 or OX40 preferably blocks binding of CD137 to its ligand, or in the case of OX40, blocks binding of OX40 to its ligand. A variable domain that binds to PD-L1 preferably blocks binding of PD-1 to PD-L1.

[0174] Further provided is an ex vivo system comprising an antibody of the present invention, or a functional portion, derivative, and / or analog thereof, or a bispecific antibody, or a functional portion, derivative, and / or analog thereof, and a first cell and a second cell. The first and second cells preferably express a first and a second membrane protein, respectively, on their cell membranes. The system is preferably a cell line suitable for the maintenance and / or expansion of the first cell. The cell line is preferably a cell line suitable for the maintenance and / or expansion of the second cell. Such a system is suitable, for example, for raising and / or expanding immune cells directed against abnormal cells. Such immune cells can then be administered to an individual in need thereof, for example, a cancer patient. The immune cells preferably comprise T cells or NK cells, preferably cytotoxic T cells. The immune cells are preferably autologous cells of the individual in need thereof.

[0175] Further provided is a method for stimulating an immune response in an individual against abnormal cells in the individual, the method comprising providing the individual with an antibody of the present invention, or a functional part, derivative, and / or analog thereof. The abnormal cells are preferably cancer cells, virus-infected cells, parasites, or parasite-infected cells. In a preferred embodiment, the cells are cancer cells or neoplastic cells. In this embodiment, the antibody, or functional part, derivative, and / or analog thereof, is preferably an antibody having a variable domain capable of binding to the extracellular portion of CD137 or OX40 and a variable domain capable of binding to PD-L1. The variable domain that binds to CD137 or OX40 preferably blocks binding of CD137 to its CD137 ligand, or, in the case of OX40, blocks binding of OX40 to its OX ligand. The variable domain that binds to PD-L1 preferably blocks binding of PD-1 to PD-L1.

[0176] A neoplasm is an abnormal growth of tissue, which, when it also forms a mass, is called a tumor. Neoplasms in the present invention typically form a mass. Neoplastic cells are cells from the neoplasm that have formed a mass. The World Health Organization (WHO) classifies neoplasms into four main groups: benign neoplasms, in situ neoplasms, malignant neoplasms, and neoplasms of unknown or uncertain nature. Malignant neoplasms are also simply known as cancer.

[0177] Stimulating an immune response includes inducing an immune response and enhancing an already existing immune response. The immune response in an individual can be measured by measuring the individual's tumor burden, the individual's viral load, the individual's parasite load, if applicable.

[0178] The virus-infected cells are preferably cells infected with an immunodeficiency virus, a herpes virus, preferably herpes simplex virus, Wasselaer-Zosteri virus, cytomegalovirus, or Epstein-Barr virus, a papillomavirus, a hepatitis virus, preferably hepatitis A, B, or C virus, measles virus, or adenovirus. Preferably, the virus is a virus known to be persistent in an individual. Persistent infection is characterized as a virus that is not eliminated but remains within specific cells of an infected individual. Persistent infection can involve both asymptomatic and productive infection stages without rapidly killing or causing excessive damage to host cells. Persistent virus-host interactions can be latent infections, chronic infections, and / or delayed infections.

[0179] Parasite-infected cells are cells infected with intracellular parasites. These parasites are parasitic microorganisms that can grow and reproduce inside host cells. Some intracellular parasites can also survive outside of cells. These parasites are so-called facultative intracellular parasites. Non-limiting examples include Listeria monocytogenes, Legionella, certain species of Mycobacterium, and Cryptococcus neoformans. Preferred intracellular parasites are those that are unable to grow outside of a host cell; preferred examples are Chlamydia and closely related species, certain species of Mycobacterium such as Mycobacterium leprae, certain protozoa (Apicomplexa, Plasmodium spp., Toxoplasma gondii, and Cryptosporidium parvum), and trypanosomes.

[0180] The present invention also provides nucleic acid molecules encoding antibody heavy chain variable regions according to the present invention. The nucleic acid molecules (typically in vitro, isolated, or recombinant nucleic acid molecules) preferably encode any one of the heavy chain variable regions shown in Figure 3, or the heavy chain variable regions shown in Figure 3 with one, two, three, four, or five amino acid insertions, deletions, substitutions, or a combination thereof. In a preferred embodiment, the nucleic acid molecule comprises a sequence as shown in Figure 3. The nucleic acid molecule preferably uses codons optimized for expression in the antibody-producing cell to be used. Preferably, the nucleic acid encoding the heavy chain variable region shown in Figure 3, or the heavy chain variable region shown in Figure 3 with one, two, three, four, or five amino acid insertions, deletions, substitutions, or a combination thereof, is codon optimized for expression in human cells, preferably Per.C6™, or Chinese hamster, preferably CHO. The present invention further provides nucleic acid molecules encoding the mentioned heavy chain variable regions together with the heavy chain constant region of Figure 2.

[0181] Nucleic acid molecules used in the present invention are typically, but not limited to, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids are available to those skilled in the art. A nucleic acid molecule according to the present invention is, for example, contained within a cell. When the nucleic acid molecule is expressed in the cell, the cell is capable of producing an antibody according to the present invention. Thus, in one embodiment, the present invention provides a cell comprising an antibody according to the present invention and / or a nucleic acid molecule according to the present invention. When the cell produces a heavy chain and a light chain, an antibody is produced. A cell capable of producing an antibody of the present invention is provided. The cell preferably comprises a nucleic acid molecule encoding an antibody heavy chain comprising an antibody heavy chain variable region, which, when combined with a common light chain, is capable of binding to a first membrane protein. The cell preferably further comprises a nucleic acid molecule encoding an antibody heavy chain comprising an antibody heavy chain variable region, which, when combined with a common light chain, is capable of binding to a second membrane protein. The cell preferably further comprises a nucleic acid molecule encoding a common light chain. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, most preferably a human cell. For the purposes of the present invention, a suitable cell is any cell that can contain, and preferably produce, an antibody according to the invention and / or a nucleic acid according to the invention.

[0182] The present invention further provides cells comprising an antibody of the present invention. Also provided are cells comprising one or more nucleic acid molecules encoding an antibody of the present invention, either alone or together. The one or more nucleic acid molecules are expressible, meaning that they contain the necessary cis signals for RNA transcription and translation of the protein-coding domain. Preferably, the cells (typically in vitro, isolated, or recombinant) produce the antibody. In preferred embodiments, the cells are hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells, or PER-C6™ cells. In particularly preferred embodiments, the cells are CHO cells. Also provided are cell cultures comprising the cells of the present invention. Various institutions and companies have developed cell lines for large-scale production of antibodies, e.g., for clinical use. Non-limiting examples of such cell lines are CHO cells, NSO cells, or PER-C6™ cells. These cells are also used for other purposes, such as protein production. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, in a preferred embodiment, the invention provides the use of cell lines developed for large-scale production of antibodies for the production of antibodies of the invention. The invention further provides cells for producing antibodies comprising nucleic acid molecules encoding the VH, VL, and / or heavy chains shown in Figures 3, 1 and 2. Preferably, the nucleic acid molecules comprise sequences as shown in Figures 1 and 2.

[0183] The present invention further provides a method for producing an antibody, comprising culturing the cells of the present invention and harvesting the antibody from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are adapted to suspension growth. Further provided is an antibody obtainable by the method for producing an antibody according to the present invention. The antibody is preferably purified from the culture medium. Preferably, the antibody is affinity purified.

[0184] The cells of the present invention may be, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells, or any other cell type known in the art for its suitability for producing antibodies for clinical purposes, particularly for producing antibodies to be administered to humans. In a particularly preferred embodiment, the cells are human cells, preferably cells transformed with an adenovirus E1 region or a functional equivalent thereof. A preferred example of such a cell line is the PER.C6™ cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or variants thereof, preferably variants that use a glutamine synthetase (GS) vector system for antibody expression.

[0185] The present invention further provides pharmaceutical compositions comprising one or more antibodies or variants thereof according to the invention. The pharmaceutical composition preferably comprises a pharmaceutically acceptable excipient or carrier.

[0186] The antibody or variant thereof of the present invention may further comprise a label, preferably a label for in vivo imaging. Such a label is typically not necessary for therapeutic applications. For example, a label may be useful in a diagnostic setting. For example, a label may be useful in visualizing target cells in the body. A variety of labels are suitable, many of which are well known in the art. In a preferred embodiment, the label is a radioactive label for detection. In another preferred embodiment, the label is an infrared label. Preferably, the infrared label is suitable for in vivo imaging. A variety of infrared labels are available to those skilled in the art. Preferred infrared labels are, for example, IRDye800, IRDye 680RD, IRDye 680LT, IRDye 750, IRDye 700DX, IRDye 800RS, IRDye 650, IRDye 700 phosphoramidite, and IRDye 800 phosphoramidite (LI-COR USA; 4647 Superior Street; Lincoln, Nebraska).

[0187] The amount of the antibody according to the present invention to be administered to a patient is typically within the therapeutic window, meaning that a sufficient amount is used to achieve a therapeutic effect, but the amount does not exceed the threshold that causes unacceptable side effects. The smaller the amount of antibody required to achieve the desired therapeutic effect, the larger the therapeutic window will typically be. Therefore, antibodies according to the present invention that exert a sufficient therapeutic effect at a low dosage are preferred. The dosage may be within the range of the administration regimen of nivolumab. The dosage may also be lower.

[0188] In a tumor-suppressive environment, PD-L1 expression on surrounding cells is expected to reach a density threshold that will result in the activation of CD137 on T cells, as described in Example 8 (Figures 28A and 28B). Therefore, bispecific antibodies could activate T cells within the tumor or, to a significantly lesser extent, cells expressing low PD-L1 cell surface levels. If a CD137xPD-L1 bispecific antibody contains a PD-L1 blocking Fab arm, the antibody would further overcome PD-1 / PD-L1 blockade. By acting "in trans," CD137xPD-L1 antibodies release PD-1 / PD-L1 blockade and simultaneously activate T cells by activating CD137. As a result, CD137xPD-L1 antibodies can enhance local T cell responses, resulting in the release of a plethora of cytokines (Example 9), which in turn can activate other immune cells within the tumor microenvironment, at least partially overcoming local immune suppression within the tumor. The inventors have shown that bispecific antibodies according to the present invention often have better T cell activation properties than prior art benchmark antibodies with the same type of specificity (e.g., urelumab-based antibodies (anti-CD137) or atezolizumab-based antibodies (anti-PD-L1)). In the Examples, stronger T cell activation activity was obtained using bispecific antibodies according to the present invention compared to a mixture of two such benchmark antibodies. This is shown, for example, in the T cell transactivation assay and SEB stimulation assay in the Examples of the present invention. It has also been demonstrated that bispecific antibodies according to the present invention can reverse immunosuppression induced by tumor-associated M2 macrophages and (re)stimulate tumor-specific T cells isolated from patient tumors in vitro. Bispecific antibodies according to the invention are able to (re)stimulate tumor-specific CD4+ effector memory T cells, tumor-specific CD8+ effector memory T cells, and tumor-specific CD8+ terminally differentiated T cells, whereas benchmark anti-PD-L1 antibodies based on atezolizumab typically (re)stimulate only CD4+ T cells.Thus, bispecific antibodies according to the invention have the potency to (re)stimulate a more variable subset of antigen-specific T cells compared to benchmark antibodies, including CD8+ T cells.

[0189] Next, the CD137xPD-L1 bispecific antibody can enhance de novo CD8+ T cell antitumor responses by activating antigen-experienced CD8+ T cells to reactivate existing cytotoxic T cell responses against tumors. Tumor (neo) antigens shed into the environment by staining tumor cells or tumor cells phagocytosed by antigen-presenting cells are transported to draining lymph nodes or tertiary lymphoid structures, which are ectopic lymphoid formations found in tumor tissue. In the local tumor environment, tumor antigens are presented to naive CD8+ T cells, which proliferate and differentiate upon antigen recognition.

[0190] As shown in the Examples, antibodies according to the invention are able to enhance T cell proliferation after CD8+ T cell priming to a greater extent than benchmark antibodies based on urelumab or atezolizumab. The Examples demonstrate that antibodies according to the invention are able to induce both higher proliferation and differentiation of antigen-specific CD8+ T cells than a mixture of benchmark antibodies based on urelumab or atezolizumab, which will promote the generation of large populations of tumor-specific memory and terminally differentiated killer T cells.

[0191] Antibodies or variants thereof according to the present invention, particularly bispecific antibodies or variants thereof, may have fewer side effects than combinations of bivalent monospecific antibodies with variable domains. Combinations of antibodies that block inhibitory and / or costimulatory molecules benefit patients who do not respond to existing immunotherapies. However, dual blockade of immunomodulatory receptors (iMODs) has been shown to increase immune-related toxicity. Antibodies or variants thereof according to the present invention, particularly bispecific antibodies or variants thereof, are suitable for addressing dual blockade of iMODs because they can exert functional activities that cannot be reproduced by combinations of monoclonal antibodies and can more selectively target specific cell populations, reducing safety hazards in patients. Without being bound by any theory, the reduced potential for adverse side effects of bispecific antibodies or variants of the present invention compared to (combinations of) monospecific antibodies is thought to be at least in part due to the fact that bispecific antibodies or variants of the present invention typically exhibit T cell activation in trans but have reduced T cell activation activity in cis. In the context of the present invention, the use of antibodies with reduced cis T cell activating activity is preferred as this reduces potential non-specific T cell responses. The antibodies or bispecific antibodies according to the present invention or functional parts, derivatives and / or analogues thereof have less immune-related toxicity than the combination of bivalent monospecific antibodies with variable domains.

[0192] In view of the above, the bispecific antibodies according to the invention or functional parts, derivatives and / or analogues thereof are preferred for therapeutic use.

[0193] These antibodies were produced as bispecific antibodies by cloning them into complementary expression vectors containing mutations in the CH3 region that promote heterodimerization of the heavy chains. Many bispecific antibodies were produced on a small scale and tested in binding and functional assays against cancer cell lines. The antibodies of the present invention, particularly the bispecific antibodies of the present invention, can combine high efficiency with a low toxicity profile. The antibodies of the present invention may be useful in various types and lines of immune-targeted therapy. The antibodies of the present invention may have an increased therapeutic window compared to antibodies that bind to the same antigen(s) with both arms.

[0194] Further provided is the use of a bispecific antibody according to the invention, or a functional part, derivative and / or analogue thereof, for the preparation of a medicament for the treatment or prevention of the formation of abnormal cells, tumors and / or metastases. The tumor from which metastases arise is preferably a tumor positive for a second cell membrane protein, preferably positive for a member of the B7 family.

[0195] The antibodies of the invention can be produced at levels of >50 mg / L after transient transfection in suspension 293F cells. The bispecific antibodies can be purified to greater than 98% purity with yields of >70%. Analytical characterization studies show a bispecific IgG1 antibody profile comparable to a bivalent monospecific IgG1.

[0196] The present invention also provides bispecific antibodies, or functional portions, derivatives, and / or analogs thereof, capable of binding to the extracellular portion of a membrane-bound member of the TNF receptor superfamily and to the extracellular portion of a membrane-bound second membrane protein, preferably a member of the B7 family. In some embodiments, the bispecific antibodies, or functional portions, derivatives, or analogs thereof, comprise one antigen-binding site capable of binding to a member of the TNF receptor superfamily and one antigen-binding site capable of binding to a second protein, preferably a member of the B7 family. In some preferred embodiments, the antigen-binding portion of the bispecific antibodies, or functional portions, derivatives, or analogs thereof, of the invention, consists of one immunoglobulin variable domain capable of binding to the extracellular portion of a member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to a member of the B7 family. The bispecific antibodies, or functional portions, derivatives, or analogs thereof, are preferably monovalent for members of the TNF receptor superfamily and monovalent for members of the B7 family. The bispecific antibodies are preferably full-length antibodies. In some embodiments, the bispecific antibody is a full-length IgG, ie, a full-length IgG1, IgG2, IgG3 or IgG4, preferably a full-length IgG1 or full-length IgG4.

[0197] The present invention also provides bispecific antibodies, or functional portions, derivatives, and / or analogs thereof, capable of binding to the extracellular portion of CD137 and the extracellular portion of PD-L1. The bispecific antibodies, or functional portions, derivatives, or analogs thereof, preferably comprise two antigen-binding sites. The bispecific antibodies, or functional portions, derivatives, or analogs thereof, preferably comprise one antigen-binding site capable of binding to CD137 and one antigen-binding site capable of binding to PD-L1. In some preferred embodiments, the antigen-binding portion of the bispecific antibodies, or functional portions, derivatives, or analogs thereof, of the invention consists of one immunoglobulin variable domain capable of binding to the extracellular portion of CD137 and one immunoglobulin variable domain capable of binding to PD-L1. The bispecific antibodies, or functional portions, derivatives, or analogs thereof, are preferably monovalent for CD137 and monovalent for PD-L1. In some embodiments, the antigen-binding site capable of binding to CD137 is capable of blocking binding of CD137 to CD137L. In some embodiments, the antigen-binding site capable of binding to CD137 is unable to block CD137 from binding to CD137L. In some embodiments, the antigen-binding site capable of binding to PD-L1 is able to block PD-L1 from binding to PD-1. In some embodiments, the antigen-binding site capable of binding to PD-L1 is unable to block PD-L1 from binding to PD-1. The bispecific antibody is preferably a full-length antibody. In some embodiments, the bispecific antibody is a full-length IgG, i.e., full-length IgG1, IgG2, IgG3, or IgG4, preferably full-length IgG1 or full-length IgG4.

[0198] The present invention also provides a method for treating an individual with cancer, comprising administering to an individual in need thereof a binding molecule of the invention, or a bispecific antibody of the invention, or a functional part, derivative or analogue thereof.

[0199] The present invention further provides a binding molecule of the invention, or a bispecific antibody of the invention, or a functional part, derivative or analogue, for use in treating an individual with cancer.

[0200] Further provided is a cell line comprising a bispecific antibody of the invention, or a functional part, derivative or analogue thereof, a first cell expressing a membrane-bound member of the TNF receptor superfamily, and a second cell expressing a membrane-bound second membrane protein, preferably a member of the B7 family.

[0201] The present invention provides a method for stimulating the activity of a member of the TNF receptor superfamily on a cell, the method comprising providing a first cell and a second cell, wherein the first cell comprises a member on its cell membrane and the second cell comprises a second membrane protein on its cell membrane, and the method comprises contacting the cell with a bispecific antibody or variant thereof comprising two variable domains, wherein one variable domain comprises a first antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and another variable domain comprises a second antigen-binding site capable of binding to the extracellular portion of a second membrane protein, thereby stimulating the activity of the member on the first cell. In some embodiments, the bispecific antibody comprises one antigen-binding site capable of binding to a member of the TNF receptor superfamily. In some embodiments, the method is an in vitro method. In some embodiments, the member of the TNF receptor superfamily is CD137 or preferably OX40. The second membrane protein is preferably not a member of the TNF receptor superfamily. The bispecific antibody is preferably monovalent for a member of the TNF receptor superfamily and for a second membrane protein, preferably for a member of the B7 family. The bispecific antibody is preferably a full-length antibody. In some embodiments, the bispecific antibody is a full-length IgG, i.e., full-length IgG1, IgG2, IgG3, or IgG4, preferably full-length IgG1 or full-length IgG4.

[0202] The first cell preferably does not significantly express the second membrane protein on its cell membrane. The second membrane protein is preferably a protein present in one or more regions on the cell membrane. The region is preferably a cluster, domain, microdomain, or compartment on the cell membrane, preferably an immune synapse. The second membrane protein is preferably present on the cell membrane as part of a multimeric protein containing two or more instances of the second membrane protein. In some embodiments, the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer. In preferred embodiments, the second membrane protein is a multimeric cytokine receptor, a member of the B7 family, a member of the CD28 family, a member of an ATP-binding cassette transporter (ABC transporter), an aquaporin, a member of the serine / threonine kinase receptor family, or a member of the receptor tyrosine kinase family. The second membrane protein is preferably a member of the B7 family, preferably PD-L1 or PD-L2, preferably PD-L1. In a preferred embodiment, the second membrane protein is a member of the EGF receptor family (ErbB), IGF receptor family, FGF receptor family, VEGF receptor family, HGF receptor family, or AXL receptor family. The second membrane protein is preferably a member of the EGF receptor family (ErbB), preferably EGFR, ErbB-2, or ErbB-3, preferably ErbB-2. Preferably, the variable domain that binds to a member of the TNF receptor superfamily blocks binding of a ligand to the member. The variable domain that binds to the extracellular portion of a member of the TNF receptor superfamily is preferably defined as a variable domain that, when in a bivalent monospecific antibody format comprising two variable domains that bind to a member of the TNF receptor superfamily, does not stimulate the activity of the TNF receptor superfamily member on cells.The method preferably further comprises providing a further bispecific antibody comprising an antigen-binding site capable of binding to the extracellular portion of a member of the TNF receptor superfamily and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein, wherein the first and second bispecific antibodies a distinct epitope on the first membrane protein, a different epitope on a second membrane protein, or A different epitope on a first membrane protein and a different epitope on a second membrane protein binds to The method further comprises stimulating the activity of a member of the TNF receptor superfamily on the first cell by incubating the first and second cells with the first and second bispecific antibodies. In some embodiments, the method is an in vitro method. In preferred embodiments, the TNF receptor superfamily member is CD137 or OX40. In some embodiments, the first and second bispecific antibodies each comprise one antigen-binding site capable of binding to a member of the TNF receptor superfamily. The second membrane protein is preferably not a member of the TNF receptor superfamily. The first and / or second bispecific antibodies are preferably monovalent for a member of the TNF receptor superfamily and monovalent for the second membrane protein, preferably monovalent for a member of the B7 family. The first and / or second bispecific antibodies are preferably full-length antibodies. In some embodiments, the first and / or second bispecific antibodies are full-length IgG, i.e., full-length IgG1, IgG2, IgG3, or IgG4, preferably full-length IgG1 or full-length IgG4.

[0203] The antigen-binding sites of the first and second bispecific antibodies capable of binding to the second membrane protein preferably bind to different epitopes on the extracellular portion of the second membrane protein. The different epitopes on the extracellular portion of the second membrane protein are preferably non-competing epitopes.

[0204] Also provided are bispecific antibodies comprising an antigen-binding site capable of binding to the extracellular portion of CD137 or OX40 and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein. In some embodiments, the bispecific antibody comprises one antigen-binding site capable of binding to CD137 or OX40. The second membrane protein is preferably not a member of the TNF receptor superfamily. The second membrane protein is preferably not expressed to a significant extent by T cells. The second membrane protein is preferably expressed on immune cells, myeloid cells, antigen-presenting cells, tumor cells, virus-infected cells, or parasite-infected cells. Preferably, the second membrane protein is a protein present in one or more zones on the cell membrane. The zone is preferably a cluster, domain, microdomain, or compartment on the cell membrane, preferably an immune synapse. In some embodiments, the second membrane protein is a protein present on the cell membrane as part of a multimeric protein comprising two or more of the second membrane proteins. In some embodiments, the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer. Preferably, the second membrane protein is a multimeric cytokine receptor, a member of the B7 family, a member of the CD28 family, a member of the ATP-binding cassette transporter (ABC transporter), an aquaporin, a member of the serine / threonine kinase receptor family, or a member of the receptor tyrosine kinase family. The second membrane protein is preferably a member of the B7 family, preferably PD-L1 or PD-L2, preferably PD-L1. In some embodiments, the second membrane protein is a member of the EGF receptor family (ErbB), insulin receptor family, IGF receptor family, FGF receptor family, VEGF receptor family, HGF receptor family, or AXL receptor family. In some embodiments, the second membrane protein is a member of the EGF receptor family (ErbB), preferably EGFR, ErbB-2, or ErbB-3, preferably ErbB-2.The variable domain that binds to CD137 or OX40 preferably blocks binding of a ligand to the member. The variable domain that binds to the extracellular portion of CD137 or OX40 is preferably defined as a variable domain that does not stimulate the activity of CD137 or OX40 on cells when in a bivalent monospecific antibody format comprising two variable domains that bind to CD137 or OX40. The bispecific antibody is preferably monovalent for CD137 or OX40 and monovalent for the second membrane protein. The bispecific antibody is preferably a full-length antibody. In some embodiments, the bispecific antibody is a full-length IgG, i.e., full-length IgG1, IgG2, IgG3, or IgG4, preferably full-length IgG1 or full-length IgG4.

[0205] The present invention also provides compositions comprising one or more bispecific antibodies according to the invention, as well as compositions or kits of parts comprising two or more bispecific antibodies of the invention, wherein the antigen-binding sites capable of binding to CD137 or OX40 of the first and second bispecific antibodies bind to different epitopes on CD137 or OX40. Also provided is a method for stimulating the activity of CD137 or OX40 on a cell, comprising providing a first cell and a second cell, wherein the first cell has CD137 or OX40 (a first membrane protein) on its cell membrane and the second cell has a second membrane protein on its cell membrane; the method comprising contacting the cells with a bispecific antibody (first bispecific antibody) according to the present invention comprising two variable domains, wherein one variable domain comprises a first antigen-binding site capable of binding to the extracellular portion of the first membrane protein and another variable domain comprises a second antigen-binding site capable of binding to the extracellular portion of the second membrane protein, thereby stimulating the activity of the first membrane protein on the first cell. In some embodiments, the bispecific antibody comprises one antigen-binding site capable of binding to the first membrane protein. In some embodiments, the method is an in vitro method. The method preferably further comprises providing a further bispecific antibody (second bispecific antibody) comprising a variable domain having an antigen-binding site capable of binding to the extracellular part of the first membrane protein and a variable domain having an antigen-binding site capable of binding to the extracellular part of the second membrane protein, wherein the first and second bispecific antibodies are a distinct epitope on the first membrane protein, a different epitope on a second membrane protein, or A different epitope on a first membrane protein and a different epitope on a second membrane protein binds to The method further comprises stimulating the activity of CD137 or OX40 on the first cell by incubating the first cell and the second cell with the first and second bispecific antibodies. The second membrane protein is preferably a member of the B7 family, more preferably PD-L1.

[0206] Antibodies defined by MF sequences as shown herein below are preferably bispecific antibodies having two different variable domains, one of which comprises the sequence shown.

[0207] The antibody or functional part, derivative and / or analogue thereof comprising a variable domain capable of binding to the extracellular portion of CD137 preferably comprises a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF6754, MF6763, MF6785, or MF6797 (Figure 3).

[0208] The antibody or functional part, derivative and / or analog thereof comprising a variable domain capable of binding to the extracellular portion of CD137 preferably comprises a heavy chain variable region having CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of the CDR1, CDR2, and CDR3 of the variable heavy chain region of one of the VHs shown in MF6754, MF6763, MF6785, or MF6797 (Figure 3). The CDR1, CDR2, and CDR3 sequences are preferably selected from the same VH region.

[0209] An antibody or functional part, derivative and / or analogue thereof comprising a variable domain capable of binding to the extracellular portion of CD137 preferably comprises the amino acid sequence of the variable heavy chain region of MF6754, MF6763, MF6785, or MF6797, having up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF. Preferably, the amino acid insertion(s), deletion(s), substitution(s), or combination(s), if any, are not in the amino acid sequence of the CDR regions.

[0210] Antibodies, or functional parts, derivatives and / or analogues thereof, comprising a variable domain capable of binding to the extracellular portion of PD-L1 preferably comprise a heavy chain variable region having a CDR3 region comprising the amino acid sequence of the CDR3 region of the variable heavy chain region of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442, or MF5361 (Figure 3).

[0211] Antibodies, or functional parts, derivatives and / or analogues thereof, comprising a variable domain capable of binding to the extracellular portion of PD-L1 preferably comprise a heavy chain variable region having CDR1, CDR2, and CDR3 regions that comprise the amino acid sequences of the CDR1, CDR2, and CDR3 of the variable heavy chain region of one of the VHs shown in MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442, or MF5361 (Figure 3). The CDR1, CDR2, and CDR3 sequences are preferably selected from the same VH region.

[0212] Antibodies, or functional parts, derivatives and / or analogues thereof, comprising a variable domain capable of binding to the extracellular portion of PD-L1 preferably comprise the amino acid sequence of the variable heavy chain region of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442, or MF5361, having up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof, relative to the amino acid sequence of the VH of the indicated MF. Preferably, the amino acid insertion(s), deletion(s), substitution(s), or combinations thereof, if any, are not in the amino acid sequences of the CDR regions.

[0213] The antibody or functional portion, derivative and / or analog thereof preferably comprises a variable domain capable of binding to the extracellular portion of CD137, thereby blocking CD137 from binding to a CD137 ligand, and a variable domain capable of binding to the extracellular portion of PD-L1, thereby blocking PD-1 from binding to PD-L1. The variable domain that binds to the extracellular portion of PD-L1 in the antibody or functional portion, derivative and / or analog thereof preferably comprises a VH region having the amino acid sequence of CDR3, or the amino acid sequences of CDR1, CDR2 and CDR3, of one of the VHs of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442, or MF5361 (Figure 3). In a preferred embodiment, the variable domain that binds to the extracellular portion of PD-L1 comprises a VH region having the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5424, MF5561, MF5439, MF5553, MF5594, MF5426, MF5442, or MF5361, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF.

[0214] The variable domain that binds to the extracellular portion of CD137 in this antibody or functional portion, derivative, and / or analog thereof preferably comprises a VH region having the amino acid sequence of CDR3, or the amino acid sequences of CDR1, CDR2, and CDR3, of one of the VHs of MF6754, MF6763, MF6785, or MF6797 (Figure 3). In a preferred embodiment, the variable domain that binds to the extracellular portion of CD137 comprises a VH region having the amino acid sequence of the VH of MF6754, MF6763, MF6785, or MF6797 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the indicated MF. Preferably, there are no amino acid insertion(s), deletion(s), substitution(s), or combinations thereof, if any, in the amino acid sequences of the CDR regions. Particularly preferred combinations in this antibody or functional part, derivative and / or analogue are combinations of variable domains comprising the following sequences as shown: MF6797 and MF7702, MF6763 and MF7702, MF6785 and MF7702, MF6797 and MF5553, MF6763 and MF5553, MF6785 and MF5553, MF6754 and MF5424, MF6763 and MF5561, MF6785 and MF5439, MF6797 and MF5553, MF6744 and MF5594, MF6744 and MF5361, MF6783 and MF5361, or MF6783 and MF5594, or variants thereof.

[0215] The antibodies described herein or functional parts, derivatives and / or analogues thereof preferably: a CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3 of VH of MF6754, or the amino acid sequences of CDR1, CDR2, and CDR3; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), or the amino acid sequences of the CDR1, CDR2, and CDR3; Includes.

[0216] The antibody or functional part, derivative and / or analogue thereof preferably comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6754, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6754; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the amino acid sequence of the VH of the indicated MF; Includes.

[0217] The antibodies described herein or functional parts, derivatives and / or analogues thereof preferably: a CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3 of VH of MF6763, or the amino acid sequences of CDR1, CDR2, and CDR3; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), or the amino acid sequences of the CDR1, CDR2, and CDR3; Includes.

[0218] The antibody or functional part, derivative and / or analogue thereof preferably comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6763, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6763; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the amino acid sequence of the VH of the indicated MF; Includes.

[0219] The antibodies described herein or functional parts, derivatives and / or analogues thereof preferably: a CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3 of VH of MF6785, or the amino acid sequences of CDR1, CDR2, and CDR3; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), or the amino acid sequences of the CDR1, CDR2, and CDR3; Includes.

[0220] The antibody or functional part, derivative and / or analogue thereof preferably comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6785; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the amino acid sequence of the VH of the indicated MF; Includes.

[0221] The antibodies described herein or functional parts, derivatives and / or analogues thereof preferably: a CD137-binding variable domain comprising a VH region having the amino acid sequence of CDR3 of VH of MF6797, or the amino acid sequences of CDR1, CDR2, and CDR3; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), or the amino acid sequences of the CDR1, CDR2, and CDR3; Includes.

[0222] The antibody or functional part, derivative and / or analogue thereof preferably comprises: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6797, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6797; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5554, MF5576, MF5578, MF9375, MF9376, MF7702, MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5439, or MF5361 (Figure 3), with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the amino acid sequence of the VH of the indicated MF; Includes.

[0223] As shown in the Examples, antibodies with a PD-L1-binding variable domain based on MF5553 provide particularly good T cell activation, as do combinations with different CD137-binding variable domains, including MF6754, MF6763, MF6785, and MF6797.

[0224] Therefore, further, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6754; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5553; Further provided is a bispecific antibody or a functional part, derivative and / or analogue thereof comprising:

[0225] Also, 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; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0226] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6754 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6754; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5553, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0227] Furthermore, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6763; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5553;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0228] Also, 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; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0229] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6763, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6763; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5553, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0230] Furthermore, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6785; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5553;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0231] Also, 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; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0232] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6785; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5553, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0233] Furthermore, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6797; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5553;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0234] Also, 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; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0235] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6797, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6797; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5553, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5553; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0236] The Examples further show that antibodies with a PD-L1-binding variable domain based on MF7702 provide particularly good T cell activation, as do combinations with different CD137-binding variable domains, including MF6763, MF6785, and MF6797.

[0237] Therefore, further, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6797; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF7702;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0238] Also, 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; 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; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0239] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6797, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6797; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF7702, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF7702; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0240] Furthermore, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6763; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF7702;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0241] Also, 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; 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; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0242] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6763, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6763; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF7702, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF7702; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0243] Furthermore, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6785; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF7702;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0244] Also, 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; 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; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0245] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6785, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6785; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF7702, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF7702; Also provided is a bispecific antibody or a functional part, derivative, and / or analog thereof, comprising:

[0246] It is further shown in the Examples that bispecific antibodies having a CD137-binding variable domain based on MF6744 and a PD-L1-binding variable domain based on MF5594 provide particularly good T cell activation, see, for example, Figures 14 to 16. Importantly, such antibodies have stronger T cell activation potential compared to antibodies based on the antibody urelumab.

[0247] Therefore, further a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6744; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5594;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0248] Also, 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 MF6744; 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 MF5594; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0249] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6744, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6744; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5594, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5594; Also provided is a bispecific antibody or a functional part, derivative, and / or analog thereof, comprising:

[0250] It is further shown in the Examples that bispecific antibodies having a CD137-binding variable domain based on MF6744 and a PD-L1-binding variable domain based on MF5361 provide particularly good T cell activation, see, for example, Figures 14 to 16. Importantly, such antibodies have stronger T cell activation potential compared to antibodies based on the antibody urelumab.

[0251] Therefore, further, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6744; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5361;

[0023] In one aspect, a bispecific antibody or a functional part, derivative and / or analog thereof is provided, comprising:

[0252] Also, 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 MF6744; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5361; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0253] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6744, which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6744; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5361, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5361; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0254] It is further shown in the Examples that bispecific antibodies having a CD137-binding variable domain based on MF6783 and a PD-L1-binding variable domain based on MF5361 provide particularly good T cell activation, see, for example, Figures 14-15. Importantly, such antibodies have stronger T cell activation potential compared to antibodies based on the antibody urelumab.

[0255] Therefore, further, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6783; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5361; Further provided is a bispecific antibody or a functional part, derivative and / or analogue thereof comprising:

[0256] Also, 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 MF6783; a PD-L1-binding variable domain comprising a VH region having the amino acid sequences of the CDR1, CDR2, and CDR3 regions of VH of MF5361; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0257] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6783, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6783; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5361, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5361; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0258] It is further shown in the Examples that bispecific antibodies having a CD137-binding variable domain based on MF6783 and a PD-L1-binding variable domain based on MF5594 provide particularly good T cell activation, see for example Figures 14-15.

[0259] Therefore, further, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF6783; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of VH of MF5594; Further provided is a bispecific antibody or a functional part, derivative and / or analogue thereof comprising:

[0260] Also, 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 MF6783; 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 MF5594; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0261] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6783, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6783; a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5594, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5594; Also provided is a bispecific antibody, or a functional part, derivative and / or analog thereof, comprising:

[0262] Additionally, the examples show that bispecific antibody combinations having a CD137-binding variable domain based on MF6744 and a PD-L1-binding variable domain based on MF5594, together with a CD137-binding variable domain based on MF6744 and a PD-L1-binding variable domain based on MF5361 (applied as dual bispecific, e.g., Oligoclonics® embodiments), provide superior T cell activation (see Figures 14-16) and are superior to urelumab-based antibodies.

[0263] therefore, a first bispecific antibody, or a functional part, derivative and / or analogue thereof, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6744; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5594; a second bispecific antibody, or a functional part, derivative, and / or analogue thereof, comprising a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6744, and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5361; and Further provided is a mixture or kit-of-parts comprising:

[0264] Also, a first bispecific antibody, or a functional part, derivative and / or analogue thereof, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6744; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5594; a second bispecific antibody, or a functional part, derivative, and / or analog thereof, comprising a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6744, and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5361; Also provided is a mixture or kit of parts comprising:

[0265] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6744 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6744; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5594 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5594; a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6744, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6744; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5361, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5361; and Also provided is a mixture or kit of parts comprising:

[0266] Additionally, the Examples show that bispecific antibody combinations having a CD137-binding variable domain based on MF6744 and a PD-L1-binding variable domain based on MF5361, together with a CD137-binding variable domain based on MF6783 and a PD-L1-binding variable domain based on MF5594 (applied as dual bispecific, e.g., Oligoclonics® embodiments), provide superior T cell activation compared to urelumab-based antibodies.

[0267] therefore, a first bispecific antibody, or a functional part, derivative and / or analogue thereof, comprising a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6783, and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5594; a second bispecific antibody, or a functional part, derivative, and / or analogue thereof, comprising a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF6744, and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR3 region of the VH of MF5361; and Further provided is a mixture or kit-of-parts comprising:

[0268] Also, a first bispecific antibody, or a functional part, derivative and / or analogue thereof, comprising: a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6783; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5594; a second bispecific antibody, or a functional part, derivative, and / or analog thereof, comprising a CD137-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF6744, and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the CDR1, CDR2, and CDR3 regions of the VH of MF5361; Also provided is a mixture or kit of parts comprising:

[0269] Also, a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6783 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6783; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5594 with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5594; a CD137-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6744, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF6744; and a PD-L1-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF5361, with up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of MF5361; and Also provided is a mixture or kit of parts comprising:

[0270] The Examples also show that the CD137-specific binding of the VH of MF6797, which has good T cell activation properties, is related to the presence of amino acids including Arg66, Gly70, and Phe72 in the CD137 amino acid sequence shown in Figure 42.

[0271] Accordingly, the present invention also provides an isolated, synthetic or recombinant antibody, or functional part, derivative or analogue thereof, capable of binding to CD137, wherein the binding of the antibody or functional part, derivative or analogue to CD137 is associated with the presence of amino acids including Arg66, Gly70, and Phe72 of the CD137 amino acid sequence as shown in Figure 42. The binding of the antibody or functional part, derivative or analogue to CD137 is preferably also associated with amino acids including Val71 of the CD137 amino acid sequence as shown in Figure 42.

[0272] The term "Arg66" refers to an arginine residue at position 66 of the CD137 sequence as shown in Figure 42. The term "Gly70" refers to a glycine residue at position 70 of the CD137 sequence as shown in Figure 42. The term "Val71" refers to a valine residue at position 71 of the CD137 sequence as shown in Figure 42. The term "Phe72" refers to a phenylalanine residue at position 72 of the CD137 sequence as shown in Figure 42.

[0273] Binding of the antibody or functional part, derivative or analog to CD137 is related to the presence of the listed amino acid residues if, when any one of these residues is substituted by alanine, binding of the antibody or functional part, derivative or analog to the resulting CD137 protein is reduced.

[0274] Some embodiments provide an isolated synthetic or recombinant antibody, or functional part, derivative or analogue thereof, capable of binding to CD137, wherein the antibody, or functional part, derivative or analogue specifically binds to amino acids Arg66, Gly70, and Phe72 of the CD137 amino acid sequence as shown in Figure 42. Preferably, the antibody, or functional part, derivative or analogue also specifically binds to amino acid Val71 of the CD137 amino acid sequence as shown in Figure 42.

[0275] Some preferred embodiments provide a bispecific antibody, or functional part, derivative or analogue thereof, capable of binding to CD137 and PD-L1, having a CD137-binding variable domain based on MF6797 and a PD-L1-binding variable domain based on MF7702. The binding of such bispecific antibodies to CD137 and PD-L1, which have particularly good T cell activation properties, is associated with amino acids including the CD137 amino acid residues listed above.

[0276] Now that the above-described CD137 amino acid residues have been identified, it is possible to generate or select antibodies or variants thereof that specifically bind to these amino acid residues. Generation and / or selection of binding molecules that specifically bind to specific amino acid residues can be performed using methods well known in the art, such as by immunizing transgenic non-human animals capable of generating antibodies with antigen fragments containing specific domains that include the target amino acid residues. Alternatively, by screening antibody phage display libraries for phages that bind to the identified amino acid residues.

[0277] Further provided are antibodies or variants thereof that compete with antibody PB17311 for binding to CD137 and / or PD-L1. Competing antibodies or variants thereof are identified, for example, using a competition assay in which cells containing CD137 and / or PD-L1 are incubated with PB17311 and with a candidate antibody or variant thereof. A candidate antibody or variant thereof that is capable of reducing the amount of PB17311 bound to cells containing CD137 and / or PD-L1 compared to a control in which cells are incubated with PB17311 without the candidate antibody or variant thereof is a competing antibody or variant.

[0278] Some embodiments provide isolated, synthetic or recombinant antibodies, or functional parts, derivatives and / or analogs thereof, that compete with antibody PB17311 for binding to CD137 and / or PD-L1.

[0279] Some embodiments provide an isolated synthetic or recombinant antibody, or a functional part, derivative and / or analog thereof, that competes with antibody PB17311 for binding to amino acids Arg66, Gly70, and Phe72 of the CD137 amino acid sequence as shown in Figure 42, more preferably for binding to amino acids Arg66, Gly70, Val71, and Phe72 of the CD137 amino acid sequence as shown in Figure 42.

[0280] Some embodiments provide isolated synthetic or recombinant antibodies, or functional parts, derivatives and / or analogs thereof, that compete with antibody PB17309 for binding to CD137 and / or PD-L1.

[0281] Some embodiments provide isolated synthetic or recombinant antibodies, or functional parts, derivatives and / or analogs thereof, that compete with antibody PB17310 for binding to CD137 and / or PD-L1.

[0282] Antibodies or variants thereof that compete with PB17309 or PB17310 for binding to CD137 and / or PD-L1 are isolated using, for example, a competition assay in which the binding of PB17309 or PB17310 to cells containing CD137 and / or PD-L1 in the absence of the candidate antibody or variant thereof is compared to the binding of PB17309 or PB17310 to cells containing CD137 and / or PD-L1 in the presence of the candidate antibody or variant thereof. A candidate antibody or variant thereof that is capable of reducing the amount of bound PB17309 or PB17310 compared to a control in which cells containing CD137 and / or PD-L1 are incubated with PB17309 or PB17310 without the candidate antibody or variant thereof is identified as a competing antibody or variant.

[0283] The OX40xPD-L1 bispecific antibodies or functional parts, derivatives and / or analogues thereof described herein preferably: MF6629, MF6630, MF6637, MF6643, MF6645, MF6648, MF6655, MF6658, MF6660, MF6675, MF6686, MF6690, MF6692, MF6700, MF6706 , MF6714, MF6721, MF6722, MF6724, MF6728, MF6729, MF6826, MF6940, MF6942, MF6943, MF6944, MF6947, MF6949, MF7331, MF733 an OX40-binding variable domain comprising a VH region having the amino acid sequence of CDR3 of VH of MF7334, MF7341, MF7345, MF7350, MF7351, MF7352, MF7353, MF7356, MF7358, MF7365, MF7366, MF7371, MF7372, MF7374, MF7378, MF7382, MF7383, MF7394, MF7395, or MF7397, or the amino acid sequences of CDR1, CDR2, and CDR3; PD-L1 binding variable domain and The PD-L1-binding variable domain preferably comprises a VH region having the amino acid sequence of the VH CDR3, or the amino acid sequences of the CDR1, CDR2, and CDR3, of a PD-L1-specific VH as shown in Figure 3. In preferred embodiments, the PD-L1-specific VH is as shown for MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5426, or MF5439 (Figure 3).

[0284] The antibody or functional part, derivative and / or analogue thereof preferably comprises: MF6629, MF6630, MF6637, MF6643, MF6645, MF6648, MF6655, MF6658, MF6660, MF6675, MF6686, MF6690, MF6692, MF6700, MF6706, MF6714, MF6721, MF6722, MF6724, MF6726, MF6728, MF6729, MF6630, MF6637, MF6643, MF6645, MF6648, MF6655, MF6658, MF6660, MF6675, MF6686, MF6690, MF6692, MF6700, MF6706, MF6714, MF6721, MF6722, MF6724 ...5, MF6726, MF6727, MF6728, MF6729, MF6730, MF6637, MF6643, MF6645, MF6648, MF6655, MF6658, MF6660, MF6675, an OX40-binding variable domain comprising a VH region having the amino acid sequence of the VH of MF6728, MF6729, MF6826, MF6940, MF6942, MF6943, MF6944, MF6947, MF6949, MF7331, MF7332, MF7334, MF7341, MF7345, MF7350, MF7351, MF7352, MF7353, MF7356, MF7358, MF7365, MF7366, MF7371, MF7372, MF7374, MF7378, MF7382, MF7383, MF7394, MF7395, or MF7397; PD-L1 binding variable domain and The PD-L1-binding variable domain preferably comprises a VH region having the amino acid sequence of the VH of MF5594, MF5424, MF5426, MF5553, MF5442, MF5561, MF5426, or MF5439 (Figure 3), which has up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, relative to the amino acid sequence of the VH of the shown MF (Figure 3).

[0285] The referenced up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid substitutions in the referenced H, VL, L, and VL regions are preferably conservative amino acid substitutions, and the insertions, deletions, substitutions, or combinations thereof are preferably not present in the CDR3 regions of the H, VL, L, and VL chains, preferably not present in the CDR1, CDR2, or CDR3 regions of the VH or VL chains, or preferably not present in the FR4 region.

[0286] Although features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the present invention may include embodiments having all or any combination of the described features.

[0287] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention but are provided merely to clarify the invention. [Brief explanation of the drawings]

[0288] [Figure 1] Common light chain used in monospecific and bispecific IgGs. Figure 1A: Common light chain amino acid sequence. Figure 1B: DNA sequence and translation of the common light chain variable domain (IGKV1-39 / jk1). Figure 1C: DNA sequence and translation of the common light chain constant region. Figure 1D: IGKV1-39 / jk5 common light chain variable domain translation. Figure 1E: V region IGKV1-39A. [Figure 2A] Figure 2 shows an IgG heavy chain for the generation of bispecific molecules. VH is a nucleic acid encoding the amino acid sequence for MF shown in Figure 3. [Figure 2B] This is the CH1 region. [Figure 2C] This is the hinge region. [Figure 2D] This is the CH2 region. [Figure 2E] CH2 containing L235G and G238R substitutions. [Figure 2F] The CH3 domain contains the substitutions L351K and T366K (KK). [Figure 2G] This is the CH3 domain containing the substitutions L351D and L368E(DE). [Figure 3-1] Figure 3A shows the amino acid sequences of the heavy chain variable regions. Figure 3A shows the VH sequences of CD137-specific clones. Figure 3B shows the VH sequences of PD-L1-specific clones. Figure 3C shows the VH sequences of OX40-specific clones. Figure 3D shows the VH sequences of PD-L1-specific clones. The notation MF refers to a fab containing the indicated heavy chain variable region and common light chain. The amino acid sequence of the light chain is shown in Figure 1A. The underlined sequences indicate the CDR1, CDR2, and CDR3 regions for each amino acid sequence, according to Kabat numbering. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Figure 3-4] This is a continuation of Figure 3-3. [Figure 3-5] This is a continuation of Figure 3-4. [Figure 3-6] This is a continuation of Figure 3-5. [Figure 3-7] This is a continuation of Figure 3-6. [Figure 3-8] This is a continuation of Figure 3-7. [Figure 3-9] This is a continuation of Figure 3-8. [Figure 3-10] This is a continuation of Figure 3-9. [Figure 3-11] This is a continuation of Figure 3-10. [Figure 3-12] This is a continuation of Figure 3-11. [Figure 3-13] This is a continuation of Figure 3-12. [Figure 3-14] This is a continuation of Figure 3-13. [Figure 4] Vector map and characteristics of pIRES-Neo3 (MV1363). [Figure 5] 1 is a vector map and characteristics of pVAX1. [Figure 6]1 is a vector map and characteristics of the phagemid vector MV1473 used to generate the "immune" phage display library. [Figure 7] Vector map and characteristics of IgG expression vectors MV1452 or MV1453, used to express CD137-, PD-1-, PD-L1-, and OX40-specific Fab arms in KK mutant or DE mutant heavy chains, respectively, for bispecific IgG generation. [Figure 8] Figure 1 shows the amino acid sequence of the VH gene present in the DE mutant heavy chain that was used to generate the PD-L1xTT bispecific IgG molecule that is specific for tetanus toxin when combined with a common light chain such as MF1337. The underlined sequences indicate the CDR1, CDR2, and CDR3 regions of each amino acid sequence. [Figure 9] Vector map and characteristics of the IgG expression vector MV1377, used to express the TT-specific Fab arm MF1337 in a DE mutant heavy chain for bispecific IgG generation. [Figure 10] PD-1 / PD-L1 blocking assay. Evaluation of the ability of a panel of anti-PD-L1 antibodies to block the interaction of PD-1 with coated PD-L1 at a bispecific IgG concentration of 10 μg / ml. Data are normalized to data obtained with the bivalent benchmark PD-L1 antibody MPDL3280A at a concentration of 10 μg / ml (100% blocking). A representative example of a PD-L1 panel is shown. Maximum binding (normalized to 0% blocking) was established by incubation with a non-PD-1 / PD-L1-specific human isotype antibody. All PD-L1 variable domains containing the MF sequence shown in Figure 3, but not represented here, block the PD-1 / PD-L1 interaction by >70%. [Figure 11] Activation of CD137 in Jurkat CD137-NFkBluc cells by bivalent CD137 antibodies. [Figure 12]Activation of CD137 in Jurkat CD137-NFkBluc cells by CD137xPD-L1 antibody in the absence (left) or presence (right) of IgG cross-linking antibody. MF numbers refer to the FaB of CD137 present in the CD137xPD-L1 bispecific antibody. [Figure 13] Activation of primary T cells by a bivalent CD137 antibody (top) or a monovalent antibody (bottom) combined with the Fab arm of PD-L1 (MF5594) as measured by IL-2 release. PG6744 is a bivalent CD137 antibody containing two MF6744 arms (also designated 6744x6744). PG6783 is a bivalent CD137 antibody containing two MF6783 arms (also designated 6783x6783). PG6860 is a bivalent CD137 antibody containing two MF6860 arms (also designated 6860x6860). 20H4.9 is the anti-CD137 reference antibody based on WO 2005 / 035584. [Figure 14] Activation of CD137 on Jurkat CD137-luc cells in the presence of PD-L1-overexpressing CHO cells or CHO wild-type cells. CD137 activation was measured by luciferase expression. PG6744 is a bivalent CD137 antibody (6744 x 6744). PB14671 is a bispecific CD137 x PD-L1 antibody (6744 x 5361). PB14580 is a bispecific CD137 x PD-L1 antibody (6744 x 5594). PB14890 is a bispecific CD137 x TT antibody (6744 x 1337). PG6783 is a bivalent CD137 antibody (6783 x 6783). PB14681 is a bispecific CD137 x PD-L1 antibody (6783 x 5361). PB14590 is a bispecific CD137xPD-L1 antibody (6783x5594). PB15855 is a bispecific CD137xTT antibody (6783x1337). 20H4.9 is an anti-CD137 reference antibody based on WO 2005 / 035584. [Figure 15]Activation of primary T cells with a combination of bivalent CD137 antibodies, CD137xPD-L1 bispecific antibodies, or CD137xPD-L1 Oligoclonics® in the presence of PD-L1-overexpressing CHO cells or wild-type CHO cells. Activation was measured by IL-2 release. PG6744 is a bivalent CD137 antibody (6744x6744). PB14671 is a bispecific CD137xPD-L1 antibody (6744x5361). PB14580 is a bispecific CD137xPD-L1 antibody (6744x5594). PB14890 is a bispecific CD137xTT antibody (6744x1337). 20H4.9 is the anti-CD137 reference antibody based on WO 2005 / 035584. MOR7480 is an anti-CD137 reference antibody based on US Pat. No. 8,337,850. [Figure 16] SEB stimulation of IL-2 production is enhanced by anti-CD137xPD-L1 bispecific antibodies or anti-CD137xPD-L1 Oligoclonics® in healthy donor blood cells. PB14580 is a bispecific CD137xPD-L1 antibody (6744x5594). PB14671 is a bispecific CD137xPD-L1 antibody (6744x5361). MPDL3280A is an anti-PD-L1 reference antibody based on WO 2010 / 077634. ​​PB9469 is a bispecific PD-L1xTT antibody (5594x1377). PB14890 is a bispecific CD137xTT antibody (6744x1337). 20H4.9 is an anti-CD137 reference antibody based on WO 2005 / 035584. Ctrl Ab is PG2708p213, anti-RSV-G. [Figure 17] We show that SEB stimulation of IL-2 production in blood cells from healthy donors is dramatically enhanced by the anti-CD137xPD-L1 bispecific antibody compared to the anti-CTLA-4 antibody 10D1 (based on ipilumumab). [Figure 18]Activation of OX-40 on Jurkat OX-40 NFkB-luc cells in the presence of PD-L1-overexpressing CHO cells (left panel) or wild-type CHO cells (right panel). Activation was determined by measuring luciferase expression. PD-L1 Fab arm MF5561, PD-1 Fab arm MF6256 (sequence shown in Figure 43). [Figure 19A] Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19B] Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19C]Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19D] Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19E] Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19F]Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Figure 19G] Screening of CD137xPD-L1 antibodies in a T cell activation assay (12 CD137 Fab arms). T cells from a single donor were stimulated for 72 hours at 37°C with a dose-dependent titration of the antibody panel shown below in the presence of PD-L1-overexpressing CHO cells (top panel) or CHO wild-type cells (bottom panel). CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was 20H4.9 (referred to in this figure as PG6619), and the anti-TT negative control antibody was PG1337 (Neg Ctrl Ab). [Table 1] [Figure 20A] Screening of CD137xPD-L1 antibodies in the SEB PBMC assay (12 CD137 Fab arms). CD137xPD-L1 antibodies were tested in the SEB PBMC assay in the presence of 2 μg / ml SEB. CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was an anti-CTLA-4 positive control antibody (based on ipilimumab, 10D1), and the anti-RSV-G negative control antibody was PG2708 (Neg Ctrl Ab). [Figure 20B]Screening of CD137xPD-L1 antibodies in the SEB PBMC assay (12 CD137 Fab arms). CD137xPD-L1 antibodies were tested in the SEB PBMC assay in the presence of 2 μg / ml SEB. CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was an anti-CTLA-4 positive control antibody (based on ipilimumab, 10D1), and the anti-RSV-G negative control antibody was PG2708 (Neg Ctrl Ab). [Table 2] [Figure 21] Screening of CD137xPD-L1 antibodies in the SEB PBMC assay (8 CD137 Fab arms). CD137xPD-L1 antibodies were tested in the SEB PBMC assay in the presence of 2 μg / ml SEB. CD137 activation was measured by IL-2 release using AlphaLISA and expressed as IL-2 counts. The positive control antibody was an anti-CTLA-4 positive control antibody (based on ipilimumab, 10D1), and the anti-RSV-G negative control antibody was PG2708 (Neg Ctrl Ab). [Table 3] [Figure 22] Figure 1 shows that bispecific anti-CD137xPD-L1 antibodies and their parent bivalent anti-CD137 antibodies bind to human and cynomolgus monkey CD137 as determined by flow cytometry. [Figure 23] Figure 2 shows that a bispecific anti-CD137xPD-L1 antibody and its parent bivalent anti-PD-L1 antibody bind to human and macaque PD-L1 as determined by flow cytometry. [Figure 24] We demonstrate that bispecific anti-CD137xPD-L1 antibodies and their parent bivalent antibodies bind to activated T cells as determined by flow cytometry. [Figure 25]Figure 1 shows that bispecific anti-CD137xPD-L1 antibodies and their parent bivalent anti-PD-L1 antibodies block PD-L1 ligand binding as determined by ELISA. [Figure 26] Figure 1 shows that bispecific anti-CD137xPD-L1 antibodies and their parent bivalent anti-PD-L1 antibodies block CD137 ligand binding as determined by flow cytometry. [Figure 27] Figure 2 shows that bispecific anti-CD137xPD-L1 antibodies and their parent bivalent antibodies block the interaction between PD-L1 and PD-1 in an in vitro blocking reporter assay. [Figure 28A] Figure 1 shows the transactivation of CD137 on Jurkat CD137-luc cells in the presence of CHO cells expressing different PD-L1 binding sites per cell compared to CHO wild-type cells. CD137 activation was measured by luciferase expression. [Figure 28B] Figure 1 shows transactivation of CD137 on Jurkat CD137-luc cells in the presence of human tumor cells expressing different PD-L1 binding sites per cell. CD137 activation was measured by luciferase expression. [Figure 28C] Figure 1 shows CD137 transactivation on Jurkat CD137-luc cells in the presence of CHO-PD-L1, ES-2, or CHO wild-type cells. IgGs were tested in triplicate at 10 μg / ml. CD137 activation was measured by luciferase expression. The antibodies tested and their compositions are listed below. [Table 4] [Figure 29] Figure 1 shows a comparison of CD137xPD-L1 antibodies with single and combined reference controls in a T cell activation assay. CD137 activation was measured as IL-2 and TNFα cytokine release, measured by Luminex analysis. [Figure 30A]Figure 1 shows the activity of the CD137xPD-L1 antibody PB17311 compared to single and combination reference control antibodies in a T cell activation assay. CD137 activation was measured as multiple cytokine release, measured by Luminex analysis (25plex). [Figure 30B] Figure 1 shows the activity of the CD137xPD-L1 antibody PB17311 compared to single and combination reference control antibodies in a T cell activation assay. CD137 activation was measured as multiple cytokine release, measured by Luminex analysis (25plex). [Figure 31] Figure 1 shows that bispecific anti-CD137xPD-L1 antibodies consistently enhance IL-2 release by PBMCs in an SEB stimulation assay, regardless of PBMC donor or SEB concentration. CD137 activation was measured as IL-2 release, measured by Luminex analysis. [Figure 32] These results show that bispecific anti-CD137 x PD-L1 antibodies are more potent than anti-CD137 benchmark antibodies or an equimolar mixture of anti-CD137 and anti-PD-L1 benchmark antibodies in enhancing cytokine release in an SEB stimulation assay. CD137 activation was measured as IL-2, IFNγ, and TNFα cytokine release, measured by Luminex analysis. [Figure 33] Figure 1 shows that PB17311 inhibits M2 macrophage-mediated suppression of anti-CD3 / CD28-stimulated PBMCs as demonstrated by enhanced IFNγ release. [Figure 34] 1 shows that PB17311 enhances T cell proliferation after CD8+ T cell priming. [Figure 35] This shows that PB17311 enhances the differentiation of naive T cells into central memory T cells and effector T cells after priming. TN / SCM: naive / stem cell memory, TCM: central memory, TEM: effector memory, and TE: terminal effector cells. [Figure 36]Shows the effect of PB17311 on the expression of CD107a and cytokines in the total T cell population. TEM is effector memory and TE is terminal effector cell. [Figure 37] Shows the effect of PB17311 on the expression of CD107a and cytokines in the total T cell subset. [Figure 38] Shows the effect of PB17311 on the proliferation of tumor infiltrating CD4 and CD8 T cells derived from liver metastasis in colorectal cancer (LM-CRC) and hepatic carcinoma (HCC). [Figure 39] Shows the identification and visualization of important residues in CD137 of PB17311. (A) For each mutated clone, the average binding value is plotted as a function of the average CD137 expression value (gray circles) of the clone measured by the binding of the control antibody. Binding is represented as a percentage obtained with the WT clone. The dotted line indicates the threshold used to identify important clones (black dots). (B) The table lists the average binding reactivity (and range) for all identified important residues. Residues important for PB17311 Ab binding (black outlined) were negative for PB17311 Ab binding (<20% of binding to WT), but positive for the control antibody, 555955 MAb (>70% of WT). (C) Important residues (surrounded by the outline) are visualized on a CD137 model (PDB ID#2HEY, Compaan et al., 2006) based on the structure of murine OX40L bound to human OX40. The unvalidated residue, C133, is shown in gray. [Figure 40] Shows the effect of the CD137×PD-L1 bispecific antibody PB17311 on the median tumor volume on day 19 in a xenograft mouse model. MTV is the median tumor volume and TGI is tumor growth inhibition. When compared to group 1, statistical significance in the Mann-Whitney test is indicated by *(0.01 < P < 0.05) and ***(P < 0.001). [Figure 41]Figure 1 shows interference of sCD137 with T cell activation. Figure 1 shows an assessment of the effect of soluble CD137 on the ability of a bispecific CD137xPD-L1 antibody to activate human primary T cells. [Figure 42] The amino acid sequence of the CD137 extracellular domain is shown. [Figure 43] The amino acid sequence of MF6256 is shown. [Example]

[0289] As used herein, "MFXXXX" (wherein X is independently a number from 0 to 9) refers to a Fab comprising a variable domain, where VH has the amino acid sequence identified by the four digits. Unless otherwise noted, the light chain variable region of the variable domain typically has the sequence of Figure 1A, typically the sequence of Figure 1B. "MFXXXX VH" refers to the amino acid sequence of VH identified by the four digits. MF further comprises a light chain constant region and a heavy chain constant region that normally interacts with the light chain constant region. PG refers to a monospecific antibody comprising identical heavy and light chains. PB refers to a bispecific antibody having two different heavy chains. The variable regions of the heavy chains (VH) are different, typically CH3 regions, with one of the heavy chains having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see reference PCT / NL2013 / 050294 (published as WO 2013 / 157954)).

[0290] Example 1 Generation of materials for selection and screening Cell line culture Human ES-2 cells (catalog no. CRL-1978) were purchased from ATCC and routinely maintained in McCoy's 5A (Gibco) supplemented with 10% FBS (Lonza). Freestyle 293F cells (catalog no. p / n51-0029) were obtained from Invitrogen and routinely maintained in 293 Freestyle medium. HEK293T (catalog no. ATCC-CRL-11268) and CHO-K1 (catalog no. DSMZ ACC110) cell lines were purchased from ATCC and routinely maintained in DMEM / F12 (Gibco) supplemented with L-glutamine (Gibco) and FBS (Lonza).

[0291] Generation of OX40, CD137, and PD-L1 expression vectors for immunization and generation of stable cell lines Full-length cDNAs for each target, including unique restriction sites for cloning and Kozak consensus sequences for efficient translation, were synthesized using specific primers that introduced unique restriction sites for cloning and Kozak consensus sequences for efficient translation, or obtained via PCR amplification on commercially available expression constructs containing the target cDNA. The cDNAs for each target were cloned via Nhel / EcoRI into eukaryotic expression constructs such as pIRES-Neo3 (Clontech; Figure 4) or pVAX1 (Thermo Fisher Scientific; Figure 5), resulting in pIRES-Neo3_[TARGET_NAME] and pVAX1_[TARGET_NAME], respectively. The insert sequences were verified by comparison with the NCBI reference amino acid sequence. The pIRES-Neo3 construct was used for stable cell line generation. The pVAX1 construct was used for immunization purposes. See Table 1 for a summary of the resulting construct names.

[0292] The amino acid sequence of the full-length huCD137 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: NP_001552.2) is: MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0293] Among these, MGNSCYNIVATLLLVLNFERTRS is the signal peptide.

[0294] LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ is the ECD of huCD137.

[0295] IISFFLALTSTALLFLLFFLTLRFSVV is the predicted TM region.

[0296] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL is the intracellular tail.

[0297] The amino acid sequence of the full-length macaque (Macaca fascicularis) CD137 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: ABY47575.1) was MGNSCYNIVATLLLVLNFERTRSLQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQIIFFLALTSTVVLFLLFFLVLRFSVVKRSRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0298] Among these, MGNSCYNIVATLLLVLNFERTRS is the signal peptide.

[0299] LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQ is the ECD of maCD137.

[0300] IIFFLALTSTVVLFLLFFLVLRFSVV is the predicted TM region.

[0301] KRSRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL is the intracellular tail.

[0302] The amino acid sequence of the full-length rat CD137 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: XP_008762505.1) was MGSSCYNMVVTVLLVVGTEEVRATRNPCDSCEAGTFCSKYPPVCTSCPPSTYSSTGGQPNCDICRVCQGYFRFKKPCSSTHNAECECVEGFHCLGPKCTRCEKDCRPGQELTEQGCKNCGLGTFNDQDGAGVCRPWTNCSLDGRSVLKNGTKEKDVVCGPPVVSLSPSTTPSAVTTPERESGERPLQVLTLFLALTLALLLFLIFIILWFSVPKWLRKKFPHIFKQPFKKAVRTAQEEDACSCRFPEEEEGGGGSYEL.

[0303] Among these, MGSSCYNMVVTVLLVVGTEEVRA is the signal peptide.

[0304] TRNPCDSCEAGTFCSKYPPVCTSCPPSTYSSTGGQPNCDICRVCQGYFRFKKPCSSTHNAECECVEGFHCLGPKCTRCEKDCRPGQELTEQGCKNCGLGTFNDQDGAGVCRPWTNCSLDGRSVLKNGTKEKDVVCGPPVVSLSPSTTPSAVTTPERESGERPLQ is the ECD of raCD137.

[0305] VLTLFLALTLALLLFLIFIILWF is the predicted TM region.

[0306] SVPKWLRKKFPHIFKQPFKKAVRTAQEEDACSCRFPEEEEGGGGSYEL is the intracellular tail.

[0307] The amino acid sequence of the full-length huPD-L1 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: AAI13735.1) is: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET.

[0308] Among these, MRIFAVFIFMTYWHLLNA is the signal peptide.

[0309] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER is the ECD of huPD-L1.

[0310] THLVILGAILLCLGVALTFIF is the predicted TM region.

[0311] RLRKGRMMDVKKCGIQDTNSKKQSDTHLEET is the intracellular tail.

[0312] The amino acid sequence of the full-length macaque (Macaca fascicularis) PD-L1 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: ABO33161.1) was MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTIECRFPVEKQLGLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLGPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKSGIRVTNSKKQRDTQLEET.

[0313] Among these, MRIFAVFIFTIYWHLLNA is the signal peptide.

[0314] FTVTVPKDLYVVEYGSNMTIECRFPVEKQLGLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLGPEENHTAELVIPELPLALPPNER is the ECD of maPD-L1.

[0315] THLVILGAIFLLLGVALTFIF is the predicted TM region.

[0316] YLRKGRMMDMKKSGIRVTNSKKQRDTQLEET is the intracellular tail.

[0317] The amino acid sequence of the full-length human OX40 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: NP_003318.1) was MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI.

[0318] Among these, MCVGARRLGRGPCAALLLLGLGLSTVTG is the signal peptide.

[0319] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA is an ECD.

[0320] VAAILGLGLVLGLLGPLAILL is the predicted TM region.

[0321] ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI is the intracellular tail.

[0322] The amino acid sequence of the full-length rat (Rattus norvegicus) OX40 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: NP_037181.1) was MYVWVQQPTAFLLLGLSLGVTVKLNCVKDTYPSGHKCCRECQPGHGMVSRCDHTRDTVCHPCEPGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTEDTVCQCRPGTQPRQDSSHKLGVDCVPCPPGHFSPGSNQACKPWTNCTLSGKQIRHPASNSLDTVCEDRSLLATLLWETQRTTFRPTTVPSTTVWPRTSQLPSTPTLVAPEGPAFAVILGLGLGLLAPLTVLLALYLLRKAWRSPNTPKPCWGNSFRTPIQEEQTDTHFTLAKI.

[0323] Among these, MYVWVQQPTAFLLLGLSLG is the signal peptide.

[0324] VTVKLNCVKDTYPSGHKCCRECQPGHGMVSRCDHTRDTVCHPCEPGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTEDTVCQCRPGTQPRQDSSHKLGVDCVPCPPGHFSPGSNQACKPWTNCTLSGKQIRHPASNSLDTVCEDRSLLATLLWETQRTTFRPTTVPSTTVWPRTSQLPSTPTLVAPEGP is an ECD.

[0325] AFAVILGLGLGLLAPLTVLLALYLL is the predicted TM region.

[0326] RKAWRSPNTPKPCWGNSFRTPIQEEQTDTHFTLAKI is the intracellular tail.

[0327] The amino acid sequence of the full-length macaque (Macaca fascicularis) OX40 insert (both pIRES-Neo3 and pVAX1) for cell surface expression (identical to GenBank: XP_005545179.1) was MCVGARRLGRGPCAALLLLGLGLSTTAKLHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVAAILGLGLALGLLGPLAMLLALLLLRRDQRLPPDAPKAPGGGSFRTPIQEEQADAHSALAKI.

[0328] Among these, MCVGARRLGRGPCAALLLLGLGLSTTAK is the signal peptide.

[0329] LHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPA is an ECD.

[0330] VAAILGLGLALGLLGPLAMLL is the predicted TM region.

[0331] ALLLLRRDQRLPPDAPKAPGGGSFRTPIQEEQADAHSALAKI is the intracellular tail.

[0332] Generation of stable cell lines expressing CD137, OX40, or PD-L1 The pIRES-Neo3_[TARGET_NAME] expression constructs (Table 1) were used to generate Freestyle 293F or CHO-K1 clones stably expressing the respective proteins. The constructs were transiently transfected into CHO-K1 cells using lipofectamine transfection or into Freestyle 293F cells using PEI transfection and screened by FACS using antibodies reactive with the respective proteins. After confirming expression, the transiently transfected cells were plated in limiting dilutions and cultured under the selection pressure associated with the expression construct used to obtain stable cell clones. After 2–3 weeks of selection, the clones were screened by FACS. Selected clones were expanded by serial passage, retested by FACS, and frozen at -150°C. Clones stably expressing heterologous proteins are designated CHO-K1_[TARGET_NAME] cells or Freestyle 293F_[TARGET_NAME] cells. See Table 1 for a summary of the constructs used to generate stable cell lines and the resulting designations.

[0333] Example 2 Immunization, selection, and screening Mice used for immunization For the generation of human antibodies that bind to huCD137, huOX40, and huPD-L1, mice transgenic for the human VK1-39 light chain (common light chain mice, see WO 2009 / 157771) and for the human heavy chain (HC) minilocus (containing a selection of human V gene segments, all human Ds, and all human Js) were immunized with either recombinant proteins or DNA encoding the proteins, as described briefly below. These mice are referred to as "MeMo®" mice.

[0334] Protein immunization MeMo® mice were immunized by subcutaneous injection with recombinant proteins and Gerbu adjuvant MM (Gerbu Biotechnik c#3001). Recombinant huPD-L1-His (SinoBiological, Catalog No. 10084-H08H), huOX40-Fc (R&D, Catalog No. 3388-OX), and huOX40-His (SinoBiological, Catalog No. 10481-H08H) proteins were used for immunization. Protein immunizations were not performed for CD137 antibody panel generation. Mice were immunized with 40 μg of recombinant protein in PBS mixed with 40 μl of adjuvant in a total volume of 100 μl. Subsequently, on days 14 and 28, mice were boosted with 20 μg of recombinant protein in PBS mixed with 20 μl of adjuvant in a total volume of 50 μl. Mouse serum was collected on day 35, and serum titers were determined. Mice with low serum titers underwent additional cycles of booster immunizations and serum analysis. Each cycle consisted of twice-weekly immunizations using 20 μg of recombinant protein in 50 μl of PBS, followed by serum collection for titer analysis one week later. Mice showing high serum titers against human and macaque targets received a final booster immunization consisting of daily injections of 20 μg of recombinant protein in 50 μl of PBS for three consecutive days. Mouse lymphoid tissues were collected one day after the final injection.

[0335] DNA immunization MeMo® mice were immunized by DNA tattooing using a micropigmentation device. DNA tattoo immunization was performed using 20 μg of plasmid DNA (pVAX1_[TARGET_NAME], Table 1) encoding the target antigen. Mice were immunized with DNA encoding the human target only (PD-L1) or by alternating immunization with DNA encoding human and rat targets (CD137, OX40) to obtain species-cross-reactive antibodies. For PD-L1 immunization, Treg cells were depleted 4 days before the start of immunization by injecting mice with 0.5 mg of the anti-CD25 antibody PC61.5 (Bioceros) to break tolerance. Mice were immunized on days 0, 3, 6, 14, 17, 28, and 31. Mouse serum was collected on day 35, and serum titers were determined. Mice with low seroreactivity to the human and / or macaque targets received additional cycles of booster immunizations with human, rat, or macaque DNA antigens and serum analysis was performed. Each cycle consisted of twice-weekly DNA immunizations followed by serum collection for titer analysis one week later. Mice showing strong seroreactivity to cells expressing the human and macaque targets received a final booster immunization, after which lymphoid tissues were collected three days later.

[0336] Combined protein and DNA immunization (OX40 only) Mice were immunized with recombinant huOX40-His (SinoBiological, catalog no. 10481-H08H) and boosted with alternating DNA (pVAX1_raOX40) and protein (huOX40-His) immunizations to obtain species-cross-reactive antibodies. Thus, mice were immunized with 40 μg of recombinant protein in PBS mixed with 40 μl of adjuvant in a total volume of 100 μl. Subsequently, on days 14 and 17, mice were boosted with 20 μg of pVAX1_raOX40 via DNA tattooing, followed by a protein immunization on day 28 with 20 μg of huOX40-His protein in PBS mixed with 20 μl of adjuvant in a total volume of 50 μl. Mouse serum was collected on day 35, and serum titers were determined. Mice with low human and / or macaque serum titers underwent additional cycles of booster immunizations and serum analysis. Each cycle consisted of twice-weekly protein or DNA immunizations with 20 μg of huOX40-His, pVAX1_raOX40, or pVAX1_maOX40, followed by serum collection for titer analysis one week later. Mice showing high serum titers against the human and macaque targets received a final boost consisting of daily injections of 20 μg of recombinant protein in 50 μl of PBS for three consecutive days. Mouse lymphoid tissues were harvested one day after the final injection.

[0337] Determination of serum titers Serum titers were determined by FACS analysis using cell lines expressing human and macaque target antigens (Table 1).

[0338] Generation of synthetic phage Fab libraries Synthetic libraries were constructed based on a repertoire of germline human VH genes selected for their frequent use in the natural repertoire and canonical sequence diversity. Synthetic HCDR3 regions were added to these VH genes using PCR. This was achieved using a forward primer that anneals to framework 1 of the VH genes and contains an Sfil restriction site for cloning. The reverse primer contained a sequence for annealing to framework 3 of the VH genes, followed by a randomized sequence encoding HCDR3 diversity and framework 4, which also contains BstEII and XhoI restriction sites for cloning. The synthetic CDR3 regions were either completely random or encoded more restricted diversity based on the frequency of amino acid residues at specific positions within the HCDR3. PCR products encoding the VH genes, including the consensus light chain-encoding gene, were cloned into a phage display vector in fusion with the phage M13 gene 3 protein using the aforementioned restriction enzymes. Large-scale ligation and transformation of E. coli TG1 resulted in a large library of synthetic Fab fragments displayed on phage that were used for panning on antigens or cells to identify antigen-specific Fab fragments.

[0339] Generation of "immune" phage Fab libraries by RT-PCR from tissues of immunized mice By removing the spleen and draining lymph nodes from mice, a significant humoral response was observed against the corresponding target protein.

[0340] Single cell suspensions were generated from both splenic and inguinal lymph nodes, and these tissues were subsequently lysed in Trizol LS reagent (Thermo Scientific c#10296028) and stored at -80°C until use.

[0341] Inguinal lymph nodes from successfully immunized mice were used to construct the "immune" phage antibody repertoire. RNA was extracted from single-cell suspensions of lymphoid tissue. 1 μg of total RNA was used in a RT reaction using IgG-CH1-specific primers. The resulting cDNA was then used to amplify a polyclonal pool of VH-encoding cDNAs using in-house adapted VH-specific primers, essentially as described by Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581-97). The resulting PCR products were then cloned into a phagemid vector (Figure 6) for displaying Fab fragments on phage, as described by de Haard et al. (J Biol Chem. 1999 Jun 25;274(26):18218-30), except that the light chain (Figure 1A and 1B) was the same for all antibodies and encoded by the vector. After ligation, the phagemids were used to transform E. coli TG1 bacteria, and the transformed bacteria were plated on LB-agar plates containing ampicillin and glucose. All phage libraries contained >4 x 10 5 The transformants contained 10 transformants and had an insert frequency of >90%. Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (de Haard et al., J Biol Chem. 1999 Jun 25;274(26):18218-30).

[0342] Selection of phages bearing Fab fragments that specifically bind to human target proteins from synthetic and "immune" phage Fab libraries using recombinant proteins The generated phage Fab library was used to select target-specific Fabs using phage display on directly coated recombinant proteins. For PD-L1, huPD-L1-His (Sinobiological, Catalog No. 10084-H08H), huPD-L1-Fc (R&D, Catalog No. 156-B7), and maPD-L1-His (Sinobiological, Catalog No. 90251-C08H) were used. For CD137, huCD137-Fc (R&D, catalog number 838-4B), raCD137-Fc (R&D, catalog number 7968-4B), moCD137-Fc (R&D, catalog number 937-4B), huCD137-His (Sinobiological, catalog number 10041-H08H), and huCD137-Fc (Enzo, catalog number ALX-522-031-C050) were used. For OX40, huOX40-Fc (R&D, catalog number 3388-OX) and huOX40-His (Sinobiological, catalog number 10481-H08H) were used.

[0343] For selections using recombinant proteins, proteins were coated onto the wells of MAXISORP™ ELISA plates. MAXISORP™ ELISA plates were blocked with 4% dry nonfat dry milk powder (Marvel) in PBS. Phage Fab libraries were also blocked with 4% Marvel, and when Fc-tagged recombinant proteins were used, an excess of human IgG was used to deplete Fc-region binders before adding the phage library to the coated antigen.

[0344] Incubation of the phage library with the coated protein was carried out for 1.5 hours at room temperature under shaking conditions. The plates or tubes were then washed 15 times with 0.05% Tween-20 in PBS, followed by 5 washes with PBS. Bound phages were eluted with trypsin for 20 minutes, after which the trypsin was neutralized with AEBSF trypsin inhibitor (Sigma).

[0345] The eluate was added to E. coli TG-1 and incubated at 37° C. for phage infection. Subsequently, the infected bacteria were plated onto agar plates containing ampicillin and glucose and incubated overnight at 37° C. Single clones from the selection output were screened for target binding in ELISA or FACS, depending on the target.

[0346] For selections using synthetic phage Fab libraries, a second round of selection was performed after rescue of the first round selection output using the same protocol outlined above for the first round selection.

[0347] Selection of phages carrying Fab fragments that specifically bind to human targets from an "immune" phage Fab library using cells stably expressing the target protein Phage Fab libraries generated from target-immunized mice were selected using phage display on cells expressing each target. Stable cell lines expressing CD137, OX40, or PD-L1 (Table 1) were used for the first round of selection. Cells were blocked with 10% FBS in PBS. After blocking, rescued phages were incubated with the blocked cells. The cells and phages were incubated at 4°C for 1 hour. Cells were washed five times with 1 ml of 10% FBS in PBS. Bound phages were eluted with trypsin for 20 minutes, after which the trypsin was neutralized with AEBSF trypsin inhibitor (Sigma). The eluate was added to E. coli TG-1 and incubated at 37°C for phage infection. Subsequently, the phage-infected bacteria were plated on agar plates containing ampicillin and glucose and incubated overnight at 37°C.

[0348] For PD-L1, a second round of selection using ES-2 cells that endogenously express huPD-L1 was performed using the same protocol as that used for the first round of selection. After selection, single clones were screened for target binding by FACS.

[0349] Screening of target-specific Fab clones in ELISA Soluble Fab or phage were prepared from single clones (J Mol Biol. 1991 Dec 5;222(3):581-97; J Biol Chem. 1999 Jun 25;274(26):18218-30). The resulting soluble Fab or phage samples were diluted (1:5 or 1:10, respectively) in 4% dried skim milk (Marvel) in PBS (blocking buffer) and tested by ELISA for binding to wells coated with the same antigen used for selection, or to wells coated with huCD137-Fc (R&D, catalog no. 838-4B) for all selection outputs performed with either raCD137-Fc (R&D, catalog no. 7968-4B) or moCD137-Fc (R&D, catalog no. 937-4B).

[0350] Bound FaB was detected by staining with anti-myc antibody (Roche, Cat. No. 11667203001) diluted 1:1000 in blocking buffer, followed by HRP-conjugated anti-mouse IgG antibody (Jackson Immunoresearch, Cat. No. 715-035-150) diluted 1:5000 in blocking buffer. Bound phage was detected by staining with HRP-conjugated monoclonal anti-M13 antibody (GE Healthcare, Cat. No. 27-9421-01) diluted 1:5000 in blocking buffer.

[0351] After each antibody staining, the wells were washed with PBS-T (PBS-0.05% (v / v) Tween 20). Bound secondary antibodies were visualized by TMB / H2O2 staining and OD 450nm Staining was quantified by measurement: clones were considered to bind to the target when the OD450nm was at least three times higher than the background signal obtained with the negative control Fab.

[0352] The VH-encoding cDNAs of all target-specific clones were sequenced, and a selection of unique clones based on sequence identity and cluster analysis were then analyzed by FACS for binding to the target expressed on cells, as described below for clones obtained from the cell selection output.

[0353] Screening of target-specific Fab clones in FACS Soluble Fabs or phages of single clones selected on cells expressing the corresponding targets were prepared as described (J Mol Biol. 1991 Dec 5;222(3):581-97; J Biol Chem. 1999 Jun 25;274(26):18218-30). Fab samples were tested for binding to cells expressing human and macaque targets by FACS by incubation with a mixture of Fab samples diluted 1:5 in FACS buffer and anti-myc antibody (Gentaur, catalog no. 04-CMYC-9E10) diluted 1:1000 (in PBS with 0.5% Hi-FBS) (Table 1). Bound Fab / anti-myc complexes were detected by incubation with APC-conjugated goat anti-mouse IgG antibody (BD Bioscience, catalog no. 550826) diluted 1:500 in FACS buffer.

[0354] Phage samples were tested for binding in FACS by diluting them 1:3 in blocking buffer and incubating with target-expressing cells for 1 hour. Bound phage were detected by staining with biotinylated anti-M13 antibody (Fitzgerald, catalog no. 61R-M101ABTB62-FEZ, 1:125 in FACS buffer, 30 minutes on ice) and PE-labeled streptavidin (Invitrogen, catalog no. SA1004-4, 1:400 in FACS buffer, 15 minutes on ice). After incubation with each antibody, wells were washed three times with FACS buffer. Stained cells were analyzed using a FACS Accuri C6 instrument (Becton and Dickinson). Clones were considered positive when the mean fluorescence intensity was at least three times greater than the background signal obtained with a negative control Fab.

[0355] result The VH sequences of 24 CD137-specific clones, 14 PD-L1-specific clones, and 50 OX40-specific clones obtained by the above-mentioned method are shown in Figure 3.

[0356] Example 3 Characterization of huCD137-, huOX40-, and huPD-L1-specific Fab clones in IgG format Recloning of human CD137, OX40, and PD-L1 specific Fabs into IgG format Unique clones selected based on differences in CDR3 sequences and VH germline sequences that bound to the human and macaque target proteins expressed on cells were then recloned into IgG expression plasmids, such as MV1452 (Figure 7), containing the consensus light chain (Figure 1), using Sfi1-BstEII digestion and ligation of the digested cDNA pool according to standard molecular biology techniques.

[0357] Expression of bispecific IgG containing human CD137, OX40, or PD-L1-specific Fab and tetanus toxin-specific Fab Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgGs with different VH domains, using a proprietary CH3 engineering technique to ensure efficient heterodimerization and formation of bispecific antibodies. A common light chain present on both heavy chain-containing plasmids is also co-transfected into the same cells. Co-pending applications of the present invention (e.g., WO 2013 / 157954 and WO 2013 / 157953, incorporated herein by reference) disclose methods and means for producing bispecific antibodies from a single cell, thereby providing a means for favoring the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can be suitably employed in the present invention. Specifically, preferred mutations for generating essentially only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbered according to EU numbering) ("KK-mutant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE-mutant" heavy chain), or vice versa. It has previously been demonstrated by a co-pending application of the present invention that negatively charged DE-mutant heavy chains and positively charged KK-mutant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-mutant heavy chains (DE-DE homodimers) or KK-mutant heavy chains (KK-KK homodimers) rarely occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0358] The VH genes encoding the antibodies binding to human CD137, OX40, and PD-L1 described above were cloned into an IgG expression vector such as MV1452, which encodes a positively charged CH3 domain. A tetanus toxin (TT)-targeting antibody (Figure 8) was cloned into the MV1377 IgG expression vector (Figure 9), which encodes a negatively charged CH3 domain. To express a CD137 antibody panel in IgG format, the entire panel can also be cloned into a negatively charged CH3 domain vector to produce a monospecific CD137xCD137 bivalent IgG.

[0359] 293F Freestyle cells adapted for suspension growth were cultured at 3.0 x 10 6 The cells were cultured in a T125 flask on a shaking plateau until a density of 0.3–0.5 × 10 cells / ml was reached. 0.3–0.5 × 10 cells were added to each well of a deep 24-well plate (24-well format). 6 Cells were seeded at a density of 1000 viable cells / ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies, cloned into a proprietary vector system. Seven days after transfection, cell supernatants were harvested and filtered through a 0.22 μM filter (Sartorius). Sterile supernatants were stored at 4°C until antibody purification.

[0360] (Bispecific) IgG purification Small-scale (<500 μg) IgG purification was performed using protein A affinity chromatography. Small-scale purification was performed under sterile conditions in 24-well filter plates using filtration. First, the pH of the medium was adjusted to pH 8.0. Subsequently, the IgG-containing supernatant was incubated with protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 hours at 25°C at 600 rpm on a shaking platform. The beads were then collected by filtration. The beads were washed twice with PBS pH 7.4. Bound IgG was then eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). Samples were finally collected in PBS pH 7.4. IgG concentration was measured using an Octet. Protein samples were stored at 4°C.

[0361] IgG quantification using Octet To determine the amount of purified IgG, total human IgG (Sigma Aldrich, catalog no. I4506) was used as a standard and the antibody concentration was determined by Octet analysis using a protein A biosensor (Forte-Bio, according to the supplier's recommendations).

[0362] Specificity analysis of huCD137×CD137 bivalent IgG and huOX40×TT and huPD-L1×TT bispecific IgG The huCD137xCD137 bivalent IgG and the huOX40xTT and huPD-L1xTT bispecific IgG were tested by FACS for binding to stable cell lines expressing the relevant human and macaque orthologues (Table 1) and wild-type cells. Ce...

Claims

1. 1. A method of stimulating the activity of a member of the TNF receptor superfamily on a cell, comprising providing a first cell and a second cell, wherein the first cell has the member on its cell membrane and the second cell has a second membrane protein on its cell membrane, the method comprising contacting the cells with a bispecific antibody comprising two variable domains, one variable domain comprising a first antigen binding site capable of binding to an extracellular portion of the member and another variable domain comprising a second antigen binding site capable of binding to an extracellular portion of the second membrane protein, thereby stimulating the activity of the member on the first cell.

2. The method of claim 1 , wherein the second membrane protein is not a member of the TNF receptor superfamily.

3. The method of claim 1 or 2, wherein the bispecific antibody comprises two antigen-binding sites.

4. 4. The method of claim 1, wherein the antigen-binding site of the bispecific antibody consists of one immunoglobulin variable domain capable of binding to the extracellular part of the member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to the second membrane protein.

5. The method of any one of claims 1 to 4, wherein the bispecific antibody is a full-length antibody.

6. The method of any one of claims 1 to 5, wherein the bispecific antibody is an IgG.

7. The method of any one of claims 1 to 6, wherein the first cell does not significantly express the second membrane protein on the cell membrane.

8. The method according to any one of claims 1 to 7, wherein the second membrane protein is a membrane protein present in one or more areas on the cell membrane.

9. 9. The method of claim 8, wherein the area is a cluster, domain, microdomain or compartment on the cell membrane, preferably an immune synapse.

10. 10. The method of any one of claims 1 to 9, wherein the second membrane protein is present on the cell membrane as part of a multimeric membrane protein comprising two or more instances of the second membrane protein.

11. The method of any one of claims 1 to 10, wherein the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

12. The method of any one of claims 1 to 11, wherein the second membrane protein is a member of the B7 family.

13. The method of claim 12, wherein the second membrane protein is PD-L1 or PD-L2, preferably PD-L1.

14. 14. The method of any one of claims 1 to 13, wherein the variable domain that binds to the member of the TNF receptor superfamily blocks binding of a ligand to the member.

15. 15. The method of any one of claims 1 to 14, wherein the variable domain that binds to the extracellular portion of the member of the TNF receptor superfamily is defined as a variable domain that does not stimulate activity of the TNF receptor superfamily member on a cell when in a bivalent monospecific antibody format comprising two of the variable domains that bind to the member of the TNF receptor superfamily.

16. providing a further bispecific antibody comprising an antigen-binding site capable of binding to the extracellular portion of said member of the TNF receptor superfamily and an antigen-binding site capable of binding to the extracellular portion of said second membrane protein, wherein said first and second bispecific antibodies a different epitope on the first membrane protein; a different epitope on the second membrane protein; or a different epitope on the first membrane protein and a different epitope on the second membrane protein binds to 16. The method of any one of claims 1 to 15, wherein the method further comprises stimulating the activity of the member of the TNF receptor superfamily on the first cell by incubating the first cell and second cell with the first and second bispecific antibodies.

17. 17. The method of claim 16, wherein the first and second bispecific antibodies each comprise one antigen-binding site capable of binding to the member of the TNF receptor superfamily.

18. 18. The method of claim 16 or 17, wherein the antigen binding sites of the first and second bispecific antibodies capable of binding to the second membrane protein bind to different epitopes on the extracellular portion of the second membrane protein.

19. 19. The method of any one of claims 16 to 18, wherein the different epitopes on the extracellular portion of the second membrane protein are non-competing epitopes.

20. The method of any one of claims 1 to 19, wherein the TNF receptor superfamily member is CD137 or OX40.

21. A bispecific antibody comprising an antigen-binding site capable of binding to the extracellular portion of CD137 and an antigen-binding site capable of binding to the extracellular portion of a second membrane protein.

22. 22. The bispecific antibody of claim 21 , wherein the second membrane protein is not a member of the TNF receptor superfamily.

23. 23. The bispecific antibody of claim 21 or 22, wherein the bispecific antibody comprises two antigen-binding sites.

24. 24. The bispecific antibody of any one of claims 21 to 23, wherein the antigen-binding site of the bispecific antibody consists of one immunoglobulin variable domain capable of binding to the extracellular part of the member of the TNF receptor superfamily and one immunoglobulin variable domain capable of binding to the second membrane protein.

25. The bispecific antibody of any one of claims 21 to 24, wherein the bispecific antibody is a full-length antibody.

26. The bispecific antibody of any one of claims 21 to 25, wherein the bispecific antibody is an IgG.

27. A bispecific antibody according to any one of claims 21 to 26, comprising one antigen-binding site capable of binding to CD137.

28. 28. The bispecific antibody of any one of claims 21 to 27, wherein the second membrane protein is not expressed to a significant extent by T cells.

29. 29. The bispecific antibody of any one of claims 21 to 28, wherein the second membrane protein is expressed on an antigen-presenting cell, a tumor cell, a virus-infected cell, or a parasite-infected cell.

30. 30. The bispecific antibody of any one of claims 21 to 29, wherein the second membrane protein is a membrane protein present in one or more areas on the cell membrane.

31. 31. The bispecific antibody of claim 30, wherein the area is a cluster, domain, microdomain or compartment on the cell membrane, preferably an immune synapse.

32. The bispecific antibody of any one of claims 21 to 31, wherein the second membrane protein is present on a cell membrane as part of a multimeric membrane protein comprising two or more of the second membrane proteins.

33. 33. The bispecific antibody of any one of claims 21 to 32, wherein the second membrane protein is present on the cell membrane as part of a homodimer or homotrimer.

34. 34. The bispecific antibody of any one of claims 21 to 33, wherein the second membrane protein is a member of the B7 family.

35. The bispecific antibody of claims 21 to 34, wherein the second membrane protein is PD-L1 or PD-L2, preferably PD-L1.

36. A bispecific antibody according to any one of claims 21 to 35, wherein the variable domain that binds to CD137 blocks binding of a ligand to said CD137.

37. 37. The bispecific antibody of any one of claims 21 to 36, wherein the variable domain that binds to the extracellular portion of CD137 is defined as a variable domain that does not stimulate the activity of CD137 on cells when in a bivalent monospecific antibody format comprising two of the variable domains that bind to CD137.

38. A composition or kit-of-parts comprising one or more bispecific antibodies according to any one of claims 21 to 37.

39. 38. A composition or kit-of-parts comprising two or more bispecific antibodies according to any one of claims 21 to 37, wherein the antigen-binding sites capable of binding to CD137 or OX40 of a first and a second bispecific antibody bind to different epitopes on CD137 or OX40.

40. 1. A method for stimulating the activity of CD137 on a cell, comprising: providing a first cell and a second cell, wherein the first cell has CD137 (a first membrane protein) on its cell membrane and the second cell has a second membrane protein on its cell membrane; the method comprising contacting the cells with a bispecific antibody (first bispecific antibody) comprising two variable domains, wherein one variable domain comprises a first antigen-binding site capable of binding to an extracellular portion of the first membrane protein and another variable domain comprises a second antigen-binding site capable of binding to an extracellular portion of the second membrane protein, thereby stimulating the activity of the first membrane protein on the first cell.

41. providing a further bispecific antibody (second bispecific antibody) comprising a variable domain comprising an antigen-binding site capable of binding to the extracellular portion of the first membrane protein and a variable domain comprising an antigen-binding site capable of binding to the extracellular portion of the second membrane protein, wherein the first and second bispecific antibodies are a different epitope on the first membrane protein; a different epitope on the second membrane protein; or a different epitope on the first membrane protein and a different epitope on the second membrane protein binds to 41. The method of claim 40, wherein the method further comprises stimulating the activity of CD137 on the first cell by incubating the first cell and the second cell with the first and second bispecific antibodies.

42. 42. The method of claim 40 or claim 41, wherein the second membrane protein is a member of the B7 family.

43. An antibody or a functional part, derivative and / or analogue thereof capable of binding to CD137 and PD-L1.

44. 44. The antibody or functional part or derivative or analogue of claim 43 which is a bispecific antibody.

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