Binding molecules for the treatment of cancer

Abstract

The present invention relates to novel binding molecules. Specifically, the present invention relates to binding molecules that bind to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and cadherin-3 (CDH3). The present invention also relates to nucleic acids encoding such binding molecules; methods for preparing such binding molecules; host cells expressing or capable of expressing such binding molecules; compositions containing such binding molecules; and the use of such binding molecules or such compositions for therapeutic purposes, particularly in the field of cancer diseases.

Description

Technical Field

[0001] Field of the Invention The present invention relates to novel binding molecules. Specifically, the present invention relates to novel binding molecules that bind to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and cadherin-3 (CDH3). The present invention also relates to nucleic acids encoding such binding molecules; methods for preparing such binding molecules; host cells expressing or capable of expressing such binding molecules; compositions comprising such binding molecules; and the use of such binding molecules or such compositions for therapeutic purposes, particularly in the field of cancer diseases.

Background Art

[0002] Background of the Invention Cancer is a disease characterized by abnormal, localized cell growth that has the potential to spread throughout the body. Cancer is a serious disease and is the second leading cause of death in developed countries.

[0003] In the past, the most frequently used means of treating neoplastic cancer has been through surgery, treatment with radiation, and the use of chemotherapeutic agents or immunotherapy. Although progress has been made in the treatment of certain cancers in recent years, there is still a need for improvement in the treatment of this disease.

[0004] Antibody-based biological molecules offer the potential to be powerful therapeutic agents for the treatment of cancer. Antibodies are designed to recognize and bind to specific proteins (their target antigens) on the cell surface, and such proteins may only be present on the surface of certain cancer cells or immune cells. This binding can elicit many different biological responses, depending on the function of their target antigen proteins, as well as the structure of the antibody itself and its binding site. For example, some antibodies trigger the immune system to attack and kill cancer cells, either by attracting immune cells to the cancer cells or by directly affecting the activity of the immune system itself. Other types of antibodies have drugs or radioactive particles attached to them, thus delivering these therapeutic agents to cancer cells. Still other types of antibodies that bind to cancer cells either activate or inhibit specific mechanisms, whereby cell division may be reduced or even tumor cells may be killed.

[0005] Apoptosis (i.e., programmed cell death) is a controlled cellular mechanism where the organism maintains cell homeostasis within normal tissue compartments and eliminates disordered cells.

[0006] There are two main signaling pathways leading to apoptosis in mammalian cells: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated at the mitochondrial level and plays a substantial role in cell death induced by chemotherapy or radiation. In contrast, the extrinsic death pathway is initiated through signals mediated by death receptors on the cell surface.

[0007] Cell death induced through the extrinsic pathway after signaling mediated by various members of the tumor necrosis factor (TNF) receptor superfamily has been well studied. The TNF superfamily is characterized by two to five cysteine-rich extracellular repeat sequences. Death receptors belonging to the TNF superfamily share a homologous intracellular death domain of approximately 80 amino acids that is essential for apoptotic signaling. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a natural protein ligand that interacts with the following two types of receptors: a death receptor that triggers TRAIL-induced apoptosis and a decoy receptor that suppresses TRAIL-induced apoptosis. To date, four human receptors specific for TRAIL have been identified: the death receptors DR4 (DR4 / TRAIL receptor 1 / TRAILR1) and DR5 (TRAIL receptor 2 / TRAIL-R2 / KILLER), and the decoy receptors DcR1 / TRAILR3 / TRID and DcR2 / TRAILR4. TRAIL can also bind with low affinity to osteoprotegerin (OPG), a soluble decoy receptor.

[0008] Targeting TRAIL receptors is considered a useful approach in the development of cancer therapy. This is because if an antibody-based molecule, i.e., a TRAIL receptor agonist molecule, can bind to and activate TRAIL receptors, it can induce apoptosis in cancer cells. As shown in many preclinical trials, TRAIL signaling efficiently induces apoptosis in numerous tumor cell lines but not in most normal cells. However, normal tissues, particularly hepatocytes in the liver, have also been reported to be sensitive to this apoptosis-inducing mechanism. Therefore, if a molecule that activates the pathway too effectively is used, severe side effects may be induced due to apoptosis induction in non-cancerous cells. On the other hand, weakly activating molecules have shown good tolerance but have been shown to have low anti-cancer activity.

[0009] One approach is to target TRAIL receptors in combination with cancer cell-specific markers, i.e., proteins that are not expressed or are minimally expressed by non-cancerous cells.

[0010] Numerous different anchor targets have been proposed as suitable combination pairs for TRAIL-binding molecules.

[0011] FAP (fibroblast activation protein) has also been proposed as an anchor target. However, FAP is expressed only on activated fibroblasts located within the tumor stroma. FAP is not expressed in epithelial cancer cells. Thus, FAP bispecific molecules function to promote apoptosis only in cancer cells that are physically proximal to activated fibroblasts (Bruenker et al., Molecular Cancer Therapeutics (2016), 15(5):946-957). Tumor cells that are not in direct contact with activated fibroblasts are not affected by this treatment and continue to proliferate. Therefore, it is clearly disadvantageous to use FAP as an anchor target for mediating TRAIL receptor-induced apoptosis in cancer cells. Furthermore, since activated fibroblasts are also found at sites of tissue remodeling, including liver fibrosis, pulmonary fibrosis, atherosclerosis, and arthritis, bispecific molecules that target FAP and TRAIL receptors may anchor on the surface of activated fibroblasts and also induce apoptosis on adjacent normal TRAIL-sensitive cells within the liver or other organs.

[0012] Similarly, MCSP (melanoma-associated chondroitin sulfate proteoglycan) and ROBO4 (roundabout homolog 4) have been proposed as anchor targets (He Yuan et al., The Journal of Investigative Dermatology (2016), 136(2):541-544; International Publication No. WO 2011 / 039126). In addition to its expression on the cell surface of melanoma, MCSP is mainly expressed on newly formed blood vessels. ROBO4 is specifically expressed in endothelial cells. Both are described at the site of angiogenesis in various types of tumors. Thus, similar to FAP, with the sole exception of melanoma expressing MSCP, a bispecific molecule targeting MCSP or ROBO4 and a TRAIL receptor will function to promote apoptosis in cancer cells only if they are in physical proximity to endothelial cells. This is not always possible because tumors grow rapidly such that their blood supply cannot keep up as they grow. Therefore, here too, using these molecules as anchor targets is disadvantageous.

[0013] Furthermore, bispecific antibodies targeting both TRAILR2 and LTβR (lymphotoxin β receptor) have been reported to be active in suppressing tumor growth in a mouse tumor xenograft model at levels equal to or higher than those of the combination of the respective parental antibodies (Michaelson et al., mAbs (2009), 1(2):128-141). LTβR signaling in mice has been shown to be important in liver regeneration, where LTβR is expressed on mature hepatocytes (Anders R.A. et al. J Immunol (2005), 175(2):1295-1300). Similar to FAP, LTβR signaling is widely activated during chronic hepatitis in patients with viral and non-viral hepatitis, cholangitis, and hepatocellular cancer. In particular, its expression in hepatocytes has been described (Haybaeck J. et al. Cancer Cell (2009), 16(4): 295-308). Targeting to LTβR and TRAIL receptors may anchor on the surface of hepatocytes expressing LTβR, which are sensitive to TRAILR2 activation, thereby potentially causing hepatotoxicity.

[0014] Finally, tenascin C has also been suggested to be a useful anchoring target. Tenascin C is a secreted protein and thus not anchored to the cell membrane. This represents another example of an indirect mechanism of inducing apoptosis in cancer cells. Similar to the anchoring strategies of MCSP or ROBO4, the tenascin C moiety must be in the correct orientation with respect to TRAILR2 molecules on the cancer cell membrane for apoptosis to occur. Furthermore, the expression of tenascin C is also upregulated in chronic liver diseases, and treatment with bispecific molecules containing TRAIL agonists is expected to worsen the condition. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0015] Accordingly, although progress has been made in the treatment of certain cancers in recent years, despite the fact that numerous different approaches are currently being explored, there remains a need to provide novel and therapeutically appropriate compounds for the treatment of cancer, in particular to develop antibody-based biological molecules that can function as TRAIL receptor agonist molecules, are effective in treatment but safe.

[0016] Accordingly, an object of the present invention is to produce TRAIL receptor agonist molecules that have an improved therapeutic profile and provide certain advantages compared to drugs, compositions and / or methods currently in use and / or known in the art. These advantages include, in particular, efficacy in vivo, improved therapeutic and pharmacological properties, fewer side effects, and / or other beneficial properties compared to molecules already known in the art, such as high stability in a particularly low pH environment, and improved ease of production or cost reduction of the product.

Means for Solving the Problems

[0017] Brief Summary of the Invention The object is solved by the subject matter of the present invention. The present invention is based on the concept of combining an antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and an antigen-binding site that specifically binds to cadherin-3 (CDH3) within a single binding molecule. As will be discussed in more detail below, one advantage of the molecules of the present invention is that apoptosis is promoted only in cells presenting both TRAILR2 and CDH3 on the cell surface.

[0018] The inventors of the present invention have further discovered that the use of an antigen-binding site that specifically binds to the extracellular domain 1 (EC1) of CDH3 significantly improves the potency of the binding molecule.

[0019] According to a first aspect of the present invention, the present invention provides a binding molecule comprising: (a) at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2); and (b) at least one antigen-binding site that specifically binds to cadherin-3 (CDH3), wherein at least one antigen-binding site that specifically binds to CDH3 is i. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2) and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3); ii. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2) and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3); iii. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2) and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3); and iv. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2) and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3) selected from the group consisting of.

[0020] In a specific embodiment, the present invention provides a binding molecule having at least one antigen-binding site (first antigen-binding site) that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site (second antigen-binding site) that specifically binds to extracellular domain 1 (EC1 domain) of cadherin-3 (CDH3).

[0021] Specifically, the binding molecules provided herein are bispecific binding molecules.

[0022] In one embodiment, the binding molecules provided herein thus recognize an antigen comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 98.

[0023] In a specific embodiment, the binding molecules provided herein specifically bind to an antigen comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 99.

[0024] In a preferred embodiment of the binding molecule of the present invention, the antigen-binding site (second antigen-binding site) that specifically binds to CDH3 is i. a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6; ii. a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16; iii. a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26; and, iv. a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36 selected from the group consisting of.

[0025] In a preferred embodiment, the binding molecules provided herein are i. a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 comprising.

[0026] In a specific embodiment, the binding molecule provided herein is i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 80, and a light chain comprising the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 84, and a light chain comprising the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85; or iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 86, and a light chain comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87 comprising.

[0027] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 2, and contains CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 4, and CDR3 of SEQ ID NO: 5, and a light-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 6, respectively, and contains light-chain CDR1 of SEQ ID NO: 7, light-chain CDR2 of SEQ ID NO: 8, and light-chain CDR3 of SEQ ID NO: 9.

[0028] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and contains CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 14, and CDR3 of SEQ ID NO: 15, and a light-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 16, respectively, and contains light-chain CDR1 of SEQ ID NO: 17, light-chain CDR2 of SEQ ID NO: 18, and light-chain CDR3 of SEQ ID NO: 19.

[0029] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 22, and contains CDR1 of SEQ ID NO: 23, CDR2 of SEQ ID NO: 24, and CDR3 of SEQ ID NO: 25, and a light-chain variable region comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 26, and contains light-chain CDR1 of SEQ ID NO: 27, light-chain CDR2 of SEQ ID NO: 28, and light-chain CDR3 of SEQ ID NO: 29.

[0030] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy-chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 32, and contains CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, and a light-chain variable region comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 36, and contains light-chain CDR1 of SEQ ID NO: 37, light-chain CDR2 of SEQ ID NO: 38, and light-chain CDR3 of SEQ ID NO: 39.

[0031] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 42, and contains the CDR1 of SEQ ID NO: 43, the CDR2 of SEQ ID NO: 44, and the CDR3 of SEQ ID NO: 45, and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 46, respectively, and contains the light chain CDR1 of SEQ ID NO: 47, the light chain CDR2 of SEQ ID NO: 48, and the light chain CDR3 of SEQ ID NO: 49.

[0032] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 52, and contains the CDR1 of SEQ ID NO: 53, the CDR2 of SEQ ID NO: 54, and the CDR3 of SEQ ID NO: 55, and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 56, respectively, and contains the light chain CDR1 of SEQ ID NO: 57, the light chain CDR2 of SEQ ID NO: 58, and the light chain CDR3 of SEQ ID NO: 59.

[0033] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 62, and contains CDR1 of SEQ ID NO: 63, CDR2 of SEQ ID NO: 64, and CDR3 of SEQ ID NO: 65, and a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 66 for each, and contains light chain CDR1 of SEQ ID NO: 67, light chain CDR2 of SEQ ID NO: 68, and light chain CDR3 of SEQ ID NO: 69.

[0034] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 80, and contains CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 4, and CDR3 of SEQ ID NO: 5, and a light chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81 for each, and contains light chain CDR1 of SEQ ID NO: 7, light chain CDR2 of SEQ ID NO: 8, and light chain CDR3 of SEQ ID NO: 9.

[0035] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and contains CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 14, and CDR3 of SEQ ID NO: 15, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83, and contains light chain CDR1 of SEQ ID NO: 17, light chain CDR2 of SEQ ID NO: 18, and light chain CDR3 of SEQ ID NO: 19.

[0036] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 84, and contains CDR1 of SEQ ID NO: 23, CDR2 of SEQ ID NO: 24, and CDR3 of SEQ ID NO: 25, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85, and contains light chain CDR1 of SEQ ID NO: 27, light chain CDR2 of SEQ ID NO: 28, and light chain CDR3 of SEQ ID NO: 29.

[0037] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 86, and contains CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87, and contains light chain CDR1 of SEQ ID NO: 37, light chain CDR2 of SEQ ID NO: 38, and light chain CDR3 of SEQ ID NO: 39.

[0038] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 88, and contains CDR1 of SEQ ID NO: 43, CDR2 of SEQ ID NO: 44, and CDR3 of SEQ ID NO: 45, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 89, and contains light chain CDR1 of SEQ ID NO: 47, light chain CDR2 of SEQ ID NO: 48, and light chain CDR3 of SEQ ID NO: 49.

[0039] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 90, and contains CDR1 of SEQ ID NO: 53, CDR2 of SEQ ID NO: 54, and CDR3 of SEQ ID NO: 55, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 91, and contains light chain CDR1 of SEQ ID NO: 57, light chain CDR2 of SEQ ID NO: 58, and light chain CDR3 of SEQ ID NO: 59.

[0040] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy chain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 92, and contains CDR1 of SEQ ID NO: 63, CDR2 of SEQ ID NO: 64, and CDR3 of SEQ ID NO: 65, and a light chain comprising an amino acid sequence that is respectively at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 93, and contains light chain CDR1 of SEQ ID NO: 67, light chain CDR2 of SEQ ID NO: 68, and light chain CDR3 of SEQ ID NO: 69.

[0041] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to TRAILR2 comprises a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 79 (CDR3).

[0042] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site (first antigen-binding site) that specifically binds to TRAILR2 is an antigen-binding site comprising a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 72 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 76.

[0043] In a specific embodiment, the binding molecule provided herein comprises an antigen-binding site that specifically binds to TRAILR2, which comprises a VH comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 72, and contains CDR1 of SEQ ID NO: 73, CDR2 of SEQ ID NO: 74, and CDR3 of SEQ ID NO: 75, and a VL comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 76, respectively, and contains light-chain CDR1 of SEQ ID NO: 77, light-chain CDR2 of SEQ ID NO: 78, and light-chain CDR3 of SEQ ID NO: 79.

[0044] In some embodiments, the binding molecules provided herein, as defined by their heavy-chain amino acid sequences (e.g., modified heavy chains having a TRAILR2-specific scFv fused to the C-terminus of an immunoglobulin heavy chain) and their light-chain amino acid sequences, comprise two heavy chains and two light chains, thereby forming a symmetric tetravalent bispecific structure.

[0045] In a further preferred embodiment, the binding molecule of the present invention comprises a modified heavy chain, preferably, the TRAILR2-specific scFv is fused to the C-terminus of an immunoglobulin heavy chain. Preferably, the modified heavy chain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO: 31. Specifically, the binding molecule of the present invention comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO: 31.

[0046] Specifically, the binding molecule of the present invention is i. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 and includes.

[0047] In a preferred embodiment of the binding molecule of the present invention, the molecule is bispecific and tetravalent.

[0048] Specifically, at least one antigen-binding site that specifically binds to TRAILR2 is linked to at least one antigen-binding site that specifically binds to CDH3.

[0049] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to CDH3 is an immunoglobulin (Ig) molecule (having a full-length antibody with the conventional Y-shaped structure having two light chains and two heavy chains), and at least one antigen-binding site that specifically binds to TRAILR2 comprises one or more scFvs.

[0050] In a preferred embodiment of the binding molecule of the present invention, one or more scFvs have a VL-VH orientation from the N-terminus to the C-terminus.

[0051] In a preferred embodiment of the binding molecule of the present invention, one or more scFvs are fused to the C-terminus of the heavy chain of the immunoglobulin molecule, thereby forming a modified immunoglobulin molecule. For example, one scFv is fused to the C-terminus of one of the heavy chains of the immunoglobulin molecule, and one scFv is fused to the C-terminus of the other heavy chain of the immunoglobulin molecule. Thus, the scFv specific for TRAILR2 is fused to each of the heavy chains of the immunoglobulin, thereby forming a symmetric, bispecific and tetravalent structure.

[0052] In a preferred embodiment, the binding molecule of the present invention comprises two antigen-binding sites that specifically bind to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) and two antigen-binding sites that specifically bind to cadherin-3 (CDH3).

[0053] Accordingly, in a specific embodiment, a bispecific and tetravalent binding molecule is provided herein, wherein the binding molecule is a modified immunoglobulin (Ig) molecule comprising two antigen-binding sites that specifically bind to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) and two antigen-binding sites that specifically bind to cadherin-3 (CDH3); wherein the two antigen-binding sites that specifically bind to CDH3 are present within the variable region (Fv) of the immunoglobulin molecule, and the two antigen-binding sites that specifically bind to TRAILR2 are scFvs; wherein the scFv is fused to the C-terminus of the heavy chain of the immunoglobulin molecule. Preferably, the first scFv is fused to the first heavy chain of the immunoglobulin molecule, respectively, and the second scFv is fused to the second heavy chain.

[0054] In a preferred embodiment of the binding molecule of the present invention, the immunoglobulin molecule is IgG1KO. In another preferred embodiment of the binding molecule of the present invention, the immunoglobulin molecule is IgG1FcRnmut.

[0055] In a preferred embodiment of the binding molecule of the present invention, one or more scFvs are fused to an immunoglobulin molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids (e.g., any one of 5, 6, 9, 12, 15 amino acids).

[0056] Further described herein is a binding molecule, particularly a bispecific binding molecule, having at least one antigen-binding site (first antigen-binding site) that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) as detailed above, and at least one antigen-binding site (second antigen-binding site) that specifically binds to the extracellular domain 2 (EC2 domain) of cadherin-3 (CDH3).

[0057] i. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3); ii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3); iii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3); iv. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3); v. A heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 49 (CDR3); vi. A heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3); or vii. A heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3) An antibody that specifically binds to CDH3 and comprises the above is further provided herein.

[0058] In a preferred embodiment, the antibody provided herein i. A heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6; ii. A VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16; iii. A VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26; iv. A VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36; v. A VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 46; vi. A VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; or vii. A VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 66 is included.

[0059] In yet another preferred embodiment of the present invention, the antibody that specifically binds to CDH3 i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87; v. A heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; vi. A heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 91; or vii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93 is included.

[0060] Specifically, the antibody is i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 80, and a light chain comprising the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 84, and a light chain comprising the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85; iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 86, and a light chain comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87; v. A heavy chain comprising the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 88, and a light chain comprising the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 89; vi. A heavy chain comprising the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 90, and a light chain comprising the amino acid sequence of SEQ ID NO: 91, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 91; or vii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 92, and a light chain comprising the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 93 comprising.

[0061] In a preferred embodiment, the antibody or antigen-binding fragment thereof provided herein is a chimeric antibody, a humanized antibody, and a human antibody or antibody fragment, and scFv, Fab fragment, monovalent antibody fragment, and F(ab’) 2 selected from the group consisting of fragments.

[0062] A further aspect of the invention provides a nucleic acid molecule encoding a binding molecule of the invention, or an expression vector containing such a nucleic acid molecule.

[0063] In a preferred embodiment, the vector is a plasmid vector or a viral vector.

[0064] A further aspect of the invention provides a host cell comprising a nucleic acid molecule of the invention in a functional relationship with an expression control sequence.

[0065] Also provided herein is a host cell comprising an expression vector comprising a nucleic acid molecule encoding a binding molecule as described herein.

[0066] A further aspect of the invention is i. culturing the host cell of claim 17 under conditions that allow expression of the molecule, and ii. recovering the molecule, and optionally iii. further purifying and / or modifying and / or formulating the molecule A method for producing a binding molecule of the invention is provided, which method comprises the steps of.

[0067] Also provided herein is a binding molecule of the invention for use in a medicament.

[0068] In a preferred embodiment, the binding molecule of the invention is provided for use in the treatment of cancer, preferably pancreatic cancer, lung cancer or head and neck cancer.

[0069] Further provided herein is a pharmaceutical composition comprising the binding molecule of the invention together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.

[0070] A further aspect of the invention provides a method of treating cancer comprising the step of administering to a patient in need thereof an effective amount of the binding molecule of the invention.

[0071] In a preferred embodiment, the pharmaceutical composition provided herein is lyophilized, stabilized and / or formulated for administration by injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0072]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

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Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Modes for Carrying Out the Invention

[0073] Detailed Description of the Invention As described above, one approach to cancer treatment has been to induce apoptosis in cancer cells by using molecules that specifically bind to and activate the TNF-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis pathway. As shown in many preclinical studies, TRAIL signaling effectively induces apoptosis in numerous tumor cell lines but not in most normal cells. However, normal tissues, particularly hepatocytes in the liver, have also been reported to be sensitive to this apoptosis-inducing mechanism.

[0074] TRAIL binds with high affinity to four distinct cell surface receptors. Two of these, TRAILR1 and TRAILR2, can trigger TRAIL-induced apoptosis through the interaction of their intracellular death domains with various adapter proteins and pro-caspase 8. Clustering of TRAILR1 or TRAILR2 molecules via the TRAIL ligand promotes autocatalytic cleavage and activation of pro-caspase 8, followed by induction of apoptosis. TRAILR3 and TRAILR4 are decoy receptors, whose extracellular domains can bind TRAIL, but the intracellular portion of these receptors does not contain a domain capable of inducing apoptosis upon binding to TRAIL.

[0075] Overexpression of decoy TRAIL receptors can cause cancer cells to become insensitive to the presence of the TRAIL ligand. Specific targeting of death-inducing TRAIL receptors that are not decoys circumvents this problem and has shown more effective tumor treatment.

[0076] The present invention focuses on the development of TRAILR2 agonist molecules. TRAILR2 is widely expressed in a broad range of cancers. Several TRAILR2-specific agonistic antibodies (including lexatumumab) (HGS-ETR2) have been developed for the treatment of cancer. However, these agonistic antibodies have lacked efficacy clinically. Without wishing to be bound by theory, this is likely due to insufficient clustering of the TRAILR2 receptor occurring, and thus, the inability to effectively induce apoptosis in cancer cells.

[0077] In various attempts to promote TRAILR2-mediated apoptosis in cancer cells, TAS266, a tetrameric TRAILR2-binding nanobody, has been developed. In preclinical experiments, it demonstrated superior antitumor efficacy compared to conventional TRAILR2-targeted antibodies. However, hepatocytes in the liver can be sensitive to TRAILR2-mediated apoptosis, and thus, an increase in non-targeted TRAILR2 clustering, such as that promoted by TAS266, has been reported to have a risk of toxicity potential. Indeed, the Phase I clinical trial of TAS266 has been aborted.

[0078] Therefore, if molecules that agonize the pathway too effectively are used, apoptosis can be induced in non-cancerous cells, potentially inducing serious side effects. On the other hand, if weakly agonistic molecules are used, they have shown good tolerance but have been shown to have low anti-cancer activity.

[0079] One approach is to target TRAIL receptors in combination with cancer cell-specific markers, i.e., proteins that are not expressed or are hardly expressed at all by non-cancerous cells (also referred to as "anchor proteins") (see, for example, WO 2018 / 115231, where CDH17 is used as an anchor protein). However, such tumor-specific target proteins are rare, and this rarity remains a major drawback typically faced in the development of cancer-specific therapeutics. Attempts have been made to target more widely expressed lineage antigens, but the value of these therapies is limited by the toxicity caused by the expression of these antigens in certain normal tissues, such as the expression of Epcam in the gastrointestinal tract (Kebenko et al., Oncoimmunology 2018, Vol. 7, No. 8). Various approaches are currently being explored to reduce the toxicity associated with the expression of antigens remote from the site, but for many compounds, toxicity remains dose-limiting.

[0080] The inventors have identified an anchor protein that is not present in substantial amounts in serum and have determined its location in cancer cells that co-express TRAILR2. Importantly, the selected anchor protein is not expressed in the liver, which is expected to be beneficial due to the above-mentioned potential for hepatotoxicity.

[0081] The inventors have identified cadherin-3 as a suitable anchor target that can be used in combination with a TRAIL receptor-binding molecule.

[0082] Cadherin-3 (CDH3) is a member of the cadherin superfamily, a gene that encodes a calcium-dependent membrane-bound glycoprotein. The encoded protein is a cadherin-like protein consisting of an extracellular region containing five cadherin domains, a transmembrane region, and an intracellular domain.

[0083] The inventors analyzed the expression of CDH3 in tumor tissues and found that TRAILR2 and CDH3 are co-expressed in a variety of tumors (i.e., pancreatic cancer (PAC), head and neck cancer, lung cancer, colorectal cancer, esophageal cancer, triple-negative breast cancer, ovarian cancer, cutaneous melanoma, and bladder urothelial cancer and cervical cancer), and are hardly or not co-expressed in non-cancerous cells. Notably, CDH3 could not be detected in normal liver tissue or hepatocytes, in which sensitivity to TRAILR2 activation has been reported.

[0084] Accordingly, provided herein is a binding molecule comprising at least one antigen-binding site that specifically binds to TRAILR2 and at least one antigen-binding site that specifically binds to CDH3.

[0085] In the attached experimental data, it can be seen that such molecules can induce apoptosis in vitro and in vivo in cells in which both CDH3 and TRAILR2 are expressed. Surprisingly, a bispecific binding molecule that binds to the EC1 domains of TRAILR2 and CDH3 induces apoptosis significantly more efficiently than such a bispecific binding molecule that specifically targets the EC2 domain of CDH3, as impressively demonstrated by the in vivo data shown in Example 6.

[0086] Importantly, as also shown in the examples, the same molecule induces substantially no apoptosis in cells that express TRAILR2 but not CDH3. Thus, the binding molecules of the present invention are therapeutically effective against cancers in which cancer cells express both CDH3 and TRAILR2. In one aspect, the binding molecules of the present invention do not affect CDH3-negative hepatocytes, thereby reducing the risk of hepatotoxicity.

[0087] Furthermore, the binding molecules of the present invention are very stable under low pH conditions, allowing for efficient production in large quantities, and thus they are excellent candidates for therapeutic proteins.

[0088] Notably, the binding molecules of the present invention show substantially no internalization activity in representative assays. As shown in Example 7, for an exemplary CDH3 / TRAILR2 EC1-specific binding molecule, no internalization could be measured. For the bispecific binding molecules of the present invention, negligible or very slow internalization is advantageous for supporting sustainable and efficient activation of TRAILR2 at the cell surface.

[0089] Accordingly, the CDH3 / TRAILR2 binding molecules of the present invention have distinct advantages over molecules known in the art and confer utility for treating cancers, including pancreatic cancer, head and neck cancer, and lung cancer. Some of these advantages include, in particular, improved in vivo efficacy, fewer side effects, high stability, and improved manufacturability as compared to molecules known in the art.

[0090] A first aspect of the present invention provides a binding molecule having at least one antigen-binding site (first antigen-binding site) that specifically binds to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site (second antigen-binding site) that specifically binds to cadherin-3 (CDH3), preferably the EC1 domain of CDH3.

[0091] Prior to the present invention, the preparation of binding molecules capable of specifically binding to TRAILR2 and CDH3, particularly the EC1 domain of CDH3, has not been disclosed or even considered to any extent. Nevertheless, each of the individual proteins and their related genes are known in the art and well represented in biological databases.

[0092] To avoid doubt, by "TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2)", the inventors mean the human protein provided in UniProt O14763 http: / / www.uniprot.org / uniprot / O14763, and the nucleic acid sequence encoding that protein.

[0093] To avoid doubt, by "cadherin-3 (CDH3)", the inventors mean the human protein provided in UniProt P22223 http: / / www.uniprot.org / uniprot / P22223, and the nucleic acid sequence encoding that protein.

[0094] The present invention relates to a binding molecule having binding specificity for at least two different targets. In the context of the present invention, the binding molecule is derived from an antibody. Techniques for producing binding molecules include recombinant co-expression of two pairs of immunoglobulin heavy-chain / light-chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering manipulations (see, for example, U.S. Patent No. 5,731,168), but are not limited thereto. The binding molecules of the present invention also include engineering of electrostatic steering effects for the production of antibody Fc heterodimer molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); generation of bispecific antibodies using leucine zippers (see, for example, Kostelny et al., Immunol., 148(5): 1547-1553 (1992)); use of the "diabody" technique for the production of bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., Immunol., 152:5368 (1994)); and can be made, for example, by the preparation of trispecific antibodies as described in Tutt et al. Immunol. 147: 60 (1991).

[0095] Terms not specifically defined herein should be given the meaning that would be ascribed to them by one of ordinary skill in the art in view of the disclosure and context. However, as used herein, unless otherwise specified, the following terms have the indicated meanings and the following conventions are adhered to.

[0096] Terms and Definitions Used A molecule (such as a binding molecule of the present invention or a fragment thereof) that can "bind", "bind to", "specifically bind", or "specifically bind to" a specific epitope, antigen, or protein (or at least one part, fragment, or epitope thereof), and that has "affinity for" and / or "specificity for" such an epitope, antigen, or protein is said to be "directed against" or "directed to" such an epitope, antigen, or protein, or is a "binding" molecule with respect to such an epitope, antigen, or protein. As used herein, the term "bispecific" when referring to a binding molecule relates to a molecule (such as a binding molecule of the present invention) that can specifically bind or has specificity for at least two different antigens or proteins (or at least a part, fragment, or epitope thereof).

[0097] As used herein, the term "antigen-binding site" relates to the domain of a binding molecule that confers binding to a particular antigen. Antigen-binding sites are originally derived from antibodies, but advances in the field have led to additional possibilities for designing and / or obtaining antigen-binding sites without the need to produce natural antibodies against the target of interest. Regardless of its origin, the "antigen-binding site" according to the present invention includes at least a minimal structural element, i.e., a necessary and sufficient structural element, that enables binding to its specific target antigen. Thus, the "antigen-binding site" according to the present invention includes at least three heavy-chain CDR sequences (in the case of single-domain antibodies), more preferably at least three light-chain and three heavy-chain CDR sequences. As discussed below, these CDRs typically reside in the so-called variable domains of the antibody, i.e., the variable regions (Fv). If the antigen-binding site contains at least a minimal structural element, it is understood that it typically also encompasses additional elements (such as framework regions, etc.). Thus, as used according to the present invention, the antigen-binding site can also be defined via permutations of the respective combinations of the heavy-chain variable domain and the light-chain variable domain. According to the present invention, it is particularly preferred that the "antigen-binding site" is contained within a polypeptide and / or that the CDRs or the variable domains are polypeptides and / or peptides.

[0098] An "antibody" or "immunoglobulin molecule" (immunoglobulin, also known as Ig for short) is a gamma globulin protein found in the blood or other body fluids of vertebrates and is used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. They typically consist of a basic structural unit (each having two large heavy chains and two small light chains), forming, for example, a monomer with one unit, a dimer with two units, or a pentamer with five units. Antibodies can bind to other molecules or structures known as antigens through non-covalent interactions. This binding is specific in the sense that the antibody binds only to a particular structure with high affinity. The specific part of the antigen recognized by the antibody is called an epitope or antigenic determinant. The part of the antibody that binds to the epitope is sometimes called a paratope and exists in the so-called variable domain or variable region (Fv) of the antibody. The variable domain contains three so-called complementarity-determining regions (CDRs) separated by framework regions (FRs).

[0099] Within the context of the present invention, references to CDRs are based on the definitions by Kabat (E.A. Kabat, T.T. Wu, H. Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)). Alternative definitions of CDRs are based on (i) CCG (as exemplified in Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al, Proteins 2014; 82:1599-1610 by Chemical Computing Group), (ii) Chothia (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917), (iii) IMGT (Lefranc MP, Dev Comp Immunol. 2003 Jan;27(1):55-77) and (iv) North (North B, J Mol Biol. (2011) 406:228-56).

[0100] As used herein, the expressions "variable domain" or "variable region", i.e., Fv, refer to each pair of a light chain and a heavy chain that is directly involved in the binding of an antibody to an antigen. The variable domain of the light chain is abbreviated as "VL" and the variable domain of the heavy chain is abbreviated as "VH". The variable domains of the light chain and the heavy chain have the same general structure, and each domain includes four framework (FR) regions with widely conserved sequences, which are connected by three hypervariable regions (or CDRs). The framework regions adopt a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structures. The CDRs within each chain have their three-dimensional structures maintained by the framework regions and together with the CDRs from other chains form the antigen-binding site. The CDR regions of the heavy and light chains of an antibody play a particularly important role in the binding specificity / affinity of the antibodies described in the present invention, and thus provide a further object of the present invention.

[0101] An immunoglobulin domain essentially consists of four "framework regions", hereinafter referred to in the art and in this specification as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively; the framework regions are interrupted by three "complementary determining regions" or "CDRs", hereinafter referred to in the art and in this specification as "complementary determining region 1" or "CDR1", "complementary determining region 2" or "CDR2", and "complementary determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be shown as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) that confer specificity for an antigen to an antibody by having an antigen-binding site.

[0102] The term "constant domain" or "constant region" as used within this application refers to the total number of domains of an antibody other than the variable region. Such constant domains and regions are well known in the state of the art, for example, as described by Kabat et al. ("Sequence of proteins of immunological interest", U.S. Public Health Service, National Institutes of Health, Bethesda, Maryland, Publication No. 91).

[0103] The "Fc portion" of an antibody is not directly involved in the binding of the antibody to an antigen, but exhibits various effector functions. The "Fc portion of an antibody" is a term well-known to those skilled in the art and is defined based on the cleavage of an antibody by papain. Depending on the amino acid sequences of the constant regions of their heavy chains, antibodies or immunoglobulins are classified into the following classes: IgA, IgD, IgE, IgG, and IgM. The heavy chain constant regions are called α, δ, ε, γ, and μ, respectively, for the different classes of immunoglobulins. Some of these can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The Fc portion of an antibody is directly involved in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) based on complement activation, binding to C1q, and binding to Fc receptors. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. The effect of an antibody on the complement system depends on specific conditions, but the binding to C1q is caused by defined binding sites in the Fc portion. Such binding is known in the state of the art, for example, as described by Boakle et al., Nature 282 (1975) 742-743, Lukas et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344, Thommesen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol.164 (2000) 4178-4184, Hezareh et al., J. Virology 75 (2001) 12161- 12168, Morgan et al., Immunology 86 (1995) 319-324, European Patent No. 0307434.Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering follows the Kabat EU index, see below). In mediating the binding of C1q and Fcγ receptors in IgG1, the most important of these residues are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168). Antibodies of subclasses IgG1 and IgG3 typically exhibit complement activation and binding to C1q and C3, while IgG2 and IgG4 do not activate the complement system and do not bind to C1q and C3.

[0104] The art has further developed antibodies and used them as versatile tools in medicine and engineering. Thus, in the context of the present invention, the terms "binding molecule", "antibody molecule" or "antibody" include not only antibodies as found in nature, which for example contain two light chains and two heavy chains, or only two heavy chains as in camelid species, but also all molecules containing at least one paratope having binding specificity for an antigen and structural similarity to the variable domains of immunoglobulins.

[0105] The terms "antibody" and "antibody molecule" are used interchangeably herein.

[0106] Accordingly, the antibody may include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments, particularly Fv, Fab, Fab’, or F(ab’)2 fragments, single-chain antibodies, particularly single-chain variable fragments (scFv), small modular immunopharmaceuticals (SMIP), domain antibodies, nanobodies and / or diabodies. The antibody may have effector functions usually mediated by the Fc portion (antibody constant region) of the antibody, such as ADCC or CDC, or it may have no effector function at all, for example, by lacking the Fc portion or having a blocked, shielded Fc portion that is not recognized or is poorly recognized by immune system components such as immune cells or the complement system. Monoclonal antibodies (mAbs) are single-specificity antibodies with identical amino acid sequences. They can be produced by hybridoma technology from hybrid cell lines (called hybridomas) that represent clones of the fusion of specific antibody-producing B cells and myeloma (B cell cancer) cells (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7). Alternatively, monoclonal antibodies may be produced by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. (May 1997). "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells." J Immunol Methods 204 (1): 77-87; see also below). A "recombinant antibody" or "recombinant binding molecule" is an antibody or binding molecule produced by a host cell that has been genetically engineered. It is optionally isolated or purified.

[0107] Polyclonal antibodies refer to a collection of antibody molecules with different amino acid sequences and can be obtained from the blood of vertebrates after immunization with an antigen by a process well known in the art.

[0108] Monoclonal antibodies (mAbs) are single-specific antibodies with identical amino acid sequences. They can be generated by hybridoma technology from hybrid cell lines (referred to as hybridomas) that represent clones in which specific antibody-producing B cells are fused with myeloma (B cell cancer) cells (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7.). Alternatively, monoclonal antibodies can be generated by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. (May 1997). "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.". J Immunol Methods 204 (1): 77-87; see also below).

[0109] For application to humans, it is often desirable to reduce the immunogenicity of antibodies originally derived from other species such as mice. This can be achieved by constructing chimeric antibodies or by a process called "humanization". In this context, a "chimeric antibody" is understood to be an antibody that contains a sequence portion (e.g., a variable domain) derived from one species (e.g., a mouse) fused to a sequence portion (e.g., a constant domain) derived from a different species (e.g., a human). A "humanized antibody" is an antibody that contains a variable domain originally derived from a non-human species, where certain amino acids have been mutated so that the overall sequence of the variable domain more closely approximates that of a human variable domain. Methods for chimerizing and humanizing antibodies are well known in the art (Billetta R, Lobuglio AF. “Chimeric antibodies”. Int Rev Immunol. 1993;10(2-3):165-76;Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature: 332:323.). As used herein, the term "human antibody" refers to an antibody made based on sequences derived from the human genome, for example by use of phage display or transgenic animals (see, e.g., WO 90 / 05144). As used herein, the term "antibody" clearly includes such humanized antibodies, chimeric antibodies, as well as human antibodies.

[0110] A "humanized" antibody refers to an antibody that contains amino acid residues derived from non-human hypervariable regions (HVRs) and amino acid residues derived from human framework regions. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., complementarity determining regions (CDRs)) correspond to those of a non-human antibody and all or substantially all of the framework regions (FRs) correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0111] Furthermore, techniques for producing antibodies based on sequences derived from the human genome have been developed, for example, by using phage display or transgenic animals (WO 90 / 05144; D. Marks, H.R. Hoogenboom, T.P. Bonnert, J. McCafferty, A.D. Griffiths and G. Winter (1991) "By-passing immunisation. Human antibodies from V-gene libraries displayed on phage." J.Mol.Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, "Concepts in antibody phage display". Briefings in Functional Genomics and Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. "Human antibodies from transgenic mice". Int Rev Immunol. 1995;13(1):65-93. ; Brueggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice”. Curr Opin Biotechnol. 1997 Aug;8(4):455-8). Such antibodies are "human antibodies" in the context of the present invention.

[0112] The term "antibody", particularly the antibody molecules described herein, can also include fragments of immunoglobulins that retain antigen-binding properties, such as Fab, Fab’ or F(ab’)2 fragments. Such fragments can be obtained, for example, by fragmentation of immunoglobulins by proteolytic digestion or by recombinant expression of such fragments. For example, digestion of immunoglobulins can be achieved using conventional techniques, such as using papain or pepsin (WO 94 / 29348). Digestion of an antibody with papain typically yields two identical antigen-binding fragments, so-called Fab fragments (each having a single antigen-binding site), and the remaining Fc fragment. Treatment with pepsin yields F(ab’)2. In Fab molecules, the variable domains are each preferably fused to an immunoglobulin constant domain of human origin. Thus, the heavy-chain variable domain can be fused to the CH1 domain (so-called Fd fragment), and the light-chain variable domain can be fused to the CL domain. Fab molecules can be produced by recombinant expression of the respective nucleic acids in host cells. See below.

[0113] Numerous techniques have been developed for placing the variable domain of an immunoglobulin, or a molecule derived from such a variable domain, in different molecular contexts. These should also be considered "antibodies" according to the present invention. Generally, these antibody molecules are smaller in size compared to immunoglobulins and can contain one amino acid chain or several amino acid chains. For example, a single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked to each other using a short linker, usually serine (S) or glycine (G) (WO 88 / 01649; WO 91 / 17271; Huston et al; International Reviews of Immunology, Volume 10, 1993, 195-217). "Single-domain antibodies" or "nanobodies" have an antigen-binding site within one immunoglobulin-like domain (WO 94 / 04678; WO 03 / 050531, Ward et al., Nature. 1989 Oct 12;341(6242):544-6; Revets et al., Expert Opin Biol Ther. 5(1):111-24, 2005). One or more single-domain antibodies having binding specificities for the same antigen or different antigens may be linked to each other. Diabodies are bivalent antibody molecules consisting of two amino acid chains and containing two variable domains (WO 94 / 13804, Holliger et al., Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6444-8). Another example of an antibody-like molecule is an immunoglobulin superfamily antibody (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). By a different concept, so-called small modular immunopharmaceuticals (SMIPs) are obtained, which contain an Fv domain linked to a single-chain hinge domain and an effector domain lacking the constant domain CH1 (WO 02 / 056910).

[0114] An antibody molecule may be fused (as a fusion protein) or otherwise linked (by covalent or non-covalent bonds) to another molecular entity such as a cytotoxic drug or to an entity that has a desirable effect on the properties of the antibody molecule. For example, particularly in the case of single-chain antibodies or domain antibodies, it may be desirable to improve the pharmacokinetic properties of the antibody molecule, such as stability in body fluids such as blood. In this regard, many techniques have been developed, such as PEGylation (WO 98 / 25971; WO 98 / 48837; WO 2004 / 081026), fusing or otherwise covalently attaching the antibody molecule to another antibody molecule having an affinity for a serum protein such as albumin (WO 2004 / 041865, WO 2004 / 003019), expression of the antibody molecule as a fusion protein with all or part of a serum protein such as albumin or transferrin (WO 01 / 79258), etc. Means and methods for the identification of lead compounds and the optimization of lead compounds in the design of antibodies are well known in the art and are reviewed, for example, in Goulet, D.R. and Atkins, W.M. J Pharm Sci 2020;109(1):74-103, or Tiller, K. E., & Tessier, P. M. (2015). Annual review of biomedical engineering, 17, 191-216.

[0115] With respect to the present invention, a first aspect of the present invention provides a binding molecule comprising at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site that specifically binds to cadherin-3 (CDH3). In particular, the CDH3-antigen-binding site of the binding molecule of the present invention specifically binds to the EC1 domain of CDH3.

[0116] "Binding specificity" means that the antibody molecule has a significantly higher binding affinity for the TRAILR2 or CDH3 target antigen than for structurally unrelated molecules.

[0117] "Specifically" or "selectively" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, refers to a binding reaction that determines the presence of a protein in a heterogeneous protein population and other biological agents. Thus, under specified conditions, a particular ligand binds to a particular receptor and does not bind in significant amounts to other proteins present in the sample.

[0118] An epitope is a region of an antigen that binds to an antibody or antigen-binding portion. The term "epitope" includes any polypeptide determinant that can specifically bind to an antibody or antigen-binding portion. In certain embodiments, the epitope determinant includes chemically active surface groups such as amino acids, glycan side chains, phosphoryl or sulfonyl, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. Structural epitopes and non-structural epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen-binding portion to an epitope of an antigen in an in vitro assay using purified wild-type antigen, preferably a plasmon resonance assay (Biacore®, GE Healthcare, Uppsala, Sweden).

[0119] The antibodies or binding molecules described herein bind to their antigen with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold greater than the affinity for an irrelevant antigen. In a preferred embodiment, the antibody has an affinity of greater than about 10 9 liters / mol as determined, for example, by Scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239).

[0120] Affinity is the interaction between a single antigen-binding site on an antibody molecule and a single epitope. It is the association constant K A =k 会合 / k 解離 or the dissociation constant K D =k 解離 / k 会合 and is expressed by

[0121] As used herein, the terms "binding" or "specific binding" refer to the binding of an antibody to an epitope of an antigen, for example, in an in vitro assay measured at room temperature, preferably a surface plasmon resonance assay (SPR, BIAcore, GE Healthcare, Uppsala, Sweden). Binding affinity is defined by the terms k 会合 (rate constant of association of the antibody from the antibody / antigen complex), k 解離 (dissociation constant), and K D (k 解離 / k 会合 ). Specific binding generally refers to the formation of a complex between a receptor molecule and its ligand. In the context of antibody-antigen binding, high-affinity antibodies typically bind to their target antigens with an affinity of 10 -9 M or less.

[0122] In one embodiment, the antibody binds to the TRAILR2 or CDH3 target antigen with an affinity as determined, for example, by surface plasmon resonance analysis (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 Apr;5(2):282-6), and K DThe value ranges from 1 pM to 100 μM, preferably from 1 pM to 1 μM. Antibody affinity may also be measured using the Kinetic Exclusion Assay (KinExA) technology (Darling, R.J., and Brault P-A., “Kinetic exclusion assay technology: Characterization of Molecular Interactions.” ASSAY and Drug Development Technologies. 2004, Dec 2(6): 647-657).

[0123] The binding affinity of the antibody molecule may be enhanced by a process known as affinity maturation (Marks et al., 1992, Biotechnology 10:779-783; Barbas, et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155). Therefore, affinity matured antibodies are also encompassed by the present invention.

[0124] In one embodiment, the bispecific binding molecule of the present invention can induce TRAILR2-mediated apoptosis in one or more cancer cell types, such as the colorectal cancer cell line GP2d or the lung cancer cell line NCI-H358, and suppress cell growth by more than 50% at a concentration of 1 nM or less, more preferably less than 0.01 nM.

[0125] In a further embodiment, the binding molecule of the present invention cannot induce TRAILR2-mediated apoptosis in CDH3-negative cells and suppresses cell growth by less than 50% at a concentration up to 1 nM, or more preferably up to 10 nM, even more preferably up to 100 nM.

[0126] Amino acid residues are shown according to the standard three-letter or one-letter amino acid codes as generally known and accepted in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion, or substitution of a specified number of amino acid residues at a position in the reference sequence as compared to the second sequence. In the case of substitution(s), such substitution(s) are preferably conservative amino acid substitution(s), which means that an amino acid residue is substituted with another amino acid residue having a similar chemical structure and having little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 98 / 49185, where conservative amino acid substitutions are preferably substitutions in which one amino acid within the following groups (i)-(v) is substituted by another amino acid residue within the same group: (i) small, aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar, positively charged residues: His, Arg, and Lys; (iv) large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: from Ala to Gly or Ser; from Arg to Lys; from Asn to Gln or His; from Asp to Glu; from Cys to Ser; from Gln to Asn; from Glu to Asp; from Gly to Ala or Pro; from His to Asn or Gln; from Ile to Leu or Val; from Leu to Ile or Val; from Lys to Arg, Gln, or Glu; From Met to Leu, to Tyr, or to Ile; From Phe to Met, to Leu, or to Tyr; From Ser to Thr; From Thr to Ser; From Trp to Tyr; From Tyr to Trp or to Phe; From Val to Ile or to Leu.

[0127] As used herein, the term "isolated" refers to a substance that has been removed from its original environment or natural surroundings (e.g., its natural environment if it occurs naturally). For example, a natural nucleic acid molecule or polypeptide present in a living animal is not isolated, but the same nucleic acid molecule or polypeptide separated by human intervention from some or all of the substances coexisting in the natural system is isolated. Such a nucleic acid molecule may be part of a vector, and / or such a nucleic acid molecule or polypeptide may be part of a composition, and such a vector or composition is still isolated in that it is not part of the environment in which the nucleic acid molecule or polypeptide is found naturally. For example, a nucleic acid molecule or polypeptide is considered to be in a "substantially isolated form" when it is separated from at least one other component with which it would normally associate in the source or medium, such as another nucleic acid molecule, another polypeptide, another biological component or macromolecule, or at least one contaminant, impurity, or minor component, as compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, a nucleic acid molecule or polypeptide is considered to be "substantially isolated" when it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, up to 1000-fold or more. A nucleic acid molecule or polypeptide in a "substantially isolated form" is preferably substantially homogeneous as determined using appropriate chromatographic techniques, such as appropriate techniques like polyacrylamide gel electrophoresis. The binding molecules and nucleic acids of the present invention are preferably isolated.

[0128] Unless otherwise specified, as used herein, the term "sequence" (e.g., in terms such as "immunoglobulin sequence", "binding molecule sequence" or "polypeptide sequence") generally should be understood to include both relevant amino acid sequences and nucleic acid sequences or nucleotide sequences encoding the same, unless a more limited interpretation is required from the context.

[0129] As used herein, the terms "identical" or "identity" in the context of two or more nucleic acid sequences or polypeptide sequences refer to two or more sequences or subsequences that have the same or a specified percentage of the same nucleotide residues or amino acid residues when compared and aligned to maximize correspondence. To determine identity, sequences are aligned for optimal comparison (e.g., gaps may be introduced into the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are of the same length after appropriate introduction of gaps into the sequences (e.g., excluding extra sequences that extend beyond the sequences being compared). For example, when variable region sequences are compared, leader sequences and / or constant domain sequences are not considered. For comparison of sequences between two sequences, "corresponding" CDRs refer to CDRs at the same location within both sequences (e.g., CDR-H1 of each sequence).

[0130] The determination of the identity or similarity between two arrays can be achieved using mathematical algorithms. Preferred non-limiting examples of mathematical algorithms utilized for the comparison of two arrays are the algorithms of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. By performing a BLAST nucleotide search using the NBLAST program, score = 100, wordlength = 12, a nucleotide sequence homologous to the nucleic acid encoding the protein of interest can be obtained. By performing a BLAST protein search using the XBLAST program, score = 50, wordlength = 3, an amino acid sequence homologous to the protein of interest can be obtained. To obtain a gapped alignment for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches, thereby detecting remote relationships between molecules (ibid.). When using the BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. In FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup = 2, similar regions within the two sequences being compared are found by looking for aligned residue pairs; when ktup = 1, single aligned amino acids are examined. Ktup can be set to 2 or 1 for protein sequences, or to 1-6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, alignment of protein sequences can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402. Preferably, the above CLUSTAL W algorithm is used.

[0131] As used herein, the term "comprising" indicates that other components and / or steps can be included in addition to the specifically recited components and / or steps. However, this term also encompasses the claimed subject matter consisting solely of the recited components and / or steps.

[0132] As used herein, the term "at least" refers to any number including the specifically recited number and any number higher than that. For example, "at least one" includes exactly one, as well as more than one, including but not limited to 2, such as 3 or 4. Further, for example, 5, 6, 7, 8, 9, 10, 15, such as 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500, and any integer between or above these specifically recited numbers is included. With respect to the term "at least one antigen-binding site", it is particularly preferred that the term includes 1, 2, 3, or 4 antigen-binding sites. Most preferably, the term relates to exactly one antigen-binding site. If more than one antigen-binding site is selected for the target, these multiple antigen-binding sites can be selected independently, i.e., they may be the same or different from each other.

[0133] As used herein, the term "polypeptide" refers to a linear amino acid molecule containing more than 30 amino acids, including single-chain polypeptides or fragments thereof. On the other hand, the term "peptide" as used in the present invention refers to a linear amino acid containing up to 30 amino acids. The term "(poly)peptide" used according to the present invention refers to a group of molecules including a group of peptides consisting of up to 30 amino acids, as well as a group of polypeptides consisting of more than 30 amino acids.

[0134] As used herein, the term "linker" encompasses both peptide linkers, i.e., amino acid sequences, and non-peptide linkers that connect the individual parts of a molecule either covalently or non-covalently. As used herein, the term "non-peptide linker" refers to a linking group that has two or more reactive groups but excludes peptide linkers as defined below. For example, a non-peptide linker can be a polymer having reactive groups at both ends, which individually binds to the reactive groups of the binding moiety of the molecule of the present invention, such as an amino terminus, a lysine residue, a histidine residue, or a cysteine residue. Examples of reactive groups of the polymer include an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group, a ketone group, a vinyl sulfone group, a thiol group, a hydrazide group, a carbonyldiimidazole (CDI) group, a nitrophenyl carbonate (NPC) group, a trysylate group, an isocyanate group, and a succinimide derivative. Examples of succinimide derivatives include succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA), succinimidyl succinate (SS), succinimidyl carbonate, and N-hydroxysuccinimide (NHS). The reactive groups at both ends of the non-peptide polymer may be the same or different. For example, the non-peptide polymer may have a maleimide group at one end and an aldehyde group at the other end.

[0135] A peptide linker as contemplated herein is a (poly)peptide linker that is at least 1 amino acid in length. Preferably, the linker is 1 to 100 amino acids in length. More preferably, the linker is 5 to 50 amino acids in length, more preferably 10 to 40 amino acids in length, and even more preferably the linker is 15 to 30 amino acids in length. Non-limiting examples of small linkers often used include the sequence of glycine and serine amino acids, referred to as the GS mini-linker. Preferred examples of linker sequences are Gly / Ser linkers of various lengths, such as (gly x sery ) z Linkers such as (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3. The number of amino acids in these linkers may be changed, for example, and they may be 4 (e.g., GGGS) (SEQ ID NO: 95), 6 (e.g., GGSGGS) (SEQ ID NO: 94), 7 (e.g., GGGSGGS (SEQ ID NO: 221)), or a multiple thereof, such as 2 or 3 or more repeats of these 4 / 6 amino acids. Most preferably, such a GS mini-linker has 20 amino acids and the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232). Further examples of linkers include the following: [Table 1]

[0136] It will be understood by those skilled in the art that when the molecule of interest is a single polypeptide chain, the linker is a peptide linker.

[0137] It is known in the art that the properties of the linker, i.e., length and / or composition, such as the amino acid sequence, can modify or enhance the stability and / or solubility of the molecule containing the linker. Typically, the length and sequence of the linker are selected according to the composition of each molecule of interest. Those skilled in the art are well aware of the methods for designing various linkers and testing their suitability. See, for example, Voelkel, T. et al. Protein Engineering, Design and Selection, Volume 14, Issue 10, 2001, Pages 815-823. For example, the properties of the molecule can be easily tested by comparing the binding affinity of the binding moiety of the molecule of the present invention. In the case of the trispecific molecule of the present invention, each measurement for each binding moiety may be carried out separately. The stability of the resulting molecule can be measured using an ELISA-based method to determine the residual binding ability of the molecule after incubation in human serum at 37°C over several periods. Other suitable tests can be found, for example, in Brian R. Miller, B.R. et al. Protein Engineering, Design and Selection, Volume 23, Issue 7, 2010, Pages 549-557, or Kuegler, M. et al. Protein Engineering, Design and Selection, Volume 22, Issue 3, 2009, Pages 135-147.

[0138] The term "nucleic acid molecule" according to the present invention is used synonymously with the term "polynucleotide" herein, which includes DNA, such as cDNA or genomic DNA, and RNA, such as mRNA. Further included are molecules mimicking nucleic acids known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such molecules or nucleic acid derivatives mimicking the nucleic acids described in the present invention include phosphorothioate nucleic acids, phosphoramidate nucleic acids, 2'-O-methoxyethyl ribonucleic acids, morpholino nucleic acids, hexitol nucleic acids (HNA) and locked nucleic acids (LNA). LNA is an RNA derivative where the ribose ring is constrained by a methylene bond between the 2'-oxygen and the 4'-carbon. They may contain additional non-natural or modified nucleotide bases, as will be readily understood by those skilled in the art.

[0139] The binding molecule of the present invention In a first aspect, the present invention provides a binding molecule comprising at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding molecule that specifically binds to cadherin-3 (CDH3). Preferably, said at least one antigen-binding site that binds to CDH3 specifically binds to the EC1 domain of CDH3.

[0140] Accordingly, the binding molecule of the present invention (also referred to herein as "the protein of the present invention" or "the binding substance of the present invention") comprises at least two different antigen-binding sites specifically listed, namely at least one binding site for TRAILR2 and at least one binding site for CDH3. Due to at least two specificities, the binding molecule of the present invention is also referred to herein as the "bispecific binding molecule" of the present invention.

[0141] As used herein, the term CDH3 refers to "cadherin-3", which is also known as "P-cadherin". CDH3, a member of the cadherin-like superfamily, is a calcium-dependent cell-cell adhesion glycoprotein composed of five extracellular cadherin repeats (EC), a transmembrane region, and a highly conserved cytoplasmic tail. Human CDH3 is shown by SEQ ID NO: 98 and in the database accession number UniProt P22223 available at http: / / www.uniprot.org / uniprot / P22223.

[0142] Cadherins are a group of transmembrane proteins that serve as the main adhesion molecules located within the adhesive junction. They can regulate cell-cell adhesion through their extracellular domains, and their cytoplasmic domains connect to the actin cytoskeleton by binding to catenins. Structurally, cadherins contain many domains: classically, these are a signal sequence; a propeptide of about 130 residues; a single transmembrane domain and five extracellular cadherin domains repeated in series (four of which are cadherin repeat sequences and the fifth typically contains four conserved cysteines), and a C-terminal cytoplasmic domain. However, proteins are designed as members of the cadherin family broadly defined if they have one or more cadherin repeat sequences. The cadherin repeat sequence is an independently folded sequence of about 110 amino acids that typically contains motifs including the conserved sequences DRE, DXNDNAPXF, and DXD.

[0143] As used herein, the term "EC1 domain of CDH3" refers to the first of the extracellular cadherin repeat sequences of CDH3. Specifically, the EC1 domain of CDH3 is shown by the amino acid sequence of SEQ ID NO: 99.

[0144] Accordingly, the term "EC2 domain of CDH3" refers to the second extracellular cadherin repeat sequence of CDH3, shown by SEQ ID NO: 100.

[0145] The binding molecule of the present invention is not particularly limited with respect to its format. However, it includes at least two different antigen-binding sites (TRAILR2 and CDH3) specifically enumerated, and can bind to these two targets. Thus, the format can be based on a natural antibody or antibody derivative, or a fragment of such an antibody, as well as the format of an antibody mimetic. Such formats can, if necessary, be modified to accommodate all three antigen-binding sites by adding, for example, further other antibody fragments, particularly Fv, Fab, Fab’, or F(ab’)2 fragments, single-chain antibodies, particularly single-chain variable fragments (scFv), small modular immunopharmaceuticals (SMIP), domain antibodies, or nanobodies. Further other non-limiting examples of suitable formats that can be used for any one or for all of the antigen-binding sites include antibody mimetics as defined above herein.

[0146] In a preferred embodiment, at least one antigen-binding site of the binding molecule described herein that specifically binds to cadherin-3 (CDH3) is an immunoglobulin (Ig) molecule (having a full-length antibody with the conventional Y-shaped structure containing two heavy chains and two light chains), and at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) includes one or more scFv, scFab, Fab or Fv binding elements. Preferably, the antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) includes one or more scFv. Even more preferably, the binding molecule described herein includes two antigen-binding sites that specifically bind to TRAILR2, each containing an scFv, i.e., such a binding molecule includes two scFv having target specificity for TRAILR2.

[0147] The "single-chain Fv fragment" (scFv) is a polypeptide comprising an antibody heavy-chain variable domain (VH), a linker, and an antibody light-chain variable domain (VL), wherein the antibody domain and the linker have one of the following orders in the direction from the N-terminus to the C-terminus: a) VH-linker-VL, b) VL-linker-VH, where the linker is a polypeptide 15 to 25 amino acids in length, preferably 20 amino acids in length.

[0148] Furthermore, these single-chain Fv fragments can be further stabilized by incorporation of disulfide bonds between the VH domain and the VL domain, within the VH domain, or within the VL domain through incorporation of cysteine residues. The term N-terminus refers to the first amino acid of the polypeptide chain, while the C-terminus refers to the last amino acid at the C-terminus of the polypeptide chain. Thus, embodiments of the present invention are those in which one or more scFvs form disulfide bonds by including additional cysteine residues.

[0149] In one embodiment of the present invention, the stability of the scFv portion can be enhanced by incorporating two cysteine residues in three-dimensional proximity to form a disulfide bond (referred to herein as scFvss) within the scFv. When the scFv is derived from the variable region sequence of TRv1 (as discussed below), examples of sites where such stabilizing disulfide bonds can potentially be engineered include (a) between position 99 of VL and position 45 of VH, (b) between position 102 of VL and position 44 of VH, (c) between position 4 of VL and position 100 of VL, and (d) between position 6 of VH and position 112 of VH. For stabilization to occur through engineered disulfide bonds, the residues at these positions are preferably replaced with cysteine residues.

[0150] Preferably, the antigen-binding site for TRAILR2 is an scFv fused to the C-terminus of the heavy chain of the immunoglobulin molecule; for example, one scFv fused to one of the heavy chains of the immunoglobulin molecule, or two scFvs (where one scFv is fused to one of the two heavy chains and one scFv is fused to the other heavy chain). Thereby, a modified heavy chain is formed.

[0151] In a preferred embodiment, the bispecific binding molecule of the present invention comprises a modified immunoglobulin molecule, wherein (i) the immunoglobulin heavy chain comprises a heavy chain variable domain that specifically binds to CDH3, an immunoglobulin heavy chain constant domain, and also the amino acid sequence of an scFv that specifically binds to TRAILR2, which comprises the amino acid sequences of the variable domains of the light and heavy chains, wherein the scFv is linked to the C-terminus of the immunoglobulin constant domain, and (ii) the immunoglobulin light chain comprises the amino acid sequences of a light chain variable domain and a light chain constant domain that specifically binds to CDH3. Preferably, the modified immunoglobulin molecule comprises two immunoglobulin heavy chains (e.g., modified heavy chains) and two immunoglobulin light chains.

[0152] The fusion of the various components to each other is well known in the art. The fusion may be via, for example, a peptide linker or a non-peptide linker. Preferably, the fusion is via (a) a peptide linker(s).

[0153] In a specific embodiment of the present invention, one or more scFvs that specifically bind to TRAILR2 are fused to an immunoglobulin molecule that specifically binds to CDH3 by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids (e.g., any one of 6, 9, 12, or 15). Preferably, the scFv is fused to the C-terminus of the heavy chain of the immunoglobulin molecule.

[0154] Methods of linking an scFv molecule to the C-terminus of the heavy chain of an immunoglobulin molecule, or of linking variable domains within an scFv molecule, are well known in the art. Typically, small linker sequences of glycine and serine (also referred to as GS mini-linkers) amino acids are used.

[0155] The number of amino acids in the linker may be varied, as described herein, from 4 to 10 or more. In fact, usually, the linker interrupts a nucleic acid molecule encoding the IgG of interest (in this case, a nucleic acid encoding the variable domain of the heavy chain and the constant domain of the IgG type for the CDH binding site) with a nucleic acid molecule encoding a linker sequence (e.g., any one of 5, 10, 15, or 20 amino acid GS mini-linkers, preferably a linker such as GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232)) to form a nucleic acid encoding the desired scFv (in this case, including nucleic acids encoding the variable domains of the heavy and light chains in either the VL-VH or VH-VL orientation for the TRAILR2 binding site). Then, as further described below, the nucleic acid molecule encoding this fully modified heavy chain is placed within an expression vector and introduced into a suitable host cell, whereby a complete IgG heavy chain-scFv single polypeptide is formed.

[0156] Preferably, the linker between the scFv molecule and the C-terminus of the heavy chain of the IgG molecule is GGSGGS (SEQ ID NO: 94) or GGGSGGS.

[0157] Preferably, the immunoglobulin molecule is a monoclonal, chimeric, humanized, or human immunoglobulin (e.g., antibody) molecule. More preferably, the heavy chain constant region of the immunoglobulin molecule is selected from the group consisting of the constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. In another preferred embodiment, the light chain constant region of the immunoglobulin molecule is κ or λ. Preferably, the immunoglobulin molecule is IgG.

[0158] In one embodiment, the present invention provides a binding molecule, which is a multispecific binding protein comprising: (i) one immunoglobulin molecule that specifically binds to CDH3 and has two heavy chains and two light chains, and (ii) two scFv molecules (scFv(s)) that each specifically bind to TRAILR2. Preferably, each heavy chain of the immunoglobulin molecule has one scFv fused to its C-terminus, thereby forming a bispecific and tetravalent binding protein.

[0159] In one embodiment, the present invention provides (i) two heavy chains (e.g., modified heavy chains), each of which, from the N-terminus to the C-terminus: - a heavy chain variable domain (e.g., a mouse, humanized, or human VH domain) specific for CDH3, preferably specific for the EC1 domain of CDH3, - a constant domain of IgG (e.g., human IgG1 or IgG4), - a peptide linker (e.g., a GS mini-linker), and - an scFv specific for TRAILR2 (e.g., an scFv containing a VH domain (e.g., a mouse, humanized, or human VH domain), a linker, and a VL domain (e.g., a mouse, humanized, or human VL domain) from the N-terminus to the C-terminus, or vice versa, a VL domain, a linker, and a VH domain) ; and, (ii) two light chains, each of which, from the N-terminus to the C-terminus: - a light chain variable domain (e.g., a mouse, humanized, or human VL domain) specific for CDH3, preferably specific for the EC1 domain of CDH3, - a light chain constant domain (e.g., a human κ chain) containing a binding molecule (also referred to herein as a multispecific binding protein or a modified immunoglobulin molecule).

[0160] Since the Fc region of an antibody interacts with many Fc receptors, resulting in many important functional capabilities (which are referred to as "effector functions"), an antibody in certain embodiments is a full-length antibody or an antibody containing a part of the Fc region, and the latter is as long as the antibody exhibits specific binding to both the relevant part of the antigen and Fc receptors and complement. The choice of the type and length of the constant region depends on whether effector functions such as binding to complement or antibody-dependent cell-mediated cytotoxicity are desired features, and the desired pharmacological properties of the antibody protein.

[0161] In one embodiment of the present invention, the binding molecule of the present invention may have an Fc region or a relevant segment thereof that is engineered to avoid unintended cross-linking by soluble Fcγ receptors or complement C1q. In one embodiment, such a binding molecule or antibody variant has a much lower affinity for Fcγ receptors and complement C1q than the parental antibody. (Hereinafter, unless otherwise specified, the term "parental" refers to an unengineered antibody molecule, Fc region or IgG in the context of an antibody molecule or in the context of an IgG or Fc region, respectively, from which the subsequently mutated (engineered) molecule is derived). Thus, embodiments of the present invention include immunoglobulin molecules that contain an Fc variant having a reduced affinity for Fcγ receptors or complement receptors or both compared to the wild-type Fc region. Such immunoglobulin molecules are referred to herein as IgG1(KO).

[0162] A further embodiment of the present invention is that the binding molecule of the present invention is engineered to optimize its interaction with the neonatal Fc receptor (FcRn) by, for example, a point mutation at position H310A in the CH2 domain to modify the serum level (half-life), and includes an Fc region or a relevant segment thereof. Such immunoglobulin molecules are referred to herein as IgG1FcRnmut.

[0163] A further embodiment of the invention comprises an immunoglobulin molecule comprising an IgG4 hinge region variant in which the exchange with the heavy chain having another IgG4 molecule is removed. Such an immunoglobulin molecule is referred to herein as IgG4Pro.

[0164] The present invention provides a bispecific binding molecule having at least one antigen-binding site that specifically binds to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site that specifically binds to cadherin-3 (CDH3).

[0165] Methods for preparing binding sites that bind to specific target antigens are well known in the art. A person skilled in the art can easily use these methods to devise antigen-binding sites having the required specificity for the target antigens of TRAILR2 or CDH3.

[0166] Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies can be produced via any one of several methods that utilize in vivo induction of antibody molecule production, screening of immunoglobulin libraries (Orlandi et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al 1991, Nature 349:293-299), or production of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and Epstein-Barr virus (EBV) hybridoma technology (Kohler et al 1975. Nature 20 256:4950497; Kozbor et al 1985. J. Immunol. Methods 81 :31-42; Cote et al 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al 1984. Mol. Cell. Biol. 62:109-120).

[0167] In some embodiments, the antigen-binding site (for CDH3 and / or TrailR2) is a "humanized" antigen-binding site (e.g., comprising a humanized VH / VL domain) that includes amino acid residues derived from non-human hypervariable regions (HVRs; e.g., complementarity-determining regions (CDRs)) and amino acid residues derived from human framework sequences. In some embodiments, the antigen-binding site (for CDH3 and / or TRAIL2) is a human antigen-binding site (e.g., comprising a human VH / VL domain) that includes CDR sequences and FR sequences, both of which are derived from human genomic sequences.

[0168] The amino acid sequences of specific antigen-binding sites are provided in the description of the invention and the Sequence Listing.

[0169] The following provides details of preferred embodiments of the invention that include specific antigen-binding sites for TRAILR2 and / or CDH3.

[0170] In a preferred embodiment, at least one antigen-binding site that specifically binds to CDH3 includes a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3).

[0171] In a more preferred embodiment, at least one antigen-binding site that specifically binds to CDH3 includes a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3).

[0172] In a more preferred embodiment, at least one antigen-binding site that specifically binds to CDH3 comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3).

[0173] In a more preferred embodiment, at least one antigen-binding site that specifically binds to CDH3 comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3).

[0174] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding molecule that specifically binds to TRAILR2 comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 79 (CDR3).

[0175] Thus, in a specific embodiment, the binding molecule described herein comprises an antigen-binding site that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (HCDR1), SEQ ID NO: 4 (HCDR2), and SEQ ID NO: 5 (HCDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (LCDR1), SEQ ID NO: 8 (LCDR2), and SEQ ID NO: 9 (LCDR3); and at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2, which comprise a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (HCDR1), SEQ ID NO: 74 (HCDR2), and SEQ ID NO: 75 (HCDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2), and SEQ ID NO: 79 (LCDR3).

[0176] In a further specific embodiment, the binding molecule described herein comprises a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2) and SEQ ID NO: 15 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (LCDR1), SEQ ID NO: 18 (LCDR2) and SEQ ID NO: 19 (LCDR3), an antigen-binding site that specifically binds to CDH3; and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (HCDR1), SEQ ID NO: 74 (HCDR2) and SEQ ID NO: 75 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2) and SEQ ID NO: 79 (LCDR3), at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2.

[0177] In a further specific embodiment, the binding molecule described herein comprises a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (HCDR1), SEQ ID NO: 24 (HCDR2) and SEQ ID NO: 25 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (LCDR1), SEQ ID NO: 28 (LCDR2) and SEQ ID NO: 29 (LCDR3), an antigen-binding site that specifically binds to CDH3; and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (HCDR1), SEQ ID NO: 74 (HCDR2) and SEQ ID NO: 75 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2) and SEQ ID NO: 79 (LCDR3), at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2.

[0178] In a further specific embodiment, the binding molecule described herein comprises a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (HCDR1), SEQ ID NO: 34 (HCDR2), and SEQ ID NO: 35 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (LCDR1), SEQ ID NO: 38 (LCDR2), and SEQ ID NO: 39 (LCDR3), an antigen-binding site that specifically binds to CDH3; and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (HCDR1), SEQ ID NO: 74 (HCDR2), and SEQ ID NO: 75 (HCDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2), and SEQ ID NO: 79 (LCDR3), at least one, preferably two antigen-binding sites that specifically bind to TRAILR2.

[0179] The CDRs disclosed herein and represented by the above SEQ ID numbers are presented according to Kabat's nomenclature and are shown in Table 1 below. As used herein, HCDR means heavy chain CDR and LCDR means light chain CDR.

[0180] Since additional nomenclatures are known in the art, CDR sequences based on the most commonly used nomenclature among them are also shown in Table 1 below. These numbering systems are based on the following: (i) CCG (Chemical Computing Group as exemplified in Almagro et al., Proteins 2011; 79:3050 - 3066 and Maier et al, Proteins 2014; 82:1599 - 1610), (ii) Chothia (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901 - 917), (iii) IMGT (Lefranc MP, Dev Comp Immunol. 2003 Jan;27(1):55 - 77), and (iv) North (North B, J Mol Biol. (2011) 406:228 - 56).

[0181]

Table 2

[0182] The positions of the amino acids shown herein for the CDRs according to Kabat, CCG, Chothia, IMGT, and North (see Table 1) are linear, i.e., the amino acids of each full-length molecular chain are numbered continuously, starting from 1 at the N-terminus and ending with a number corresponding to the total number of amino acids in the molecule. For example, a heavy chain consisting of 118 amino acids starts from 1 at the N-terminus and ends with 118 for the amino acid on the most C-terminal side. Thus, any reference to position 25, for example, means that the 25th amino acid from the N-terminus of this molecule is being referred to.

[0183] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to CDH3 comprises an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and an immunoglobulin light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6.

[0184] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to CDH3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0185] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to CDH3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0186] In a preferred embodiment of the binding molecule of the present invention, at least one antigen-binding site that specifically binds to CDH3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0187] According to the present invention, the terms "immunoglobulin heavy chain variable domain" and "immunoglobulin light chain variable domain" are used according to their definitions in the art.

[0188] Specifically, in the binding molecule of the present invention, at least one antigen-binding site for CDH3 is selected from the above antigen-binding sites herein, where the antigen-binding site for TRAILR2 is selected by those skilled in the art from such TRAILR2-specific antigen-binding sites available in the art or from those disclosed herein.

[0189] Preferably, the antigen-binding site that specifically binds to TRAILR2 is a single-chain variable fragment (scFv). More preferably, the scFv is arranged such that the heavy chain variable domain is at its N-terminus and the light chain variable domain is at its C-terminus.

[0190] Methods for linking a polypeptide of interest, such as an scFv molecule, to the C-terminus of the heavy chain of an IgG molecule, for example, are well known in the art. It is understood that the fusion of the scFv to the immunoglobulin molecule may be a direct fusion or via a linker, preferably a peptide linker as described above. In fact, the linkage is typically achieved by combining a nucleic acid molecule encoding the desired IgG with a nucleic acid encoding the desired polypeptide, such as an scFv, interrupted if necessary by a nucleic acid molecule encoding a linker sequence, thereby forming a single nucleic acid molecule containing all three elements. This complete HC-scFv-encoding nucleic acid molecule is then placed within an expression vector and introduced into a suitable host cell, thereby forming a single polypeptide of the complete IgG heavy chain-scFv, and optionally, the same is also carried out for the IgG light chain-scFv counterpart.

[0191] In a specific embodiment of the binding molecule of the present invention, the antigen-binding site (first antigen-binding site) that specifically binds to TRAILR2 is an antigen-binding site comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 72 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 76.

[0192] Thus, in a specific embodiment, the binding molecule described herein comprises an antigen-binding site that specifically binds to CDH3, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 2 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 6; and at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 72 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 76.

[0193] In a further specific embodiment, the binding molecule described herein comprises an antigen-binding site that specifically binds to CDH3, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 16; and at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 72 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 76.

[0194] In a further specific embodiment, the binding molecule described herein comprises an antigen-binding site that specifically binds to CDH3, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 22 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 26; and at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2, comprising an immunoglobulin heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 72 and an immunoglobulin light chain variable domain containing the amino acid sequence of SEQ ID NO: 76.

[0195] In a further specific embodiment, the binding molecule described herein comprises an antigen-binding site that specifically binds to CDH3, which comprises an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 32 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 36; and at least one, preferably two, antigen-binding sites that specifically bind to TRAILR2, which comprise an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 72 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 76.

[0196] In a preferred embodiment of the present invention, the binding molecule comprises the following: i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87.

[0197] In a specific embodiment, the binding molecule provided herein is i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 80, and a light chain comprising the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 84, and a light chain comprising the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85; or iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 86, and a light chain comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87 is included.

[0198] In a specific embodiment, therefore, the binding molecule described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 80, and a light chain comprising the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81; and at least one, preferably two scFvs that have target specificity for TRAILR2 and comprise an immunoglobulin heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 72, and an immunoglobulin light chain variable domain having the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO: 71.

[0199] In a further specific embodiment, thus, the binding molecule described in the present specification comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 82, and a light chain comprising the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83; and at least one, preferably two, scFvs that have target specificity for TRAILR2 and comprise an immunoglobulin heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 72, and an immunoglobulin light chain variable domain having the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO: 71.

[0200] In a further specific embodiment, therefore, the binding molecule described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 84, and a light chain comprising the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85; and at least one, preferably two scFvs, which have target specificity for TRAILR2 and comprise an immunoglobulin heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 72, and an immunoglobulin light chain variable domain having the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO: 71.

[0201] In a further specific embodiment, therefore, the binding molecule described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 86, and a light chain comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87; and at least one, preferably two scFvs that have target specificity for TRAILR2 and comprise an immunoglobulin heavy chain variable domain having the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 72, and an immunoglobulin light chain variable domain having the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 76. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO: 71.

[0202] In a specific embodiment, the binding molecule of the present invention comprises a modified heavy chain, and preferably, the TRAILR2-specific scFv is fused to the C-terminus of the immunoglobulin heavy chain. Preferably, the modified heavy chain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO: 31. Specifically, the binding molecule of the present invention comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, or SEQ ID NO: 31.

[0203] Specifically, the binding molecule of the present invention i. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 comprising.

[0204] In a preferred embodiment, thus, the binding molecule described herein comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1, and a light chain comprising the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 81.

[0205] In a further preferred embodiment, thus, the binding molecule described herein comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 11, and a light chain comprising the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 83.

[0206] In a further preferred embodiment, thus, the binding molecule described herein comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 85.

[0207] In a further preferred embodiment, therefore, the binding molecule described herein comprises a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 31, and a light chain comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 87.

[0208] Also described herein is a bispecific binding molecule having at least one antigen-binding site (first antigen-binding site) that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) as detailed above, and at least one antigen-binding site (second antigen-binding site) that specifically binds to the extracellular domain 2 (EC2 domain) of cadherin-3 (CDH3).

[0209] In one embodiment, therefore, the binding molecule provided herein binds to an antigen comprising or consisting of the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 100.

[0210] Preferably, said at least one antigen-binding site that specifically binds to the EC2 domain of CDH3 is i. an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 49 (CDR3); ii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3), an antigen-binding site; and, iii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), an antigen-binding site selected from the group consisting of.

[0211] More preferably, the antigen-binding site that specifically binds to the EC2 domain of CDH3 is i. VH comprising the amino acid sequence of SEQ ID NO: 42 and VL comprising the amino acid sequence of SEQ ID NO: 46; ii. VH comprising the amino acid sequence of SEQ ID NO: 52 and VL comprising the amino acid sequence of SEQ ID NO: 56; and, iii. VH comprising the amino acid sequence of SEQ ID NO: 62 and VL comprising the amino acid sequence of SEQ ID NO: 66 selected from the group consisting of.

[0212] Preferably, the binding molecule described herein and having target specificity for the EC2 domain of CDH3 is i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 91; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93 comprising.

[0213] In a specific embodiment, the binding molecule targeting the EC2 domains of TRAILR2 and CDH3 comprises a modified heavy chain. Preferably, the TRAILR2-specific scFv is fused to the C-terminus of the immunoglobulin heavy chain. Preferably, the modified heavy chain comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 51, or SEQ ID NO: 61. Specifically, the binding molecule comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 41, SEQ ID NO: 51, or SEQ ID NO: 61.

[0214] Specifically, the binding molecule is: i. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; ii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 91; or iii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 93 and includes.

[0215] The monospecific antibody molecule of the present invention As used herein, antibody molecules that specifically bind to CDH3 (e.g., full-length antibodies / immunoglobulin molecules having a Y-shaped structure with two heavy chains and two light chains, or fragments thereof, such as Fv, Fab, Fab’, or F(ab’)2 fragments, single-chain antibodies, single-chain variable fragments (scFv)) are further provided. Specifically, antibody molecules that specifically bind to the EC1 domain or EC2 domain of CDH3 are provided herein. In some embodiments, the antibody molecule specific for CDH3 is a recombinant monoclonal antibody, chimeric, humanized, or human antibody molecule.

[0216] In some embodiments, the antibody molecule specific for CDH3 comprises any one of the following combinations of CDRs shown in (i)-(vii): i. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3); ii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3); iii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3); iv. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3); v. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 49 (CDR3); vi. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3); vii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3).

[0217] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3).

[0218] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3).

[0219] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3).

[0220] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3).

[0221] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 49 (CDR3).

[0222] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3).

[0223] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3).

[0224] In some embodiments, the antibody molecule specific for CDH3 is i. VH comprising the amino acid sequence of SEQ ID NO: 2 and VL comprising the amino acid sequence of SEQ ID NO: 6; ii. VH comprising the amino acid sequence of SEQ ID NO: 12 and VL comprising the amino acid sequence of SEQ ID NO: 16; iii. VH comprising the amino acid sequence of SEQ ID NO: 22 and VL comprising the amino acid sequence of SEQ ID NO: 26; iv. VH comprising the amino acid sequence of SEQ ID NO: 32 and VL comprising the amino acid sequence of SEQ ID NO: 36; v. VH comprising the amino acid sequence of SEQ ID NO: 42 and VL comprising the amino acid sequence of SEQ ID NO: 46; vi. VH comprising the amino acid sequence of SEQ ID NO: 52 and VL comprising the amino acid sequence of SEQ ID NO: 56; or vii. VH comprising the amino acid sequence of SEQ ID NO: 62 and VL comprising the amino acid sequence of SEQ ID NO: 66 and comprises.

[0225] Specifically, in the present specification, there is provided an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy-chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light-chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6.

[0226] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0227] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26.

[0228] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0229] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 46.

[0230] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56.

[0231] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 66.

[0232] In some embodiments, a CDH3-specific antibody as defined above further comprises a human heavy chain constant domain (e.g., an IgG constant domain) and a human light chain constant domain (e.g., a κ or λ light chain constant domain).

[0233] In a specific embodiment of an antibody molecule specific for CDH3, the antibody molecule i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87; v. A heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; vi. A heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 91; vii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93 are included.

[0234] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81.

[0235] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83.

[0236] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0237] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3 and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87.

[0238] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0239] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 91.

[0240] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0241] The CDH3-specific antibodies provided herein can be used to label, localize, identify, or target cells expressing CDH3 in vitro, in vivo, or ex vivo (e.g., in ELISA assays, FACS (fluorescence-activated cell sorting) assays, immunohistochemical examinations, etc.) by attaching them to dyes, drugs, or other molecules having binding specificities for different antigens. In some embodiments, the CDH3-specific antibody specifically binds to the surface of CDH3-expressing cells, which is used for the localization and / or identification of such cells. In some embodiments, the CDH3 antibody provided herein is used for the identification of cells (e.g., tumor cells) expressing CDH3. In some embodiments, the CDH3 antibody provided herein is used to deliver a drug or a cytotoxic agent to target cells (e.g., tumor cells expressing CDH3) by attaching such a drug or cytotoxic agent to the CDH3 antibody, thereby killing the target cells, for example.

[0242] Also provided herein is a method for detecting cadherin-3 (CDH3) in a sample, the method comprising (a) contacting the sample with an anti-CDH3 antibody molecule as defined hereinabove; (b) A step that enables the formation of antibody-antigen complexes in the sample; and, (c) A step of detecting the anti-CDH3 antibody comprises.

[0243] Means and methods for detecting antibodies are well known in the art, and these include, for example, immunohistochemical examination, immunoblotting, and ELISA.

[0244] Methods for identifying whether a specific tumor expresses TRAILR2 and / or CHD3 are well known in the art. For example, immunohistochemical examination can be used to determine whether a tumor tissue expresses TRAILR2 and / or CDH3 (for example, using the TRAILR2 and / or CDH3 antibody molecules described herein).

[0245] As used herein, a kit for detecting cadherin-3 (CDH3) is further provided, where the kit includes an anti-CDH3 antibody molecule as defined above herein, and instructions for use.

[0246] As used herein, a kit for detecting cadherin-3 (CDH3) and TRAILR2 is further provided, where the kit preferably includes an anti-CDH3 antibody molecule and an anti-TRAILR2 antibody molecule as defined above herein, and instructions for use.

[0247] The nucleic acid molecules, expression vectors, and host cells of the present invention The present invention further relates to a nucleic acid molecule or a part thereof encoding the bispecific binding molecule or antibody molecule described herein. The present invention further encompasses a set of nucleic acid molecules encoding the bispecific binding molecule or antibody molecule described herein.

[0248] According to the present invention, the nucleic acid molecule "encodes" the binding molecule of the present invention or a part thereof, which means that the nucleic acid molecule is provided in an expressible form, that is, in a form that can reliably express the binding molecule of the present invention (or each part thereof) therefrom.

[0249] In some embodiments, the binding molecule of the invention or the antibody molecule of the invention comprises a polypeptide of the heavy chain and / or light chain of an antibody. As can be understood by those skilled in the art, nucleic acid molecules encoding the heavy chain polypeptide, the light chain polypeptide, or both the heavy chain polypeptide and the light chain polypeptide can be readily prepared.

[0250] The term "a portion thereof" reflects the fact that not all components of the binding molecule of the invention need to be encoded on a single nucleic acid molecule, as is understood by those skilled in the art. Instead, two or more nucleic acid molecules may be relied upon to separately encode specific portions of the binding molecule of the invention. Accordingly, the invention also encompasses a set of isolated nucleic acid molecules, where the set together encodes all portions of the binding molecule of the invention, such that expression of this set of isolated nucleic acid molecules generates the complete binding molecule of the invention. In other words, provided herein are one or more nucleic acid molecules encoding the individual polypeptide chains of the binding molecule of the invention, including the heavy chain, light chain, scFv, and combinations thereof, either separately on individual nucleic acid molecules or combined within one nucleic acid molecule.

[0251] Preferably, the nucleic acid molecule is a DNA molecule comprising a coding sequence. More preferably, the DNA molecule further comprises regulatory sequences and optionally natural or artificial introns (such as the β-globin intron from Homo sapiens into which the miRNA-557 expression cassette is embedded). It may have its original codons or an optimized codon usage frequency specifically adapted for expression in the target host cell or host organism. Such nucleic acid molecules of the invention can be readily prepared or obtained by those skilled in the art relying on methods known per se, such as automated DNA synthesis, isolation from natural sources, and / or recombinant DNA techniques, based on the information regarding the amino acid sequences of the binding molecules of the invention shown herein.

[0252] The nucleic acid molecules of the present invention include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the Sequence Listing. The present invention further contemplates nucleic acid molecules complementary to the DNA molecules defined above, and nucleic acid molecules that hybridize thereto under high stringency binding and washing conditions as defined in WO 2007 / 042309. Preferred molecules (from the perspective of mRNA) have a homology or sequence identity of at least 75% or 80% (preferably at least 85%, more preferably at least 90%, most preferably at least 95%) to one of the DNA molecules described herein. For example, if the goal is to express the bispecific binding molecule or antibody molecule of the present invention in eukaryotic cells, the DNA sequence must be designed to match the codon usage frequency in eukaryotic cells. If it is desired to express an antibody in E. coli or other prokaryotic cell lines, these sequences must be designed to match the codon usage frequency of E. coli or the respective prokaryotic cell line. Variants of the DNA molecules of the present invention can be constructed in several different ways, for example, as described in WO 2007 / 042309.

[0253] Preferably, the nucleic acid(s) is / are isolated, and the term "isolated" is further defined above.

[0254] The present invention further relates to expression vectors containing the nucleic acid molecule(s) of the present invention.

[0255] To generate the binding molecule or antibody of the present invention, a DNA molecule or a portion thereof encoding the binding molecule or antibody molecule described herein is inserted into an expression vector such that the sequence is operably linked to transcriptional and translational control sequences.

[0256] To produce the binding molecule or antibody of the present invention, one of ordinary skill in the art can select from a variety of expression systems well known in the art, such as those reviewed by Kipriyanov and Le Gall, Curr Opin Drug Discov Devel. 2004 Mar;7(2):233-42.

[0257] According to the present invention, the vector is an expression vector, i.e., a vector that can effect the expression of the respective polypeptide from the encoding nucleic acid molecule in vitro and / or in vivo (e.g., in a suitable host cell, host organism, and / or expression system). Examples of expression vectors include plasmids, retroviruses, cosmids, Epstein-Barr virus-derived episomes, and the like. The expression vector and the expression control sequences are typically selected to be compatible with the host cell. The expression vector generally contains at least one nucleic acid molecule of the present invention operably linked to one or more appropriate regulatory sequences (singular or plural), such as a promoter(s), enhancer(s), terminator(s), etc. Specific examples of such regulatory sequences and other sequences useful or necessary for expressing the polypeptide of the present invention, such as integrase(s), selectable marker(s), signal sequence or leader sequence(s), reporter gene(s), etc., are disclosed, for example, on pages 131 to 133 of WO 2006 / 040153.

[0258] Non-limiting examples of promoter sequences (exemplified for expression in mammalian cells) include promoters and / or enhancers derived from cytomegalovirus (e.g., the cytomegalovirus promoter / enhancer of human cytomegalovirus, or the cytomegalovirus simian virus 40 (SV40) promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters. Examples of polyadenylation signals include hamster growth hormone or bovine growth hormone polyA, SV40 late or early polyA; or the 3' untranslated region (UTR) of the immunoglobulin gene, etc., may be used.

[0259] The recombinant expression vector may also have sequences that regulate the replication of the vector in the host cell (e.g., an origin of replication, such as the ColE1 (pUC) origin of replication) and a selectable marker gene (e.g., the β-lactamase gene that confers ampicillin resistance for plasmid amplification in E. coli, etc.). The recombinant expression vector may also encode a signal peptide that promotes the secretion of the resulting polypeptide. The nucleic acid molecule encoding each polypeptide chain can be cloned into the vector such that the signal peptide is ligated in-frame to the amino terminus of the mature full-length nucleic acid molecule chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide derived from a non-immunoglobulin protein. Alternatively, the DNA sequence encoding the full-length chain of the protein of the present invention may already contain a signal peptide sequence.

[0260] As indicated above, the coding sequences inserted into the vector may be generated, for example, by standard methods of synthesis, or isolated from natural sources, or semi-synthetically, i.e. by combining chemical synthesis and recombinant techniques. Ligation of the coding sequences to transcriptional regulatory sequences and / or to other amino acid coding sequences can be carried out using established methods. One approach often used is, for example, to use a vector encoding a functionally complete human CH (constant heavy chain) immunoglobulin sequence having appropriate restriction enzyme sites engineered such that any antigen-binding site, for example a single-chain Fab sequence or any heavy / light chain variable domain, can be readily inserted and expressed. For the heavy chain of the antibody, it can be, but is not limited to, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulin (including allelic variants).

[0261] If more than one nucleic acid molecule is required for the construction of the binding molecule of the invention, these more than one nucleic acid molecules may be inserted into different expression vectors or into the same expression vector. In the latter case, they may be under the control of the same regulatory sequences, such as promoters, enhancers, terminators, etc., or they may each have a unique set of regulatory sequences. According to the invention, when more than one nucleic acid molecule encodes the individual sequences of the binding molecule of the invention, it is particularly preferred that all the individual nucleic acid molecules required to form the binding molecule of the invention are present on a single expression vector, preferably each nucleic acid molecule having a unique set of regulatory sequences.

[0262] Expression vectors containing these DNA molecules can be introduced into host cells, such as bacterial cells or (higher) eukaryotic cells, such as mammalian cells, according to transfection methods well known in the art (including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or introduction by viral vectors).

[0263] Accordingly, the present invention also relates to a host cell transfected with the expression vector(s) of the present invention.

[0264] The host cell can be any suitable cell known in the art, including prokaryotic cells such as bacteria, and eukaryotic cells such as yeast cells or mammalian cells. Non-limiting examples of mammalian cells include, but are not limited to, human, mouse, rat, monkey, and rodent cell lines. Specific mammalian cell lines available as host cells for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., HepG2 and A-549 cells), 3T3 cells, or derivatives / descendants of any such cell line. Suitable culture media and culture conditions for the above host cells are known in the art.

[0265] Manufacturing and preparation methods To produce the antibodies of the present invention, those skilled in the art can select from a variety of methods well known in the art.

[0266] For the production of antibodies containing two complete heavy chains and two complete light chains, such as IgG1 or IgG4 antibodies, see Norderhaug et al., J Immunol Methods 1997, 204 (1): 77-87; Kipriyanow and Le Gall, Molecular Biotechnology 26: 39-60, 2004; Shukla et al., 2007, J. Chromatography B, 848(1): 28-39.

[0267] Fab molecules can be produced by the expression of nucleic acids encoding such constructs in host cells such as E. coli, Pichia pastoris, or mammalian cell lines (e.g., CHO or NS0). Processes that allow for the proper folding, association, and disulfide bonding of these chains into functional Fab molecules containing the Fd fragment and the light chain are known in the art (Burtet et al., J. Biochem. 2007, 142(6), 665 - 669; Ning et al., Biochem. Mol. Biol. 2005, 38: 204 - 299; Quintero - Hernandez et al., Mol. Immunol. 2007, 44: 1307 - 1315; Willems et al. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2003;786:161 - 176).

[0268] Processes for producing scFv antibodies by recombinant expression of nucleic acids encoding scFv constructs in host cells (such as E. coli, Pichia pastoris, or mammalian cell lines, e.g., CHO or NS0) that result in functional scFv molecules are also known (Rippmann et al., Applied and Environmental Microbiology 1998, 64(12): 4862 - 4869; Yamawaki et al., J. Biosci. Bioeng. 2007, 104(5): 403 - 407; Sonoda et al., Protein Expr. Purif. 2010, 70(2): 248 - 253).

[0269] Specifically, provided herein is a method for generating the binding molecules or antibody molecules described herein, the method comprising (a) culturing a host cell of the invention under conditions that allow for the expression of a binding molecule of the invention; (b) optionally, recovering the molecule; and, optionally (c) the step of further purifying and / or modifying and / or formulating the binding molecule comprises.

[0270] The protein of the present invention is produced by culturing a host cell for a time sufficient to permit expression of the protein by the host cell.

[0271] Conditions suitable for culturing a prokaryotic or eukaryotic host are well known to those skilled in the art. To increase the yield and solubility of the expression product, the medium may be buffered or supplemented with appropriate additives known to enhance or facilitate or both. Generally, those skilled in the art also know that these conditions must be adapted to the needs of the host and the requirements of the molecule to be expressed. When an inducible promoter controls the nucleic acid molecule(s) of the present invention within a vector(s) present in the host cell, expression of the molecule of interest can be induced by the addition of an appropriate inducer. Appropriate expression protocols and strategies are known to those skilled in the art.

[0272] Subsequently, the binding molecules of the present invention are recovered and, if necessary, further purified. Preferably, they are recovered from the culture medium as secreted molecules. However, if they are expressed, for example, without a secretion signal, they can also be recovered from the host cell lysate. The term "recovering the molecule" refers to the isolation of the binding molecule of the present invention encoded by the nucleic acid molecule(s) of the present invention, i.e., the binding molecule present in the host cell of the present invention due to transformation or transfection of the host cell with the nucleic acid molecule or vector of the present invention.

[0273] The optional step of purifying the binding molecule of the present invention is further useful in obtaining a substantially homogeneous preparation of the molecule. Means and methods for purifying the molecule of interest are well known, and those skilled in the art can use, for example, standard protein purification methods used for recombinant proteins and host cell proteins, and can adapt them in a manner suitable for each molecule. For example, state-of-the-art purification methods in the art useful for obtaining the binding molecule of the present invention include, as a first step, removal of cells and / or particulate cell debris from the culture medium or lysate, followed by fractionation on, for example, an immunoaffinity column or an ion exchange column, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, silica chromatography or cation exchange resin chromatography to purify from contaminating soluble proteins, polypeptides and nucleic acids.

[0274] As a final optional step in the process for obtaining the binding molecule of the present invention, the purified protein molecule may be dried, for example lyophilized, or formulated in other ways as desired, as described below for therapeutic applications. Further, the resulting binding molecule of the present invention may be subjected to further modification, for example to remove unwanted post-translational modifications and the like.

[0275] Pharmaceutical compositions, and medical use of the binding molecule or pharmaceutical composition of the present invention The present invention further relates to a pharmaceutical composition comprising or consisting of one or more of the binding molecules or antibody molecules described herein. In one embodiment, the binding molecule(s) is the sole pharmaceutically active agent(s). In alternative embodiments, the composition comprises, in addition to the binding molecule(s), one or more further pharmaceutically active agents, as further defined, for example, below.

[0276] According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical compositions of the present invention contain the compounds listed above, either alone or in combination. Optionally, it may further contain other molecules which can, for example, stabilize, modulate and / or activate their functions by changing the characteristics of the compounds of the present invention. The composition can be in solid, liquid or gaseous dosage forms, and in particular, can be in the dosage forms of powders (single or plural), for example lyophilized powders, solutions (single or plural), tablets (single or plural), aerosol agents (single or plural). Preferably, the composition is a lyophilized powder or a solution.

[0277] For use in therapy, the bispecific binding molecules described herein are formulated into pharmaceutical compositions suitable for facilitating administration to an animal or a human. Thus, the pharmaceutical compositions of the invention preferably also contain a pharmaceutically acceptable carrier. Compositions containing such carriers can be formulated by well-known conventional methods. Typically, a pharmaceutical composition containing a binding molecule of the invention can be formulated by mixing the binding molecule with such a pharmaceutically acceptable carrier and (optionally) an excipient or stabilizer. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type. Also, other excipients, modifiers, or stabilizers are non-toxic at the dosages and concentrations employed. Pharmaceutically acceptable carriers, excipients, modifiers, and stabilizers include buffer systems such as phosphates, citrates, acetates, and other inorganic or organic acids and their salts; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosaccharides, or polysaccharides and other carbohydrates such as glucose, mannose, sucrose, trehalose, dextrin or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or ionic or non-ionic surfactants such as TWEEN™ (polysorbate), pluronics™ or fatty acid esters, fatty acid ethers, or sugar esters, but are not limited thereto. Also, organic solvents such as ethanol or isopropanol may be contained in the formulation.The excipient may also have a release modification function or an absorption modification function.

[0278] Generally, an aqueous solution or suspension is preferred. Generally, a formulation suitable for a therapeutic protein such as the binding molecule of the present invention is a buffered protein solution, for example, a protein at an appropriate concentration (e.g., 0.001 - 400 mg / ml, preferably 0.005 - 200 mg / ml, more preferably 0.01 - 200 mg / ml, more preferably 1.0 - 100 mg / ml, e.g., 1.0 - 40.0 mg / ml, preferably 10.0 - 30 mg / ml, most preferably 30 mg / ml (for intravenous administration) or 100 mg / ml (for subcutaneous administration)) and an aqueous buffer, for example: - Phosphate buffered saline, pH 7.4, - Other phosphate buffers, pH 6.2 - 8.2, - Acetate buffer, pH 3.2 - 7.5, preferably pH 4.8 - 5.5, - Histidine buffer, pH 5.0 - 7.0, preferably pH 5.3 - 5.5, - Succinate buffer, pH 3.2 - 6.6, or - Citrate buffer, pH 2.1 - 6.2, and optionally, salts (e.g., sodium chloride) and / or stabilizers (e.g., sucrose, trehalose, lysine) and / or other polyalcohols (e.g., mannitol and glycerol) to render the solution isotonic, and optionally a detergent (e.g., 0.02% Tween - 20 or Tween - 80) for example to prevent aggregation is a solution containing the above.

[0279] A buffered protein solution preferred for intravenous administration is a solution containing about 10 mg / ml of the binding molecule of the present invention dissolved in 10 mM citrate buffer, pH 5.5, 207 mM sucrose, 25 mM lysine hydrochloride, and 0.02% polysorbate 20.

[0280] A particularly preferred buffered protein solution for intravenous administration is a solution containing about 30 mg / ml of the binding molecule of the present invention dissolved in about 20 mM His / His hydrochloride buffer, pH 5.3 - 5.5, preferably pH 5.3, 220 mM sucrose, 0.02% polysorbate 29, and water for injection (WFI).

[0281] Formulations for subcutaneous application may contain significantly higher concentrations of the antibodies of the present invention, for example, concentrations up to and including 100 mg / ml or even exceeding 100 mg / ml. However, it will be apparent to those skilled in the art that the components and amounts as set forth above are merely illustrative of one preferred option. Alternative options and variations thereof will be readily understood by those skilled in the art or can be readily conceived starting from the above disclosure.

[0282] The pharmaceutical composition of the present invention can be administered to a subject using any suitable mode of administration, including, for example, parenteral administration (intravenous, intra-articular, intramuscular, subcutaneous, intraperitoneal, intradermal) by infusion or injection, as well as transdermal, intranasal, buccal, or oral administration, or administration by inhalation. For administration of a solution or reconstituted lyophilized powder, a parenteral mode of administration is preferred.

[0283] Generally, the efficacy of the specific binding molecule of the present invention intended to be used, the specific route of administration, and the specific pharmaceutical formulation or pharmaceutical composition used for the treatment, prevention, and / or alleviation of the diseases, disorders, and conditions described herein, and according to the specific disease, disorder, or condition to be treated, affect the actual dose to be administered. Further, the actual pharmaceutically effective amount or therapeutic dose also depends on factors known to those skilled in the art, such as the age and weight of the patient. In any case, the binding molecule of the present invention or the pharmaceutical composition of the present invention is administered in a dose and manner that enables delivery of a pharmaceutically effective amount based on the specific condition of the patient. Preferably, the binding molecule of the present invention or the pharmaceutical composition of the present invention is administered in an amount of 0.005 to 20.0 mg per kg of body weight, preferably 0.05 to 10.0 mg / kg / dose, more preferably 0.5 to 5 mg / kg / dose, either continuously (e.g., by infusion) or more preferably as a single dose. The dosing interval can be, for example, twice a week, once a week, or once a month, but specifically can vary significantly depending on the above parameters. Thus, in some cases, it may be sufficient to use a dose less than the minimum dose shown above, while in other cases, it may be necessary to exceed the upper limit. When administering a large amount, it may be advisable to divide it into multiple smaller doses dispersed throughout the day. Preferably, the administration is once a week, in a dose range of 0.005 to 20.0 mg per kg of body weight, and preferably 0.05 to 10.0 mg / kg / dose, more preferably 0.5 to 5 mg / kg / dose.

[0284] The effectiveness of the binding molecule of the present invention and the composition containing the same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, and / or animal model known per se, or any combination thereof, according to the specific disease that has occurred. Suitable assays and animal models will be apparent to those skilled in the art and include, for example, the assays and animal models used in the following examples.

[0285] The binding molecule of the present invention or the pharmaceutical composition of the present invention may be used alone or in combination with other pharmacologically active ingredients such as compounds of the state-of-the-art technology or standard treatment, for example, cell division inhibitors or cytotoxic substances, cell growth inhibitors, angiogenesis inhibitors, steroids, immune modulators / checkpoint inhibitors, and the like.

[0286] Accordingly, a further aspect of the present invention provides a binding molecule of the present invention or a pharmaceutical composition containing the binding molecule of the present invention together with one or more further active ingredients and optionally a pharmaceutically acceptable carrier.

[0287] According to the present invention, cell division inhibitors and / or cytotoxic substances that can be administered as a combination pair include, but are not limited to, hormones, hormone analogs and antihormones, aromatase inhibitors, LHRH (luteinizing hormone-releasing hormone) agonists and antagonists, growth factor inhibitors (growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, such as HER2, HER3, HER4), and hepatocyte growth factor (HGF)). The inhibitors include, for example, (anti)growth factor antibodies, (anti)growth factor receptor antibodies, and tyrosine kinase inhibitors such as cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib, and trastuzumab; antimetabolites (such as folic acid antimetabolites such as methotrexate, raltitrexed, pyrimidine analogs such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine and gemcitabine, purine and adenosine analogs such as mercaptopurine, thioguanine, cladribine, and pentostatin, cytarabine (araC), fludarabine); antitumor antibiotics (such as anthracyclines); platinum derivatives (such as cisplatin, oxaliplatin, carboplatin); alkylating agents (such as estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as carmustine, and lomustine, thiotepa); mitotic inhibitors (such as vinca alkaloids such as vinblastine, vindesine, vinorelbine, and vincristine; and taxanes such as paclitaxel, docetaxel); angiogenesis inhibitors such as bevacizumab, ramucirumab, and aflibercept, tubulin inhibitors;DNA synthesis inhibitors, PARP (poly ADP ribose polymerase) inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK (phosphoinositide-dependent kinase) 1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR (mammalian target of rapamycin) inhibitors, mTORC1 / 2 inhibitors, PI3K (phosphatidylinositol 3 kinase) inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g., volasertib), CDK (cyclin-dependent kinase) inhibitors such as CDK9 inhibitors, aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors, MCL-1 inhibitors, c-FLIP inhibitors, or KRAS inhibitors such as KRASG12c, KRAS G12D, or KRAS G13d), protein-protein interaction inhibitors, MEK inhibitors, ERK (extracellular signal-regulated kinase) inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF (insulin-like growth factor)-1R inhibitors, Bcl-xL inhibitors, Bcl-2 inhibitors, Bcl-2 / Bcl-xL inhibitors, ErbB receptor inhibitors, BCRABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin analogs (e.g., everolimus, temsirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT (DNA methyltransferase) inhibitors, HDAC (histone deacetylase) inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals (including but not limited to whole-body targeted biopharmaceuticals), immunotherapeutic agents such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3 and TIM-binding molecules / immunoglobulins such as ipilimumab, nivolumab, pembrolizumab), interferon, interferon α, or rituximab; oncolytic viruses; anti-cancer vaccines and various chemotherapeutic agents such as amifostine, anagrelide, clodronic acid, filgrastim, leucovorin, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer;Protease inhibitors (e.g., bortezomib); Smac and BH3 mimetics; agents that restore p53 function, such as mdm2-p53 antagonists; Wnt / β-catenin signaling pathway inhibitors; stromal modulators, such as (preferably bispecific) molecules that target CD137 and FAP; and / or cyclin-dependent kinase 9 inhibitors.

[0288] Preferred according to the invention is treatment using the binding molecule of the invention or the pharmaceutical composition of the invention in combination with a drug selected from the following: (i) Anti-VEGF (vascular endothelial growth factor) antibodies (bevacizumab and other angiogenesis inhibitors), with or without combination with chemotherapy (such as the combination of doxorubicin / cyclophosphamide and / or the combination of capecitabine / docetaxel in the neoadjuvant setting; including taxane / platinum regimens for primary and subsequent treatments), especially in breast cancer patients; (ii) Chemotherapeutic agents (including 5-fluorouracil (5-FU), irinotecan, oxaliplatin, and TAS-102); (iii) Anti-EGFR (epidermal growth factor receptor) antibodies (cetuximab and panitumumab in KRAS wild-type tumors) with or without combination with chemotherapy (including irinotecan) and / or with or without combination with anti-VEGF antibodies (bevacizumab and other angiogenesis inhibitors) or regorafenib, especially for the treatment of colorectal cancer patients; and / or (iv) EGFR inhibitors, such as gefitinib, afatinib, nintedanib, lapatinib, erlotinib, osimertinib (asimertinib); and / or (v) ALK (activin receptor-like kinase) inhibitors; and / or (vi) ROS1 inhibitors; and / or (vii) Immunotherapeutic agents, such as anti-PD-1 antibody drugs and anti-PD-L1 antibody drugs, and anti-LAG3 antibody drugs, such as ezabenlimab, pembrolizumab, and nivolumab, and other antibodies as disclosed in WO 2017 / 198741; and / or (viii) A (preferably bispecific) molecule that targets a stromal modulator, such as CD137 and FAP (fibroblast activation protein).

[0289] In a particularly preferred embodiment, the binding molecule or pharmaceutical composition of the invention is used for the treatment of cancer in combination with an immune checkpoint inhibitor, preferably a PD-1 antagonist, such as an anti-PD-1 antibody or an anti-PDL-1 antibody. Preferably, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, or PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 as described in WO 2017 / 198741 (incorporated herein by reference), and more preferably the anti-PD-1 antibody is ezabenlimab. Preferably, the anti-PDL-1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab. Specifically, such a therapy may also be combined with a chemotherapeutic agent, such as cisplatin, carboplatin, or gemcitabine.

[0290] In a further preferred embodiment, the binding molecule or pharmaceutical composition described herein is used for the treatment of cancer in combination with one chemotherapeutic agent, such as cisplatin, carboplatin, paclitaxel, docetaxel, 5-FU, methotrexate, cetuximab, capecitabine, afatinib, irinotecan, oxaliplatin, gemcitabine, paclitaxel, docetaxel, capecitabine, pemetrexed, or cetuximab.

[0291] In a particularly preferred embodiment, the binding molecule or pharmaceutical composition described herein is - Cetuximab, platinum (cisplatin or carboplatin), and 5-FU; - Cisplatin and cetuximab; - Cisplatin or carboplatin in combination with docetaxel or paclitaxel; - Cisplatin and 5-FU; - Cisplatin or carboplatin in combination with docetaxel and cetuximab; - Cisplatin or carboplatin in combination with paclitaxel and cetuximab; - Pembrolizumab, platinum (cisplatin or carboplatin), and paclitaxel; - Pembrolizumab, platinum (cisplatin or carboplatin), and docetaxel; - Cisplatin and etoposide; - Carboplatin and etoposide; and, - Cyclophosphamide in combination with doxorubicin and vincristine is used for cancer treatment in combination with a combination regimen selected from the group consisting of.

[0292] In a preferred embodiment, the cancer to be treated with the binding molecule described herein is gastric cancer or esophageal adenocarcinoma, and the treatment method includes a combination of the binding molecule described herein and a chemotherapeutic agent selected from the group consisting of irinotecan, oxaliplatin, paclitaxel, and capecitabine.

[0293] In a further preferred embodiment, the cancer to be treated with the binding molecule described herein is pancreatic ductal adenocarcinoma (PDAC), and the treatment method includes a combination of the binding molecule described herein and a chemotherapeutic agent selected from the group consisting of irinotecan, gemcitabine, paclitaxel, and capecitabine.

[0294] In a further preferred embodiment, the cancer to be treated with the binding molecule described herein is lung adenocarcinoma, and the treatment method includes a combination of the binding molecule described herein and a chemotherapeutic agent selected from the group consisting of oxaliplatin, docetaxel, and pemetrexed. Preferably, lung adenocarcinoma is treated with a combination therapy comprising the binding molecule of the present invention, oxaliplatin, and cisplatin.

[0295] In a further preferred embodiment, the cancer to be treated with the binding molecule described herein is squamous cell lung cancer (lung SCC), and the treatment method comprises a combination of the binding molecule described herein and a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin and docetaxel. Preferably, lung SCC is treated with a combination therapy comprising the binding molecule of the present invention, oxaliplatin and cisplatin.

[0296] In yet a further preferred embodiment, the cancer to be treated with the binding molecule described herein is squamous cell carcinoma of the head and neck (SCCHN or HNSCC), and the treatment method comprises a combination of the binding molecule described herein and a chemotherapeutic agent selected from the group consisting of oxaliplatin, paclitaxel, docetaxel and cetuximab.

[0297] In a further embodiment, the binding molecule or pharmaceutical composition described herein is used in combination with radiotherapy for the treatment of cancer.

[0298] The present invention further relates to the binding molecule of the present invention, or the pharmaceutical composition of the present invention, for use in medicine. The present invention further relates to the binding molecule of the present invention, or the pharmaceutical composition of the present invention, for use in the preparation of a medicament.

[0299] Furthermore, the present invention also relates to the binding molecule of the present invention, or the pharmaceutical composition of the present invention, for use in a method of treating, remitting, or preventing cancer. The present invention further relates to a method of treating, preventing or remitting cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of the binding molecule of the present invention or the pharmaceutical composition of the present invention.

[0300] The "therapeutically effective amount" of the molecule to be administered is the minimum amount required to prevent, remit, or treat the clinical symptoms of cancer, in particular the minimum amount effective against the specific cancer to be treated.

[0301] As used herein, the term "cancer" means all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues or organs, regardless of histopathologic type or stage of invasion. Thus, all cancers, tumors, neoplasms, etc. described below, characterized by their specific location / origin within a living body, are meant to include both primary tumors and metastatic tumors derived therefrom.

[0302] Cancers, tumors, and other proliferative diseases that can have their growth inhibited using the multispecific binding molecules described herein are cancers that express TRAILR2 / CDH3, including, but not limited to, head and neck cancer, preferably head and neck squamous cell carcinoma; lung cancer; preferably non-small cell lung cancer; pancreatic cancer; cervical cancer; ovarian cancer; endometrial cancer; breast cancer, preferably triple negative breast cancer; liver cancer (hepatoblastoma or hepatocellular carcinoma); prostate cancer; gastric sarcoma; gastrointestinal stromal tumor, esophageal cancer; colorectal cancer; colon-rectal cancer; kidney cancer; skin cancer; or gastrointestinal cancer. Gastrointestinal cancers include, but are not limited to, esophageal cancer (e.g., esophagogastric junction cancer), gastric cancer, hepatocellular carcinoma, cholangiocarcinoma (e.g., cholangiocarcinoma), bladder cancer, pancreatic cancer, or colorectal cancer (CRC).

[0303] In preferred embodiments of the binding molecule or pharmaceutical composition for use according to the invention, or of the method of treating, preventing or alleviating cancer according to the invention, or of the use according to the invention, the cancer is lung cancer, particularly lung adenocarcinoma, lung squamous cell carcinoma (lung SCC), and non-small cell lung cancer (NSCLC); head and neck cancer, particularly head and neck squamous cell carcinoma (HNSCC); pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC); breast cancer, particularly triple negative breast cancer (TNBC); gastric cancer (GC); ovarian cancer; endometrial cancer, or esophageal cancer.

[0304] In a particularly preferred embodiment of the invention, the cancer is pancreatic cancer, non-small cell lung cancer (NSCLC); or head and neck squamous cell carcinoma (HNSCC).

[0305] Pancreatic cancer (PAC) is a malignant disease that causes more than 400,000 deaths worldwide each year. It is one of the causes of the high frequency of cancer-related deaths in industrialized countries. Despite treatment interventions such as surgery and chemotherapy, pancreatic adenocarcinoma, which accounts for about 90% of all cases of pancreatic cancer, typically has a very poor prognosis, with about 25% of people surviving for one year and only 5% of patients surviving for five years. Pancreatic ductal adenocarcinoma (PDAC) is the most frequently seen neoplastic disease of the pancreas, accounting for more than 90% of all pancreatic malignancies.

[0306] Lung cancer is the leading cause of cancer-related mortality in the United States. The five-year relative survival rate for lung cancer patients from 2011 to 2017 was 22%. Non-small cell lung cancer is any type of epithelial lung cancer other than small cell lung cancer (SCLC). The most common types of non-small cell lung cancer are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, although there are also several less frequent types. Non-small cell lung cancer as a class is generally less sensitive to chemotherapy and radiation therapy compared to small cell lung cancer. Patients with resectable disease may be cured by surgery or chemotherapy following surgery. However, many patients have unresectable disease, and local control can be achieved using radiation therapy, but cures are seen in only a minority of patients. Therefore, there is a great need for additional therapeutic agents to treat this disease.

[0307] Most head and neck cancers originate from the mucosal epithelium of the oral cavity, pharynx, and larynx and are collectively known as head and neck squamous cell carcinoma (HNSCC). Oral cancer and laryngeal cancer are generally associated with smoking, alcohol abuse, or both, while pharyngeal cancer is increasingly caused by infection with the human papillomavirus (HPV), mainly HPV-16. Head and neck cancers also begin in the salivary glands, sinuses, or muscles or nerves within the head and neck, but these types of cancers are much less prevalent than squamous cell carcinoma.

[0308] Oral head and neck squamous cell carcinoma is generally treated, depending on the stage of the disease, by surgical resection followed by adjuvant radiotherapy, i.e., chemotherapy and radiation (also known as chemoradiation or chemoradiotherapy (CRT)). Except for early-stage oral cancer, which is treated only surgically, the treatment of the majority of patients with head and neck squamous cell carcinoma is complex and requires a multi-modal approach, thus requiring multidisciplinary treatment. Therefore, there is an urgent need for targeted therapeutic agents for treating patients with head and neck squamous cell carcinoma, especially those in the advanced stage.

[0309] Colorectal cancer (CRC) is an individual malignant disease listed in ICD (International Classification of Diseases)-10 and is one of the main causes of cancer incidence and mortality worldwide. Approximately 25% of colorectal cancer patients present with overt metastases, and metastatic disease develops in 40 - 50% of newly diagnosed patients. Recent improvements in chemotherapy have extended the survival of patients with metastatic colorectal cancer, but the majority of patients succumb to these diseases. Therefore, there is a great need for additional therapeutic agents for treating this disease.

[0310] Approximately 30 - 50% of colorectal cancers are known to have a mutated (abnormal) KRAS gene. The KRAS mutations frequently seen in neoplasms include mutations in exon 2 (codons 12 and 13) and exon 3 (codon 61), which can be analyzed from tumor biopsy materials. They include activating mutations, which result in continuous signaling that stimulates downstream signaling pathways involved in cell growth, cell proliferation, invasion, and metastasis. Thus, in one embodiment, the binding molecules of the present invention are for use in the treatment of KRAS mutant colorectal cancer, i.e., patients having KRAS mutant tumors. In an alternative embodiment, the binding molecules of the present invention are for use in the treatment of KRAS wild-type colorectal cancer, i.e., patients having KRAS wild-type tumors.

[0311] Esophageal cancer is one of the most frequently diagnosed cancers worldwide. Similar to pancreatic cancer, it is difficult to diagnose, tends to be diagnosed at an advanced stage, and this symptom leads to a very poor prognosis. As a result, it accounts for about 5% of cancer-related deaths, making it the sixth leading cause of cancer-related death.

[0312] Breast cancer has approximately 2.3 million new cases worldwide (GLOBOSCAN 2020) and is the most frequently diagnosed malignant disease among women. Triple-negative breast cancer (TNBC) constitutes 15-20% of breast cancer and occurs more frequently in women over 40 years old. Due to its high degree of invasiveness, distant metastasis occurs in nearly 50% of patients with triple-negative breast cancer. Triple-negative breast cancer patients show a shorter survival period compared to other types of breast cancer, with a 5-year mortality rate of approximately 40% and a mortality rate of up to 75% after disease recurrence. Endocrine therapy or molecular targeted therapy currently used in other subtypes of breast cancer is not an option for triple-negative breast cancer due to its molecular phenotype. Since the effectiveness of chemotherapy-based systemic triple-negative breast cancer treatment, such as postoperative adjuvant chemoradiotherapy, is very low, there is a high need to develop new treatment modalities for this disease.

[0313] Ovarian cancer is the seventh most common type of cancer in women worldwide and has the highest incidence rate among women's malignant diseases. Among epithelial cancer types, serous ovarian cancer is the most frequently seen cancer. Ovarian cancer is diagnosed at an advanced stage in about 80% of cases and therefore usually has a poor prognosis.

[0314] Particularly in recurrent ovarian cancer, current treatments are characterized by high intensity, low quality of life, and low cure probability. Therefore, new treatment strategies are urgently needed for the treatment of this disease.

[0315] As described above, the inventors have identified that the binding molecules described herein have great utility for targeting cancer cells and can therefore be used for the treatment of cancers that express both TRAILR2 and CDH3. Methods for identifying whether a particular tumor expresses TRAILR2 and CDH3 are well known in the art. For example, immunohistochemical assays can be used to determine whether a tumor tissue expresses TRAILR2 and CDH3 (e.g., using the TRAILR2 and / or CDH3 antibody molecules described herein), and thus whether it is suitable for treatment with the binding molecules of the invention.

[0316] The binding molecules of the invention can be used in a treatment regimen in the context of a primary treatment, a secondary treatment, or any further subsequent treatment, and a maintenance treatment.

[0317] In a further aspect, the binding molecules of the invention are used in combination with an instrument useful for the administration of the binding molecule, such as a syringe, a pen-type syringe, a micropump, or another instrument. In a further aspect, the binding molecules of the invention are included in a kit of parts that also includes, for example, instructions for use of the binding molecule.

[0318] The present invention further includes the following items: 1. (a) at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and (b) at least one antigen-binding site that specifically binds to cadherin-3 (CDH3) A binding molecule comprising, wherein at least one antigen-binding site that specifically binds to CDH3 is i. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3); ii. An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3); iii. An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3); and iv. An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3) selected from the group consisting of

[0319] 2. At least one antigen-binding site that specifically binds to TRAILR2 is a binding molecule of item 1 comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 79 (CDR3).

[0320] 3. At least one antigen-binding site that specifically binds to CDH3 is an immunoglobulin (Ig) molecule, and at least one antigen-binding site that specifically binds to TRAILR2 is a binding molecule of item 1 or 2 comprising one or more scFvs.

[0321] 4. The binding molecule of item 3, wherein one or more scFvs have a VL-VH orientation from the N-terminus to the C-terminus.

[0322] 5. One or more scFvs are fused to the C-terminus of an immunoglobulin molecule, preferably the first scFv is fused to the first heavy chain of the immunoglobulin molecule and the second scFv is fused to the second heavy chain respectively, the binding molecule of item 3 or 4.

[0323] 6. Each of one or more scFvs is fused to an immunoglobulin molecule by a peptide linker, preferably a peptide linker having a length of about 4 to 20 amino acids, the binding molecule of any one of items 3 to 5.

[0324] The immunoglobulin molecule of the binding molecule of any one of items 3 to 6 is IgG.

[0325] 8. The antigen-binding site that specifically binds to CDH3 is i. A heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 6; ii. A VH containing the amino acid sequence of SEQ ID NO: 12 and a VL containing the amino acid sequence of SEQ ID NO: 16; iii. A VH containing the amino acid sequence of SEQ ID NO: 22 and a VL containing the amino acid sequence of SEQ ID NO: 26; and, iv. A VH containing the amino acid sequence of SEQ ID NO: 32 and a VL containing the amino acid sequence of SEQ ID NO: 36 selected from the group consisting of, the binding molecule of any one of items 1 to 7.

[0326] 9. i. A heavy chain containing the amino acid sequence of SEQ ID NO: 80 and a light chain containing the amino acid sequence of SEQ ID NO: 81; ii. A heavy chain containing the amino acid sequence of SEQ ID NO: 82 and a light chain containing the amino acid sequence of SEQ ID NO: 83; iii. A heavy chain containing the amino acid sequence of SEQ ID NO: 84 and a light chain containing the amino acid sequence of SEQ ID NO: 85; or iv. A heavy chain containing the amino acid sequence of SEQ ID NO: 86 and a light chain containing the amino acid sequence of SEQ ID NO: 87 comprising, the binding molecule of any one of items 1 to 8.

[0327] At least one antigen-binding site that specifically binds to TRAILR2 is an antigen-binding site comprising a heavy-chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 72 and a light-chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 76, and is a binding molecule according to any one of items 1 to 9.

[0328] 11.i. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. A modified heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 The binding molecule according to item 10, comprising

[0329] 12.i. Heavy-chain CDRs comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3) and light-chain CDRs comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3); ii. Heavy-chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3) and light-chain CDRs comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3); iii. Heavy-chain CDRs comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3) and light-chain CDRs comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3); iv. Heavy-chain CDRs comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3) and light-chain CDRs comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3); v. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 43 (CDR1), SEQ ID NO: 44 (CDR2), and SEQ ID NO: 45 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 49 (CDR3); vi. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 54 (CDR2), and SEQ ID NO: 55 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3); or vii. A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 63 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3) An antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising

[0330] 13.i. A heavy-chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light-chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6; ii. A VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16; iii. A VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26; iv. A VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36; v. A VH comprising the amino acid sequence of SEQ ID NO: 42 and a VL comprising the amino acid sequence of SEQ ID NO: 46; vi. A VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; or vii. A VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 66 An antibody or an antigen-binding fragment thereof according to item 12, comprising

[0331] 14.i. A heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; iv. A heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87; v. A heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; vi. A heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 91; or vii. A heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93 An antibody or antigen-binding fragment thereof according to item 12 or 13, comprising:

[0332] 15. An antibody or antigen-binding fragment thereof according to any one of items 12 to 14, selected from the group consisting of chimeric, humanized, and human antibodies or antibody fragments, and scFv, Fab fragments, monovalent antibody fragments, and F(ab') 2 fragments.

[0333] 16. An isolated nucleic acid encoding a binding molecule according to any one of items 1 to 11, or an antibody or antigen-binding fragment according to any one of items 12 to 15.

[0334] 17. An expression vector comprising the nucleic acid of item 16.

[0335] 18. The vector of item 17, wherein the vector is a plasmid.

[0336] 19. The vector of item 17, wherein the vector is a viral vector.

[0337] 20. A host cell comprising an expression vector according to any one of items 17 to 19.

[0338] 21. i. Culturing the host cell of item 20 under conditions that allow expression of the molecule, and ii. Recovering the molecule, and optionally iii. The step of further purifying and / or modifying and / or formulating the molecule A method for producing a binding molecule according to any one of items 1 to 11, or an antibody or an antigen-binding fragment thereof according to any one of items 12 to 15, comprising:

[0339] 22. A binding molecule according to any one of items 1 to 11 for use in medicine.

[0340] 23. A binding molecule according to any one of items 1 to 11 for use in the treatment of cancer.

[0341] 24. The binding molecule for use according to item 23, wherein the cancer is pancreatic cancer, lung cancer, or head and neck cancer.

[0342] 25. A pharmaceutical composition comprising a binding molecule according to any one of items 1 to 11 and a pharmaceutically acceptable carrier.

[0343] 26. The pharmaceutical composition according to item 25, which is lyophilized, stabilized, and / or formulated for administration by injection.

[0344] 27. A bispecific binding molecule comprising at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site that specifically binds to the EC1 domain of cadherin-3 (CDH3).

[0345] The features and advantages of the present invention will become apparent from the following more detailed examples that illustrate the principles of the present invention by way of examples.

Example

[0346] Example 1: High-frequency occurrence of both CDH3 and TRAILR2 in the symptoms of selected tumors Immunohistochemical examination (IHC) for TRAILR2 and CDH3 was performed as follows: Sections of each 2-μm-thick formalin-fixed paraffin-embedded block were prepared with a microtome, placed on glass slides, and defatted. CDH3 sections were stained using an anti-CDH3 rabbit monoclonal primary antibody (Abcam ab242060, diluted 1:50 in casein, Roche 760-219) with program 183 on an automated platform Ventana Discovery Ultra. Immunohistochemical examination of DR5 (death receptor 5) was performed using the following antibodies with Ventana Discovery program 148: rabbit anti-DR5 antibody (Cell Signaling 69400S, lot: 1, diluted 1:50 with Discovery antibody diluent (Roche 760-108)). After the automated staining procedure, the slides were washed with distilled water in a neutral detergent, then rinsed thoroughly with distilled water, placed in a 90% ethanol bath for 1 minute, then transferred through three 1-minute baths of 100% ethanol, then transferred through two 30-second baths of xylene, and finally covered with a coverslip with mounting medium. The slides were scanned (3DHistech) and analyzed.

[0347] The expression of CDH3 in non-neoplastic tissues (n = 40) including the liver was examined by immunohistochemistry, and no expression of CDH3 was seen (data not shown). Co-expression of TRAILR2 and CDH3 at the mRNA level was detected in several signatures such as pancreatic cancer (PAC), head and neck cancer, lung cancer, colorectal cancer, esophageal cancer, triple-negative breast cancer, and in bladder urothelial cancer, ovarian cancer, endometrial cancer, and cervical cancer (Cancer Genome Atlas), as shown in Table 2 below for some of these signatures.

[0348]

Table 3

[0349] For example, FIGS. 2A and B show protein expression and membrane localization correlated with mRNA expression of TRAILR2 and CDH3 in colorectal cancer.

[0350] FIG. 2C shows images of representative immunohistochemical examinations of CDH3 and TRAILR2 in tissue sections of head and neck squamous cell carcinoma and esophageal squamous cell carcinoma.

[0351] Example 2: Design and production of a binding molecule that recognizes human cadherin 3 (CDH3) and human TRAIL receptor 2 (TRAILR2) In this study, a binding molecule was developed that binds to CDH3 and TRAILR2 and induces apoptosis in cancer cells that express both CDH3 and TRAILR2. The molecular design includes an IgG antibody specific for CDH3 and an scFv specific for TRAILR2 conjugated to the C-terminus of the heavy chain. The bispecific molecule contains a flexible peptide sequence between the variable heavy chain (VH) domain and the variable light chain (VL) domain of the scFv, and the scFv domain is further linked to the IgG antibody via another series of linkers.

[0352] The binding molecule is bispecific and tetravalent. The design diagram is shown in FIG. 3. An example of the binding molecule of the present invention is shown, which includes (i) an immunoglobulin molecule that specifically binds to CDH3, including two heavy chains and two light chains, and (ii) two scFvs that specifically bind to TRAILR2. The N-terminus of the scFv is fused to the C-terminus of each heavy chain of the immunoglobulin molecule, thereby forming a symmetric, bispecific, and tetravalent antibody-like molecule.

[0353] The following examples illustrate the methods used to produce bispecific molecules that bind to CDH3 and TRAIL2, and the biological activities of these molecules.

[0354] 2.1 Preparation of binding domains that recognize CDH3 and TRAILR2 using high-throughput recovery of V genes from hybridomas and cultured single B cells To prepare a bispecific molecule that binds to human CDH3 and TRAILR2, it is necessary to obtain variable domains that bind to the individual target antigens (anti-CDH3 antibody and anti-TRAILR2 antibody). To achieve this, clonal hybridomas or single B cells derived from AlivaMab humanized mice (Ablexis, San Francisco, CA, USA: AlivaMab transgenic mouse platform having the human immunoglobulin locus) immunized with CDH3 or TRAILR2 were cultured in vitro. The supernatants were screened by AlphaLISA (PerkinElmer, Waltham, MA, USA) for reactivity against recombinant human CDH3 or TRAILR2 and by flow cytometry for reactivity against binding to GP2d cells.

[0355] The VH and VL genes of immunoglobulin (Ig) were then amplified from the identified positive clones. To isolate RNA from hybridomas, approximately 2×10 6 cells from one clone were pelleted and used as starting material. For one B cell, 100 - 500 cells amplified from one isolated B cell were used as starting material. RNA was isolated using RNeasyPlus (Qiagen, Hilden, Germany). cDNA was then synthesized using the Smarter cDNA synthesis kit (Clontech, Mountain View, CA) according to the manufacturer's instructions.

[0356] To facilitate cDNA synthesis, reverse transcription of all messenger RNAs was initiated using oligo dT, followed by "5' capping" using SmarterIIA oligonucleotides. Subsequent amplification of the VH and VL fragments was performed using two-step PCR amplification with a 5' primer targeting the SmarterIIA cap and a 3' primer targeting a common region within CH1. Briefly, each 50 μl PCR reaction consisted of a mix of 20 μM forward and reverse primers, 25 μl of PrimeStar Max DNA polymerase premix (Clontech), 2 μl of unpurified cDNA, and 21 μl of redistilled water. The cycling program started at 94 °C for 3 minutes, followed by 35 cycles (94 °C for 30 seconds, 50 °C for 1 minute, 68 °C for 1 minute), and ended at 72 °C for 7 minutes. The second PCR was performed using second primers for VL and VH that contained 15 bp complementary extension sequences that "overlapped" each respective region (VH and VL) within their respective pTT5 parental vectors. The second PCR was performed using the following program: 94 °C for 3 minutes; 35 cycles (94 °C for 30 seconds, 50 °C for 1 minute, 68 °C for 1 minute), and ended at 72 °C for 7 minutes.

[0357] The In-Fusion® HD Cloning Kit (Clontech, USA) was used for directional cloning of the VL gene into the pTT5hulgK vector and the VH gene into the pTT5hulgG1KO vector. To facilitate In-Fusion® HD cloning, the PCR products were purified and treated with a cloning enhancer prior to In-Fusion HD cloning. Cloning and transformation were performed according to the manufacturer's protocol (Clontech, USA). Mini-prep DNA was subjected to Sanger sequencing to confirm that complete V gene fragments were obtained.

[0358] Using this method, a large number of pairs of VH and VL genes of immunoglobulins encoding binding domains specific for CDH were prepared.

[0359] 2.2 Verification Screening of Recombinant Antibodies Recombinant antibodies were produced by transient transfection of CHO-E37 cells using plasmids encoding the corresponding heavy and light chains. The supernatant containing the expressed recombinant antibody was assayed by flow cytometry for binding to cell lines expressing human or cynomolgus CDH3. Briefly, cells were incubated with the recombinant supernatant, washed, and bound monoclonal antibodies from the supernatant were detected using anti-human-IgG-APC (allophycocyanin) (Jackson ImmunoResearch 109-136-098). The median fluorescence intensity (MFI) of the sample was divided by that of an isotype control (variable regions against unrelated proteins and frameworks of various constant regions) to calculate the signal-to-background ratio (S / B). Clones of interest were selected for multiplex-specific formatting. Multiplex-specific binding proteins were produced and further evaluated in mechanical and functional screenings (such as cell binding assays, cytotoxicity assays, and caspase activation assays).

[0360] 2.3 Construction of Bispecific Molecules That Bind to CDH3 and TRAIR2 To construct the gene segment encoding TRAILR2scFv, a pair of VL and VH genes encoding the TRAIL2-binding variable domains (SEQ ID NO: 76 and SEQ ID NO: 72, respectively) were connected by a gene segment encoding a flexible linker of the peptide sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232). The resulting gene segment encoding the scFv was then cloned in-frame at the 3'-end of the gene encoding the heavy chain of the human IgG antibody. These coding segments were synthesized by the overlap PCR method and cloned into the expression vector pTT5. The pair of VL and VH genes encoding the CDH3-binding variable domain prepared in Example 2.1 was then formatted into the bispecific format exemplified in Figure 3. The VH gene was cloned into the pTT5 expression vector as an in-frame fusion at the 5'-end of the gene encoding human immunoglobulin γ. The gene encoding the TRAILR2-binding scFv was cloned in-frame at the 3'-end of the segment encoding the same immunoglobulin γ. Similarly, the VL gene was cloned into the pTT5 expression vector as an in-frame fusion with the gene encoding the human IgGκ light chain.

[0361] The pair of VL and VH genes encoding the CDH3-binding variable domain prepared in Example 2.1 was further used to prepare an antibody molecule (a full-length antibody molecule containing two light chains and two heavy chains) that specifically binds to CDH3 (and those called CDH3v1, CDH3v2, CDH3v3, CDH3v4, CDH3v5, CDH3v6, or CDH3v7, see Tables 11-17) using methods known in the art, and this was used for the detection / labeling of cells or as a control antibody in the following examples. Thereafter, the anti-TRAILR2 scFv (Table 10) was linked via the above linker to the C-terminus of each heavy chain of the anti-CDH3 antibody, thereby generating a bispecific and tetravalent CDH3 / TRAILR2-binding molecule (Tables 3-9).

[0362] Each expression vector contains a eukaryotic cell promoter sequence for the gene encoding the chain, a signal sequence and the gene encoding the heavy or light chain, an expression cassette for a prokaryotic cell selectable marker gene such as ampicillin, and an origin of replication. These DNA plasmids were amplified in ampicillin-resistant E. coli colonies and purified.

[0363] 2.4 Expression and purification of a bispecific and tetravalent molecule that recognizes human TRAILR2 and human CDH3 The bispecific molecule that binds to CDH3 and TRAILR2 was produced by transient transfection of CHO-E cells using the pTT5 vector having the gene encoding the CDH3 / TRAILR2 chain. The transfected CHO-E cells growing in serum-free suspension medium were cultured in a shake flask with stirring at 140 rpm, 37 °C and 5% carbon dioxide and maintained under exponential growth conditions. On the day of transfection, the cells were chemically transfected with 1 mg of the light chain plasmid and 0.5 mg of the heavy chain plasmid (mass ratio of 1:3). Then, the CHO-E cells were seeded at 1-2×10 6 cells / ml in 1 L of Gibco® Freestyle™ CHO expression medium (Life Technologies, New York, USA). Then, the cells were incubated for 10-12 days with swirling under shaking while feeding once with 200 ml of a commercially available feed solution to enable protein expression. The antibody titer in the cell culture supernatant was determined according to the manufacturer's instructions using an Octet® instrument (Pall30 Fortebio, California, USA) and a Protein A biosensor chip.

[0364] The recombinant CDH3 / TRAILR2 binding molecule or antibody was purified from the culture supernatant by a two-step process: a first purification step by protein A affinity chromatography using a MabSelect™ column (GE Healthcare), followed by a second purification step by cation exchange chromatography using a Poros 50HS column (Applied Biosystems, Carlsbad, CA, USA). The material purified in the two steps was stored in a final buffer of 50 mM sodium acetate and 100 mM sodium chloride (pH 5.0). The purity and heterogeneity of the sample were evaluated by mass spectrometry, analytical size exclusion chromatography, and analytical ultracentrifugation. The sample was confirmed to have a monomer content of more than 90% and to contain less than 10% impurities prior to functional testing.

[0365]

Table 4

[0366]

Table 5

[0367]

Table 6

[0368]

Table 7

[0369]

Table 8

[0370]

Table 9

[0371]

Table 10

[0372]

Table 11

[0373]

Table 12

[0374]

Table 13

[0375]

Table 14

[0376]

Table 15

[0377]

Table 16

[0378]

Table 17

[0379]

Table 18

[0380] 2.5: Determination of the binding affinity of bispecific molecules against recombinant CDH3 based on surface plasmon resonance The binding affinity of the purified CDH3 / TRAILR2 bispecific construct against recombinant human CDH3 (either full-length, EC1-EC2 or EC2-EC3) was determined by surface plasmon resonance (SPR) using a Proteon XPR36 instrument (Bio-Rad). Running buffer and all dilutions (unless otherwise specified) were performed in PBS-TEDTA. The GLM sensor chip (Bio-Rad) was standardized and preconditioned according to the manufacturer's recommendations. The sensor chip was activated with an equal mixture of EDC / s-NHS for 300 seconds at a flow rate of 30 μl / min in the horizontal direction, and immobilized with Protein A / G (60 μg / ml in 10 mM acetate pH 4.5) for 300 seconds at a flow rate of 30 μl / min in the horizontal direction, resulting in 5000 RU of Protein A / G on the surface. The sensor chip was deactivated with 1 M ethanolamine for 300 seconds at a flow rate of 30 μl / min in the horizontal direction. The sensor chip was stabilized horizontally 3 times and vertically 3 times with 0.85% phosphoric acid at a flow rate of 100 μl / min for 18 seconds each.

[0381] For determination of the binding kinetics against CDH3, the bispecific molecule was individually captured onto the Protein A / G surface vertically for 150 seconds at a flow rate of 30 μl / min. The baseline was stabilized by injecting PBS-T-EDTA horizontally at a flow rate of 100 μl / min for 60 seconds. Analytes (human full-length CDH3, CDH3[EC1-EC2], or CDH3[EC2-EC3]) were injected horizontally onto the captured antibody for 300 seconds at a flow rate of 30 μl / min and dissociated for 1200 seconds. The concentration of the injected analyte was 200 nM for full-length CDH3 and 500 nM for EC1-EC2 and EC2-EC3. The surface was regenerated by injecting 0.85% phosphoric acid solution at a flow rate of 100 μl / min, once horizontally and once vertically, for 18 seconds. The data was processed by subtracting the inter-spot (interaction with the sensor surface) and then fitting to a 1:1 Langmuir kinetic model to obtain the rate constant and affinity.

[0382] The affinity for CDH3 (full-length or EC1-EC2) was in the low nM range. The binding affinity for the EC2-EC3 peptide was either in the low nM range (mapping the epitope to EC2) or below the detection level (mapping the epitope to EC1).

[0383] The results are shown in Table 18 below, and the generated binding molecules containing CDH3v1, CDH3v2, CDH3v3, or CDH3v7 as EC1-binding substances and those containing CDH3v4, CDHv5, or CDHv6 as EC2-binding substances are classified.

[0384]

Table 19

[0385] Example 3: In vitro cell killing assay The examples shown above demonstrate the preparation of bispecific molecules that recognize TRAILR2 and human CDH3. To investigate whether these molecules can cause a decrease in cell viability and whether any such decrease is caused by apoptosis, many different bispecific molecules based on the format shown in Figure 3 were prepared.

[0386] 3.1 Characterization of target expression in the GP2d cell line model The cell line GP2d derived from human colorectal adenocarcinoma was selected for the implementation of the cell killing assay. The surface expression of TRAILR2 and CDH3 proteins on GP2d cells was confirmed by flow cytometry as follows. Cells were detached using Versene solution (Gibco 15040066) and washed twice with FACS buffer (PBS, Gibco 14190; 3% fetal bovine serum, Gibco 26140; and 0.09% NaN3, Sigma-Aldrich S2002). Cells were counted using Vicell (Beckman Coulter Life Sciences), and the cell number was 2×10 6Adjusted to cells / ml. After seeding 100 μl of the cell suspension per well in a 96-well round-bottom plate, the plate was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Then, the cells were resuspended in the primary antibody dilution (1 μg / ml) and incubated at 4°C for 60 minutes. The cells were washed twice with FACS buffer, an appropriate dilution of the secondary / conjugated antibody at 100 μl / well was added, and incubated in the dark at 4°C for 45 minutes. After washing twice with FACS buffer, the cells were resuspended in 100 μl of FACS buffer per well and analyzed with a FACS Canto (BD Biosciences). For the detection of TRAILR2, a conjugated anti-human CD262 (DR5) PE (phycoerythrin) antibody (eBioscience, 12-9908-42) was used. For the detection of CDH3, a secondary anti-mouse IgG FITC (fluorescein isothiocyanate) was used following the anti-P-cadherin antibody (GeneTex GTX52961). As a control, a mouse IgG isotype control was used. In Figure 4, the surface protein expression of TRAILR2 and CDH3 in GP2d cells is shown, and both proteins show significant expression.

[0387] 3.2 Effect of the bispecific CDH3 / TRAILR2 molecule on GP2d cells (in vitro two-dimensional) Since GP2d cells were identified as a suitable cancer cell line for evaluating the function of the bispecific CDH3 / TRAILR2 binding molecule, the following assay was devised. GP2d cells were seeded in culture medium (RPMI1640 / Glutamax, Gibco 61870-010; additionally 10% fetal bovine serum, Gibco 26140). After resting overnight at 37 °C and 5% carbon dioxide, the cells were incubated for 24 hours with 50 μl dilutions of various antibodies or binding molecules at the desired concentrations. Subsequently, cell viability was evaluated by using the CellTiter-Glo Luminescent cell viability assay (Promega G7571) according to the instructions provided by the manufacturer. Finally, luminescence was recorded using a PerkinElmer VICTOR X4 2030 multi-label plate reader. Two TRAILR2-binding substances, typically an anti-TRAILR2 IgG (HGS-ETR2, International Publication No. WO 2003 / 054216), typically called lexatumumab, and an anti-TRAILR2 nanobody (International Publication No. WO 2011 / 098520), were used as controls (both synthesized in-house). The source of the sequence of lexatumumab was obtained from the World Health Organization as listed in the Recommended International Nonproprietary Names for Pharmaceutical Substances (World Health Organization, Recommended International Nonproprietary Names for Pharmaceutical Substances, List 57, WHO Drug Information 2007; 21: 53-83). The expression vector of the anti-TRAILR2 nanobody was prepared as described in International Publication No. WO 2011 / 098520, SEQ ID NO: 032.

[0388] GP2d cells were incubated for 24 hours with (i) the bispecific molecule (CDH3 / TR2v1), (ii) anti-TRAILR2 antibody only (lexatumumab), (iii) anti-CDH3 antibody only (anti-CDH3v1 antibody or anti-CDH3v2 antibody), or (iv) an equivalent combination of anti-TRAILR2 antibody and CDH3 (CDH3v1 or CDH3v2) antibody.

[0389] Figure 5 shows the effects of these molecules on cell viability. Two CDH3 molecules (anti-CDH3v1 antibody or anti-CDH3v2 antibody) alone had no effect on cell viability. The TRAILR2 antibody (lexatumumab) alone could significantly reduce cell viability (EC50: 5.802 nM), and the addition of either anti-CDH3 antibody (either anti-CDH3v1 antibody or anti-CDH3v2 antibody) in free combination did not change the effect observed with the anti-TRAILR2 antibody alone. The above-mentioned bispecific CDH3 / TRAILR2-binding molecule CDH3 / TRAILR2v1 brought about potent killing of GP2d cells (EC50: 0.337 nM), with a 17-fold improvement in potency compared to the anti-TRAILR2 antibody alone (lexatumumab) or the anti-TRAILR2 antibody combined with either anti-CDH3 antibody (either anti-CDH3v1 antibody or anti-CDH3v2 antibody).

[0390] Many different bispecific molecules were compared in the same assay using GP2d cells against the tetravalent anti-TRAILR2 nanobody.

[0391] Figure 6 shows the potencies of four bispecific molecules (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, CDH3 / TRAILR2v7) that bind to the EC1 domains of TRAILR2 and CDH3, which were approximately 55-fold, 11-fold, or 7-fold less potent than the anti-TRAILR2 nanobody.

[0392] Figure 7 shows the potencies of three bispecific molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) that bind to the EC2 domains of TRAILR2 and CDH3, which were approximately 15-fold, 327-fold, or 8-fold less potent than the anti-TRAILR2 nanobody.

[0393] 3.3 Effect of bispecific CDH3 / TRAILR2 molecules on GP2d cells lacking CDH3 (two-dimensional in vitro) Next, it was confirmed that the increase in apoptosis regulated by the bispecific CDH3 / TRAILR2-binding molecule of the present invention was specifically mediated by CDH3 present on the surface of GP2d cells. To demonstrate this, GP2d cells lacking CDH3 on the cell surface (CDH3 knockout cells) were generated by gene editing using the CRISPR / Cas9 system. As shown in Figure 8, flow cytometry confirmed the absence of CDH3 protein on GP2d-CDH3 knockout (CDH3-KO) cells, and the cells retained the same TRAILR2 protein on the surface as isogenic GP2d (CDH3 wild-type) cells. The same bispecific molecules previously tested on GP2d cells were tested on GP2d CDH3 knockout cells.

[0394] Figure 9 shows that the TRAILR2 antibody (lexatumumab) alone significantly reduced the cell viability of GP2d CDH3 knockout cells, which was similar to the effect observed in GP2d wild-type cells (EC50: 5.802 nM; Figure 5), and the addition of either anti-CDH3 antibody (either anti-CDH3v1 antibody or anti-CDH3v2 antibody) freely combined did not change the effect observed with the anti-TRAILR2 antibody alone. In contrast, the bispecific CDH3 / TRAILR2-binding molecule CDH3 / TRAILR2v1 had little effect on the cell viability of GP2d CDH3 knockout cells and did not reach a 50% cell death rate even at the highest concentration tested (200 nM).

[0395] Figure 10 shows the potency of the anti-TRAILR2 nanobody against GP2d CDH3 knockout cells, which was similar to that previously detected in GP2d wild-type cells. In contrast, four bispecific molecules (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, CDH3 / TRAILR2v7) that bind to the EC1 domains of TRAILR2 and CDH3 showed a lack of significant potency, which was approximately 41666-fold, 12602-fold, or 21504-fold less potent than the anti-TRAILR2 nanobody.

[0396] Figure 11 shows the potencies of three bispecific molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) that bind to the EC2 domains of TRAILR2 and CDH3, all of which were more than 30,000-fold less potent than the anti-TRAILR2 nanobody.

[0397] This confirms that the bispecific molecules produced in the tests of the present invention induce apoptosis only in the presence of CDH3, while both the anti-TRAILR2 antibody lexatumumab and the anti-TRAILR2 nanobody induce apoptosis regardless of the presence of CDH3.

[0398] Example 4: Effect of bispecific CDH3 / TRAILR2 molecule on NCI-H358 cells (3D in vitro) To extend the in vitro analysis, the bispecific CDH3 / TRAILR2 molecule was tested against cell lines grown as spheroids (3D culture model) to mimic in vivo tumor growth conditions. NCI-H358 cells derived from lung adenocarcinoma were seeded at 2,000 cells per well in 2% Cultrex 3D low growth factor basement membrane extract (R+D Systems, 3445-005-01) in 96-well clear round bottom ultra-low attachment surface microplates (Corning, 7007). The cells were treated over 48 hours with serial dilutions of the same molecules tested in Example 3 against GP2d cells. The NCI-H358 cells were incubated with (i) the bispecific molecule (CDH3 / TRAILR2v1), (ii) anti-TRAILR2 antibody alone (lexatumumab or anti-TRAILR2 nanobody), (iii) anti-CDH3 antibody alone (anti-CDH3v1 antibody or anti-CDH3v2 antibody), or (iv) a combination of anti-TRAILR2 antibody (lexatumumab) and anti-CDH3 (CDH3v1 or CDH3v2) antibody.

[0399] Figure 12 shows the effects of these molecules on cell viability in the 3D model of NCI-H358. Using anti-CDH3 antibody molecules alone (anti-CDH3v1 antibody or anti-CDH3v2 antibody), rituximab alone, or a combination of anti-CDH3 antibody and rituximab, no effect on cell viability was detected. Similar efficacy was detected only using CDH3 / TRAILR2v1 and the anti-TRAILR2 nanobody. Figure 13 shows tests of EC1-binding CDH3 / TRAILR2 molecules (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, and CDH3 / TRAILR2v7), all of which showed efficacy similar to TAS266. In contrast, EC2-binding CDH3 / TRAILR2 molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) showed reduced efficacy compared to EC1-binding substances and TAS266 (Figure 14).

[0400] This surprisingly shows that only CDH3-expressing cells are targeted, resulting in a significantly higher accuracy, and that EC1-binding bispecific molecules alone can induce apoptosis with efficacy similar to that of the anti-TRAILR2 nanobody. In contrast, the EC2-binding bispecific molecules CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, and CDH3 / TRAILR2v6 did not reach the efficacy level of the anti-TRAILR2 nanobody.

[0401] Example 5: CDH3 / TRAILR2 molecules induce activation of caspase-8 in GP2d cells Next, it was examined whether the decrease in cell viability was caused by the induction of TRAIL-induced apoptosis mediated by the mobilization and activation of caspase-8. To determine whether the antibodies and binding molecules prepared herein can specifically activate the apoptotic pathway, the inventors measured caspase-8 activity in the GP2d cell line. To assemble the experiment, GP2d cells were rested overnight at 37 °C and 5% carbon dioxide and then incubated the next day with dilutions of various antibodies or binding molecules in the range of 0.001 - 10 nM. Caspase-8 activity was measured from the collected cell extracts 7 hours after treatment using the Promega Caspase-Glo 8 assay (product number G8201) respectively. Thereafter, the luminescence of each sample was measured using a PerkinElmer VICTOR X4 2030 multi-label plate reader.

[0402] Figure 15 shows caspase activity 7 hours after treatment with either the bispecific CDH3 / TRAILR2 binding molecule, anti-TRAILR2 nanobody, or lexatumumab of the present invention compared to untreated controls. Lexatumumab increased activated caspase 8 by only about 2-fold even at the highest concentration (10 nM). In contrast, an increase in activated caspase 8 was detected using CDH3 / TRAILR2v1 and anti-TRAILR2 nanobody at a concentration of 0.1 nM (or higher). The levels of activated caspase 8 were similarly increased using higher concentrations of the bispecific CDH3 / TRAILR2 binding molecule and anti-TRAILR2 nanobody, and were about 15-fold above background with a 10 nM treatment. This data demonstrates that the decrease in cell viability observed in the cell death assay is not due to an unspecified mechanism and that the bispecific CDH3 / TRAILR2 molecule can efficiently and specifically induce apoptosis in target cells. Consistent with the cell viability data, it was observed that incubation of GP2d cells with the bispecific CDH3 / TRAILR2 binding molecule resulted in superior activation of caspase-8 compared to the anti-TRAILR2 antibody lexatumumab.

[0403] Example 6: In Vivo Antitumor Activity of the CDH3 / TRAILR2 Molecule in a GP2d Xenograft Model The in vivo efficacy of three EC1 domain-binding and three EC2 domain-binding CDH3 / TRAILR2 molecules was investigated. For this purpose, GP2d cancer cells were transplanted into immunodeficient mice, and the effect of administration of the molecules of the present invention on tumor volume was measured. Female BomTac:NMRI-Foxn1nu mice were subcutaneously transplanted with 5.0 × 10 6 GP2d cells (in 0.1 mL of 5% fetal bovine serum at a ratio of 1:1 (v / v) PBS:Matrigel), and tumor growth was monitored until it reached approximately 200 mm 3 . The mice were randomized into seven groups and administered vehicle control, an EC1-binding substance (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, or CDH3 / TRAILR2v3), or an EC2-binding substance (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, or CDH3 / TRAILR2v6) intravenously (i.v.) at 1 mg / kg. Subsequently, tumor growth was monitored for 34 days, except for the vehicle-treated cohort, which was euthanized 24 days after reaching the upper limit of tumor volume of 1000 mm 3 .

[0404] The data presented in Figure 16 demonstrate that the binding molecules of the present invention can induce a significant and prolonged decrease in tumor volume compared to the control group. Surprisingly, the EC1-binding molecules showed superior efficacy compared to the EC2-binding molecules. The EC1-binding substances brought about a long-lasting regression, showing 132%, 125%, and 125% inhibition of tumor growth, respectively. In contrast, a single treatment with the EC2-binding molecules resulted in an initial regression, but tumor regrowth was observed from day 10, resulting in only 70%, 61%, and 102% inhibition of tumor growth, respectively.

[0405] Example 7: Internalization Ability of the EC1-Binding Bispecific CDH3 / TRAILR2 Molecule The rate of internalization mediated by endocytosis and degradation of surface receptor-binding biopharmaceuticals by lysosomes affects their stability, and thus their plasma half-life and duration of response. CDH3 EC1-binding compounds previously developed by other researchers exhibit high internalization ability (International Publication No. 12 / 057328), and thus may have a lower ability to support sustainable and effective activation of TRAILR2 at the cell surface when used in the setting of bispecific compounds.

[0406] The internalization ability of an exemplary EC1-binding bispecific CDH3 / TRAILR2 compound, the TRAILR2-binding antibody lexatumumab, or an anti-CDH3 EC1-specific antibody was analyzed and compared to a commercially available internalizing anti-CDH3 antibody (MAB861, bio-techne®, R&D Systems). To achieve this, GP2d colorectal cancer cells were seeded in 96-well plates (3340, Corning, Inc., New York, USA) one day prior to exposing the cells to the compounds. Prior to the addition of the compounds, the antibodies were incubated with the Incucyte® Fabfluor-pH antibody labeling reagent (4722, Sartorius AG, Göttingen, Germany) according to the manufacturer's protocol. Next, the Fabfluor-labeled antibodies were added to the cells at a concentration of 1 nM in a total volume of 100 μl, and the cells were analyzed over 24 hours in an Incucyte® S3 live cell analysis instrument (Sartorius AG) at 10x magnification, taking four pictures per well per hour, for the red Fabfluor signal as a surrogate for internalization. The data presented in FIG. 17 demonstrate that for the bispecific CDH3 / TRAILR2 compound, the anti-TRAILR2 antibody lexatumumab, and the EC1-binding CDH3 compound, at the applied concentrations, there is no measurable internalization (no detectable red Fabfluor signal at all) compared to the internalizing anti-CDH3 antibody MAB861, which internalizes moderately. The lack of measurable internalization of the exemplary bispecific EC1-binding CDH3 / TRAILR2 antibodies prepared herein is consistent with the long-lasting tumor regression observed in the GP2d xenograft model, as demonstrated in FIG. 16.

[0407] Example 8: Stability of Bispecific CDH3 / TRAILR2-Binding Molecules under Low pH Conditions The stability of the bispecific CDH3 / TRAILR2 molecule was tested under low pH conditions. The biological preparation was exposed to low pH conditions during production to achieve elution from the affinity column (the "capture step"). Low pH conditions are also required to inactivate possible contaminants such as bacteria, fungi, and viruses. Effective elimination of possible contaminants is an important step during the manufacture of biological preparations using mammalian cell lines to ensure the safety of the drug. However, for many recombinant proteins, incubation at low pH induces aggregation and reduces the yield of the recovered protein. Thus, the stability of the protein at low pH is an important factor for the successful and efficient production of therapeutic proteins.

[0408] To test the stability of the bispecific CDH3 / TRAILR2 binding molecule at low pH, the pH was adjusted to pH 3.5 using 1 M acetic acid, followed by incubation at room temperature for 90 minutes, and then neutralized to pH 5.0 using 1 M Tris buffer (pH 9.0). The concentration of the sample was measured, and the possible formation of aggregates (high molecular weight species, HMWS) or fragments (low molecular weight species, LMWS) was determined by analytical size exclusion chromatography (aSEC).

[0409] The results are summarized in Table 19 below, demonstrating that none of the binding molecules CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6, or CDH3 / TRAILR2v7 showed any significant increase in HMWS (less than 1.06%) or LMWS (0%) or significant decrease in the main peak (less than 1.06%) compared to the starting material, which demonstrates good stability after incubation at pH 3.5 for 90 minutes.

[0410]

Table 20

Claims

**Claim 1** (a) at least one antigen-binding site that specifically binds to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2); and (b) at least one antigen-binding site that specifically binds to cadherin-3 (CDH3), a binding molecule comprising: wherein the at least one antigen-binding site that specifically binds to CDH3 i. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 3 (CDR1), SEQ ID NO: 4 (CDR2), and SEQ ID NO: 5 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3); ii. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2), and SEQ ID NO: 19 (CDR3); iii. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 23 (CDR1), SEQ ID NO: 24 (CDR2), and SEQ ID NO: 25 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3); and iv. an antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 33 (CDR1), SEQ ID NO: 34 (CDR2), and SEQ ID NO: 35 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3) a binding molecule selected from the group consisting of. **Claim 2** The binding molecule according to claim 1, wherein the at least one antigen-binding site that specifically binds to TRAILR2 comprises a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 79 (CDR3). **Claim 3** The binding molecule according to claim 1 or 2, wherein the at least one antigen-binding site that specifically binds to CDH3 is an immunoglobulin (Ig) molecule, and the at least one antigen-binding site that specifically binds to TRAILR2 comprises one or more scFvs. **Claim 4** The one or more scFvs are fused to the C-terminus of the Ig molecule, preferably, a first scFv is fused to the first heavy chain of the immunoglobulin molecule and a second scFv is fused to the second heavy chain, respectively, the binding molecule according to claim 3.

5. The at least one antigen-binding site that specifically binds to CDH3 is i. a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6; ii. a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16; iii. a VH comprising the amino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acid sequence of SEQ ID NO: 26; and iv. a VH comprising the amino acid sequence of SEQ ID NO: 32 and a VL comprising the amino acid sequence of SEQ ID NO: 36 The binding molecule according to any one of claims 1 to 4, selected from the group consisting of.

6. The at least one antigen-binding site that specifically binds to TRAILR2 is an antigen-binding site comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 76, the binding molecule according to any one of claims 1 to 5.

7. The at least one antigen-binding site that binds to CDH3 is i. a heavy chain comprising the amino acid sequence of SEQ ID NO: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 The binding molecule according to any one of claims 1 to 6, comprising.

8. The at least one antigen-binding site that binds to TRAILR2 is the binding molecule according to any one of claims 1 to 7, comprising the amino acid sequence of SEQ ID NO:

71.

9. i. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 81; ii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 83; iii. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; or iv. a modified heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 87 The binding molecule according to any one of claims 1 to 8, comprising

10. An isolated nucleic acid encoding the binding molecule according to any one of claims 1 to 9.

11. An expression vector comprising the nucleic acid according to claim 10.

12. A host cell comprising the expression vector according to claim 11.

13. A method for producing the binding molecule according to any one of claims 1 to 9, comprising i. culturing the host cell according to claim 12 under conditions that allow expression of the molecule, and ii. recovering the molecule, and optionally iii. further purifying and / or modifying and / or formulating the molecule A method comprising

14. The binding molecule according to any one of claims 1 to 9 for use in medicine, preferably for the treatment of cancer.

15. The binding molecule for use according to claim 14, wherein the cancer is pancreatic cancer, lung cancer or head and neck cancer.

16. A pharmaceutical composition comprising the binding molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

17. A bispecific binding molecule comprising at least one antigen-binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and at least one antigen-binding site that specifically binds to the EC1 domain of cadherin-3 (CDH3).

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