Binding molecules for treatment of cancer

Abstract

The present invention relates to novel binding molecules. In particular, the present invention relates to binding molecules that bind to TNF-related apoptosis inducing ligand receptor 2 (TRAILR2) and Cadhexin-3 (CDH3). The invention also relates to nucleic acids encoding such binding molecules; processes 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, in particular for therapeutic purposes in the field of cancer diseases.

Description

Technical Field

[0001] The present invention relates to novel binding molecules. The present invention specifically 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 comprising such binding molecules; and the use of such binding molecules or such compositions, especially for therapeutic purposes in the field of cancer diseases. Background of the Invention

[0003] Cancer is a disease characterized by abnormal localized cell growth that has the potential to spread throughout the body. It is a serious illness and the second most common cause of death in developed countries.

[0004] In the past, the most common ways to treat tumor cancers have been through surgery, radiation therapy, and the use of chemotherapy drugs or immunotherapy. Although advances have been made in the treatment of certain cancers in recent years, there is still a need for improved treatments for this disease.

[0005] Antibody-based biomolecules offer the possibility of being a powerful therapeutic agent for treating cancer. Antibodies are designed to recognize and bind to specific proteins (their target antigens) on the cell surface, and such proteins can only be present on the surface of specific cancer cells or on immune cells. This combination can provoke a variety of different biological responses, depending on the function of its target antigen protein and the structure of the antibody itself and its binding site. For example, some antibodies trigger the immune system to attack and kill cancer cells by attracting immune cells to cancer cells or by directly affecting the activity of the immune system itself. Other types of antibodies are attached with drugs or radioactive particles, so these therapeutic agents can be delivered to cancer cells. Other types of antibodies combined with cancer cells can activate or inhibit specific mechanisms that can reduce cell division or even kill tumor cells.

[0006] Apoptosis (or programmed cell death) is a controlled cellular mechanism by which organisms maintain cellular homeostasis in normal tissue compartments and eliminate disordered cells.

[0007] There are two major signaling pathways that lead to apoptosis in mammalian cells: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway begins at the mitochondrial level and plays an important role in chemotherapy- or irradiation-induced cell death. In contrast, the extrinsic death pathway is initiated by signals mediated by death receptors on the cell surface.

[0008] Cell death induced via the extrinsic pathway has been extensively studied following signals mediated by multiple members of the tumor necrosis factor (TNF) receptor superfamily. The TNF superfamily is characterized by sequences of two to five extracellular repeats rich in cysteine. Death receptors belonging to the TNF superfamily share a homologous intracellular death domain of approximately 80 amino acids necessary for transducing apoptotic signals. TNF-related apoptosis-inducing ligand (TRAIL) is a natural protein ligand that interacts with two types of receptors: death receptors that trigger TRAIL-induced apoptosis and decoy receptors that inhibit TRAIL-induced apoptosis. To date, four human receptors specific for TRAIL have been identified: death receptors DR4 (DR4 / TRAIL receptor 1 / TRAILR1) and DR5 (TRAIL receptor 2 / TRAIL-R2 / KILLER); and decoy receptors DcR1 / TRAILR3 / TRID and DcR2 / TRAILR4. TRAIL can also bind with low affinity to the soluble decoy receptor osteoprotegerin (OPG).

[0009] Targeting TRAIL receptors has been considered as a useful approach for developing cancer therapy, because if antibody-based molecules can bind to TRAIL receptors and activate (i.e., TRAIL receptor agonist molecules), they can induce apoptosis in cancer cells. As shown in many preclinical studies, TRAIL signaling effectively induces apoptosis in a variety of tumor cell lines but not in most normal cells. However, it is reported that normal tissues (especially hepatocytes in the liver) are also susceptible to this apoptosis induction mechanism. Therefore, if molecules that over-effectively activate pathways are used, severe side effects may be induced due to apoptosis induction in non-cancerous cells. On the other hand, weak activation molecules have been shown to have poor anti-cancer activity, although they have good tolerance.

[0010] One approach is the combined targeting of TRAIL receptors with cancer cell specific markers, ie proteins that are not expressed or very poorly expressed by non-cancerous cells.

[0011] A variety of different anchoring targets have been proposed as suitable combinatorial partners for TRAIL receptor binding molecules.

[0012] FAP (fibroblast activation protein) is also proposed as an anchoring target. However, FAP is only expressed on activated fibroblasts located in the tumor stroma. FAP is not expressed in epithelial cancer cells. Therefore, FAP bispecific molecules will only be used to promote apoptosis in these cancer cells that are in close physical contact with activated fibroblasts (Brünker et al., Molecular Cancer Therapeutics (2016), 15 (5): 946-957). Tumor cells that are not in direct contact with activated fibroblasts will not be affected by this treatment and will continue to proliferate. Therefore, there are obvious disadvantages in using FAP as an anchoring target to mediate TRAIL receptor-induced apoptosis in cancer cells. In addition, since activated fibroblasts are also present in sites of tissue remodeling including liver fibrosis, pulmonary fibrosis, atherosclerosis and arthritis, bispecific molecules targeting FAP and TRAIL receptors can potentially anchor to the surface of activated fibroblasts and induce apoptosis on adjacent normal TRAIL-sensitive cells in the liver or other organs.

[0013] Similarly, MCSP (melanoma-associated chondroitin sulfate proteoglycan) and ROB04 (circuit homolog 4) have been proposed as anchoring targets (He Yuan et al., The Journal of Investigative Dermatology (2016), 136 (2): 541-544; WO2011039126A1). In addition to its expression on the cell surface of melanoma, MCSP is mainly expressed on new blood vessels. ROB04 is specifically expressed in endothelial cells. Both are described at sites of angiogenesis in different tumor types. Therefore, like FAP is effective, and only except for melanomas expressing MSCP, if the cancer cells are in close physical contact with endothelial cells, the bispecific molecules targeting MCSP or ROB04 and the TRAIL receptor will only act to promote apoptosis of cancer cells. Since tumors will quickly exceed their blood supply as they grow, this will not always be possible. Therefore, there are also disadvantages in using these molecules as anchoring targets.

[0014] In addition, bispecific antibodies targeting TRAILR2 and LTβR (lymphotoxin β receptor) were reported to be active in inhibiting tumor growth in a murine tumor xenograft model, with inhibition levels comparable to or greater than the combination of the respective parent antibodies (Michaelson et al., mAbs (2009), 1(2): 128-141). LTβR signaling in mice has been shown to be essential for liver regeneration, with LTβR expressed on mature hepatocytes (Anders RA 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 HCC. In particular, its expression in hepatocytes has been described (Haybaeck J. et al. Cancer Cell (2009), 16(4): 295-308). Targeting LTβR and TRAIL receptors could potentially anchor onto the surface of LTβR-expressing hepatocytes that are sensitive to TRAILR2 activation and thus could potentially cause hepatotoxicity.

[0015] Finally, TENASCIN C has also been shown to be a useful anchoring target. TENASCIN C is a secreted protein and therefore not anchored to the cell membrane. This represents another example of an indirect mechanism for inducing apoptosis in cancer cells. Like the MCSP or ROBO4 anchoring strategy, the TENASCIN C portion must be appropriately oriented with the TRAILR2 molecule on the cancer cell membrane to achieve apoptosis. In addition, TENASCIN C expression is also upregulated in chronic liver disease, and it is expected that treatment with a bispecific molecule comprising a TRAIL agonist will worsen the condition.

[0016] Therefore, despite the progress that has been made in the treatment of certain cancers in recent years and even though a number of different approaches are currently being pursued, there is still a need to provide novel, therapeutically suitable compounds for the treatment of cancer, and in particular to develop therapeutically effective but safe antibody-based biomolecules that can be used as TRAIL receptor agonist molecules.

[0017] Therefore, the object of the present invention is to produce TRAIL receptor agonist molecules that have an improved therapeutic profile and provide certain advantages over the agents, compositions and / or methods currently used and / or known in the art. These advantages include in vivo efficacy, improved therapeutic and pharmacological properties, fewer side effects and / or other favorable properties, such as high stability (especially in low pH environments) and improved manufacturing convenience or reduced cost of goods, especially compared to molecules known in the art. Summary of the invention

[0018] 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) with an antigen binding site that specifically binds to homocadherin-3 (CDH3) in a single binding molecule. As discussed in more detail below, one advantage of the molecules of the present invention is that apoptosis is promoted only in cells that simultaneously present TRAILR2 and CDH3 on their surface.

[0019] 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 efficacy of the binding molecule.

[0020] According to a first aspect of the present invention, the present invention provides a binding molecule comprising:

[0021] (a) at least one antigen binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and

[0022] (b) at least one antigen binding site that specifically binds to E-cadherin-3 (CDH3), wherein the at least one antigen binding site that specifically binds to CDH3 is selected from the group consisting of:

[0023] i. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3);

[0024] ii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3);

[0025] iii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3); and

[0026] iv. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

[0027] In a specific aspect, 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 the extracellular domain 1 (EC1 domain) of cadherin-3 (CDH3).

[0028] In particular, the binding molecules provided herein are bispecific binding molecules.

[0029] In one aspect, a binding molecule provided herein therefore recognizes an antigen comprising SEQ ID NO:98 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:98.

[0030] In a specific aspect, the binding molecules provided herein specifically bind to an antigen comprising SEQ ID NO:99 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:99.

[0031] In a preferred embodiment of the binding molecule of the present invention, the antigen binding site that specifically binds to CDH3 (second antigen binding site) is selected from the group consisting of:

[0032] 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;

[0033] 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;

[0034] 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

[0035] 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.

[0036] In a preferred embodiment, the binding molecules provided herein comprise:

[0037] 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;

[0038] 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;

[0039] 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

[0040] 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.

[0041] In certain embodiments, the binding molecules provided herein comprise:

[0042] i. a heavy chain comprising an 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 an 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;

[0043] ii. a heavy chain comprising an 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 an 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;

[0044] iii. a heavy chain comprising an 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 an 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

[0045] 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.

[0046] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:3, CDR2 of SEQ ID NO:4, and CDR3 of SEQ ID NO:5; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO:7, a light chain CDR2 of SEQ ID NO:8, and a light chain CDR3 of SEQ ID NO:9.

[0047] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 14, and CDR3 of SEQ ID NO: 15; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO: 17, a light chain CDR2 of SEQ ID NO: 18, and a light chain CDR3 of SEQ ID NO: 19.

[0048] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:23, CDR2 of SEQ ID NO:24, and CDR3 of SEQ ID NO:25; and comprising 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:26, and comprising a light chain CDR1 of SEQ ID NO:27, a light chain CDR2 of SEQ ID NO:28, and a light chain CDR3 of SEQ ID NO:29.

[0049] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:34, and CDR3 of SEQ ID NO:35; and comprising 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:36, and comprising a light chain CDR1 of SEQ ID NO:37, a light chain CDR2 of SEQ ID NO:38, and a light chain CDR3 of SEQ ID NO:39.

[0050] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:43, CDR2 of SEQ ID NO:44, and CDR3 of SEQ ID NO:45; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO:47, a light chain CDR2 of SEQ ID NO:48, and a light chain CDR3 of SEQ ID NO:49.

[0051] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:53, CDR2 of SEQ ID NO:54, and CDR3 of SEQ ID NO:55; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO:57, a light chain CDR2 of SEQ ID NO:58, and a light chain CDR3 of SEQ ID NO:59.

[0052] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO:67, a light chain CDR2 of SEQ ID NO:68, and a light chain CDR3 of SEQ ID NO:69.

[0053] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:3, CDR2 of SEQ ID NO:4, and CDR3 of SEQ ID NO:5; and comprising 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, and comprising a light chain CDR1 of SEQ ID NO:7, a light chain CDR2 of SEQ ID NO:8, and a light chain CDR3 of SEQ ID NO:9.

[0054] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:14, and CDR3 of SEQ ID NO:15; and comprising 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:83, and comprising a light chain CDR1 of SEQ ID NO:17, a light chain CDR2 of SEQ ID NO:18, and a light chain CDR3 of SEQ ID NO:19.

[0055] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:23, CDR2 of SEQ ID NO:24, and CDR3 of SEQ ID NO:25; and comprising 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:85, and comprising a light chain CDR1 of SEQ ID NO:27, a light chain CDR2 of SEQ ID NO:28, and a light chain CDR3 of SEQ ID NO:29.

[0056] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:34, and CDR3 of SEQ ID NO:35; and comprising 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:87, and comprising a light chain CDR1 of SEQ ID NO:37, a light chain CDR2 of SEQ ID NO:38, and a light chain CDR3 of SEQ ID NO:39.

[0057] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:43, CDR2 of SEQ ID NO:44, and CDR3 of SEQ ID NO:45; and comprising 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:89, and comprising a light chain CDR1 of SEQ ID NO:47, a light chain CDR2 of SEQ ID NO:48, and a light chain CDR3 of SEQ ID NO:49.

[0058] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:53, CDR2 of SEQ ID NO:54, and CDR3 of SEQ ID NO:55; and comprising 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:91, and comprising a light chain CDR1 of SEQ ID NO:57, a light chain CDR2 of SEQ ID NO:58, and a light chain CDR3 of SEQ ID NO:59.

[0059] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to CDH3, the antigen binding site comprising 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 comprising CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65; and comprising 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:93, and comprising a light chain CDR1 of SEQ ID NO:67, a light chain CDR2 of SEQ ID NO:68, and a light chain CDR3 of SEQ ID NO:69.

[0060] 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 sequence 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 sequence of SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2) and SEQ ID NO:79 (CDR3).

[0061] In a preferred embodiment of the binding molecule of the present invention, at least one antigen binding site that specifically binds to TRAILR2 (first antigen binding site) is an antigen binding site comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:72 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:76.

[0062] In a specific embodiment, the binding molecules provided herein comprise an antigen binding site that specifically binds to TRAILR2, which antigen binding site 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 comprises CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:74, and CDR3 of SEQ ID NO:75; and comprises 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, and comprises a light chain CDR1 of SEQ ID NO:77, a light chain CDR2 of SEQ ID NO:78, and a light chain CDR3 of SEQ ID NO:79.

[0063] In some embodiments, the binding molecules provided herein defined by their heavy chain amino acid sequence (e.g., a modified heavy chain in which a TRAILR2-specific scFv is fused to the C-terminus of an Ig heavy chain) and their light chain amino acid sequence comprise two heavy chains and two light chains, thereby forming a symmetrical tetravalent and bispecific structure.

[0064] In another preferred embodiment, the binding molecule of the present invention comprises a modified heavy chain, preferably wherein a TRAILR2-specific scFv is fused to the C-terminus of an Ig 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.

[0065] Specifically, the binding molecule of the present invention comprises:

[0066] 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;

[0067] 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;

[0068] 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

[0069] 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.

[0070] In a preferred embodiment of the binding molecules of the invention, said molecules are bispecific and tetravalent.

[0071] Specifically, the at least one antigen binding site that specifically binds to TRAILR2 is connected to at least one antigen binding site that specifically binds to CDH3.

[0072] In a preferred embodiment of the binding molecules of the present invention, at least one antigen binding site that specifically binds to CDH3 is an immunoglobulin (Ig) molecule (having the conventional Y-shaped structure of a full-length antibody containing two light chains and two heavy chains), and at least one antigen binding site that specifically binds to TRAILR2 comprises one or more scFvs.

[0073] In preferred embodiments of the binding molecules of the invention, one or more scFvs have a VL-VH orientation from N-terminus to C-terminus.

[0074] 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 an Ig molecule to form a modified Ig molecule. For example, one scFv is fused to the C-terminus of one heavy chain of an Ig molecule, and one scFv is fused to the C-terminus of another heavy chain of an Ig molecule. Thus, a scFv specific for TRAILR2 is fused to each heavy chain of an Ig molecule to form a symmetrical, bispecific and tetravalent structure.

[0075] In a preferred embodiment, the binding molecule of the invention comprises two antigen binding sites that specifically bind to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) and two antigen binding sites that specifically bind to cadherin-3 (CDH3).

[0076] Therefore, in a particular aspect, 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 TNF-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 in the variable region (Fv) of the Ig molecule, and the two antigen binding sites that specifically bind to TRAILR2 are scFv; and wherein the scFv is fused to the C-terminus of the heavy chain of the Ig molecule. Preferably, respectively, the first scFv is fused to the first heavy chain of the Ig molecule, and the second scFv is fused to the second heavy chain of the Ig molecule.

[0077] In a preferred embodiment of the binding molecule of the present invention, the Ig molecule is IgG1KO. In another preferred embodiment of the binding molecule of the present invention, the Ig molecule is IgG1FcRnmut.

[0078] In a preferred embodiment of the binding molecules of the invention, one or more scFvs are fused to the Ig molecule via a peptide linker, preferably a peptide linker of about 4 to 20 amino acids in length (e.g., any one of 5, 6, 9, 12, 15 amino acids).

[0079] The present invention further describes binding molecules, in particular bispecific binding molecules, which have at least one antigen binding site (first antigen binding site) that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) as described in detail above, and at least one antigen binding site (second antigen binding site) that specifically binds to the extracellular domain 2 (EC2 domain) of E-cadherin-3 (CDH3).

[0080] The present invention further provides an antibody that specifically binds to CDH3, comprising:

[0081] i. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3);

[0082] ii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3);

[0083] iii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3);

[0084] iv. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3);

[0085] v. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2) and SEQ ID NO: 49 (CDR3);

[0086] vi. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2) and SEQ ID NO: 59 (CDR3); or

[0087] vii. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3).

[0088] In preferred embodiments, the antibodies provided herein comprise:

[0089] 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;

[0090] 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;

[0091] 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;

[0092] 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;

[0093] 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;

[0094] 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

[0095] 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.

[0096] In another preferred embodiment of the present invention, the antibody that specifically binds to CDH3 comprises:

[0097] 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;

[0098] 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;

[0099] 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;

[0100] 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;

[0101] 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;

[0102] 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

[0103] 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.

[0104] Specifically, the antibody comprises:

[0105] i. a heavy chain comprising an 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 an 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;

[0106] ii. a heavy chain comprising an 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 an 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;

[0107] iii. a heavy chain comprising an 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 an 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;

[0108] iv. a heavy chain comprising an 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 an 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;

[0109] 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;

[0110] vi. a heavy chain comprising an 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 an 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

[0111] 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.

[0112] In a preferred embodiment, the antibodies or antigen-binding fragments thereof provided herein are selected from the group consisting of chimeric antibodies, humanized antibodies and human antibodies or antibody fragments, and scFv, Fab fragments, monovalent antibody fragments and F(ab')2 fragments.

[0113] Another aspect of the present invention provides a nucleic acid molecule encoding a binding molecule of the present invention or an expression vector containing such a nucleic acid molecule.

[0114] In a preferred embodiment, the vector is a plasmid or a viral vector.

[0115] A further aspect of the invention provides a host cell comprising a nucleic acid molecule of the invention in functional association with an expression control sequence.

[0116] Further provided herein is a host cell comprising an expression vector comprising a nucleic acid molecule encoding a binding molecule as described herein.

[0117] Another aspect of the present invention provides a method for producing a binding molecule of the present invention, the method comprising i. culturing a host cell as described in technical solution 17 under conditions that allow expression of the molecule, and

[0118] ii. recovering the molecule, and optionally

[0119] iii. further purifying and / or modifying and / or formulating the molecule.

[0120] Further provided herein are binding molecules of the invention for use in medicine.

[0121] In a preferred embodiment, the binding molecules of the invention are provided for use in the treatment of cancer, preferably pancreatic cancer, lung cancer or head and neck cancer.

[0122] Further provided herein is a pharmaceutical composition comprising a binding molecule of the invention together with a pharmaceutically acceptable carrier and optionally one or more other active ingredients.

[0123] Other aspects of the invention provide a method of treating cancer comprising administering an effective amount of a binding molecule of the invention to a patient in need thereof.

[0124] In preferred embodiments, the pharmaceutical compositions provided herein are lyophilized, stabilized and / or formulated for administration by injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 : Amino acid sequence of CDH3.

[0127] Figure 2 : Expression of CDH3 and TRAILR2. Protein expression and membrane localization of TRAILR2 and CDH3 were confirmed in PDXs derived from colorectal cancer and the correlation with mRNA expression (A and B). In addition, membrane staining of CDH3 and TRAILR2 in surgical tumor tissues from head and neck squamous cell carcinoma and esophageal squamous cell carcinoma was shown (C).

[0128] Figure 3 : Schematic representation of molecular design. An example of a binding molecule of the invention is depicted, comprising (i) an Ig molecule that specifically binds to CDH3, comprising two heavy chains and two light chains, and (ii) two scFv molecules that specifically bind to TRAILR2. The N-terminus of the scFv is fused to the C-terminus of each heavy chain of the Ig molecule, thereby forming a symmetrical, bispecific and tetravalent antibody-like molecule.

[0129] Figure 4 : Flow cytometric analysis of protein surface expression of TRAILR2 and CDH3 in GP2d cells. Results are shown as mean fluorescence intensity.

[0130] Figure 5 : Effect of antibody incubation on cell viability. GP2d cells were treated for 24 h with different concentrations of (i) CDH3 / TRAILR2 bispecific molecule (CDH3 / TR2v1), (ii) anti-TRAILR2 (Lexatumumab) alone, (iii) anti-CDH3v1 (or anti-CDH3v2) alone, or (iv) an equivalent combination of separate anti-TRAILR2 (Lexatumumab) antibodies and anti-CDH3v1 (or anti-CDH3v2) antibodies. Data are expressed as mean relative values ​​compared to untreated controls.

[0131] Figure 6 : Effect of antibody incubation on cell viability. GP2d cells were treated with different concentrations of the following for 24 h: (i) bispecific CDH3 / TRAILR2 molecules (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, CDH3 / TRAILR2v7) in EC1 bound to CDH3, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobodies). Data are expressed as the average relative value compared to untreated controls.

[0132] Figure 7 : Effect of antibody incubation on cell viability. GP2d cells were treated with different concentrations of the following for 24 h: (i) bispecific CDH3 / TRAILR2 molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR22v5, CDH3 / TRAILR2v6) in EC2 bound to CDH3, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobodies). Data are expressed as the average relative value compared to untreated controls.

[0133] Figure 8: Flow cytometric analysis of protein surface expression of TRAILR2 and CDH3 in GP2d CDH3 knockout cells generated by CRISPR / Cas9. Results are shown as mean fluorescence intensity.

[0134] Fig. 9 : Effect of antibody incubation on cell viability in the absence of CDH3 target. GP2d (CDH3 knockout) cells were treated for 24 h with different concentrations of (i) exemplary CDH3 / TRAILR2 bispecific molecules of the invention, (ii) anti-TRAILR2 (lesatumumab) alone, (iii) anti-CDH3v1 alone, (iv) anti-CDH3v2 alone, or (iv) an equivalent combination of separate anti-TRAILR2 (lesatumumab) antibodies and anti-CDH3v1 (or anti-CDH3v2) antibodies. Data are expressed as mean relative values ​​compared to untreated controls.

[0135] Fig.10 : Effect of antibody incubation on cell viability in the absence of CDH3. GP2d (CDH3 knockout) cells were treated with different concentrations of the following for 24 h: (i) CDH3 / TRAILR2 bispecific molecules (CDH3 / TR2v1, CDH3 / TR2v2, CDH3 / TR2v3) binding to EC1, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobody). Data are expressed as the average relative value compared to untreated controls.

[0136] Fig.11 : Effect of antibody incubation on cell viability in the absence of CDH3. GP2d (CDH3 knockout) cells were treated with different concentrations of the following for 24 h: (i) CDH3 / TRAILR2 bispecific molecules (CDH3 / TR2v4, CDH3 / TR2v5, CDH3 / TR2v6) binding to EC2, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobodies). Data are expressed as the average relative value compared to untreated controls.

[0137] Fig.12 : Effect of antibody incubation on cell viability in a 3D cell culture model. NCI-H358 cells were treated for 48 h with different concentrations of (i) CDH3 / TRAILR2 bispecific molecule (CDH3 / TR2v1), (ii) anti-TRAILR2 (lesatumumab) alone, (iii) anti-CDH3v1 (or anti-CDH3v2) alone, or (iv) an equivalent combination of anti-TRAILR2 (lesatumumab) antibody alone and anti-CDH3v1 (or anti-CDH3v2) antibody. Data are expressed as mean relative values ​​compared to untreated controls.

[0138] Fig.13 : Effect of antibody incubation on cell viability in a 3D cell culture model. NCI-H358 cells were treated for 48 h with different concentrations of the following: (i) TRAILR2 / CDH3 bispecific molecules (CDH3 / TR2v1, CDH3 / TR2v2, CDH3 / TR2v3) bound to EC1, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobodies). Data are expressed as the average relative value compared to untreated controls.

[0139] Fig.14 : Effect of antibody incubation on cell viability in a 3D cell culture model. NCI-H358 cells were treated for 48 h with different concentrations of the following: (i) CDH3 / TRAILR2 bispecific molecules (CDH3 / TR2v4, CDH3 / TR2v5, CDH3 / TR2v6) binding to EC2, or (ii) anti-TRAILR2 alone (anti-TRAILR2 nanobodies). Data are expressed as the average relative value compared to the untreated control.

[0140] Fig.15 : Analysis of apoptosis induction after antibody incubation (apoptosis proteinase 8 activation analysis). GP2d cells were treated with different concentrations of the following for 7 h: (i) an exemplary bispecific CDH3 / TRAILR2 binding molecule of the invention, or an anti-TRAILR2 molecule, (ii) an anti-TRAILR2 nanobody, or (iii) lesatumumab, and apoptosis proteinase-8 activation was measured. Data are expressed as the average fold change relative to the untreated control.

[0141] Fig.16 : In vivo data on the GP2d xenograft model. In vivo efficacy of bispecific molecules that recognize human TRAILR2 and human CDH3: CDH3 / TRAILR2 molecules CDH3 / TRAILR2v1 (black circles), CDH3 / TRAILR2v2 (black squares), CDH3 / TRAILR2v3 (black triangles) that bind EC1, or CDH3 / TRAILR2 molecules CDH3 / TRAILR2v4 (white circles), CDH3 / TRAILR2v5 (white squares) and CDH3 / TRAILR2v6 (white triangles) that bind EC2, or vehicle control (grey crosses) were administered to mice bearing GP2d tumor cells. Tumor volume (mm3) was measured on the indicated days after administration. Data are expressed as the median tumor volume. Each group included at least 8 animals.

[0142] Fig.17: Internalization of CDH3 / TRAILR2 antibodies and single antigen binding antibodies that bind to EC1. GP2d apoptotic proteinase 8 knockout cells were exposed to 1 nM Fabfluor labeled antibodies: exemplary CDH3 / TRAILR2 molecules that bind to EC1 (white circles), exemplary CDH3 molecules that bind to EC1 (crossed white circles), TRAILR2 binding molecule lesatumumab (white squares), or commercially available CDH3 binding molecule CDH3 (MAB861, R&D systems) (black circles). Fabfluor counts per image were analyzed as a quantitative measure of internalization at the indicated hours after antibody administration. Mean values ​​of measurements are shown. Error bars represent standard deviations. DETAILED DESCRIPTION OF THE INVENTION

[0144] As described above, a method for treating cancer is to induce apoptosis in cancer cells by using molecules that specifically bind 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 a variety of tumor cell lines but not in most normal cells. However, it has been reported that normal tissues (especially hepatocytes in the liver) are also susceptible to this apoptosis-inducing mechanism.

[0145] TRAIL binds to four different cell surface receptors with high affinity. Two of them (TRAILR1 and TRAILR2) can trigger TRAIL-induced apoptosis via the interaction of its intracellular death domain with different adapter proteins and pro-apoptotic proteinase 8. TRAILR1 or TRAILR2 molecules promote the autocatalytic cleavage and activation of pro-apoptotic proteinase 8 via the clustering of TRAIL ligands, which in turn leads to induction of apoptosis. TRAILR3 and TRAILR4 are decoy receptors, and although their extracellular domains can bind TRAIL, the intracellular part of the receptor does not contain a domain that can induce apoptosis after TRAIL binding.

[0146] Overexpression of decoy TRAIL receptors can render cancer cells insensitive to the presence of TRAIL ligands. Specific targeting of non-decoy death-inducing TRAIL receptors avoids this problem and represents a more effective tumor therapy.

[0147] The present invention focuses on the development of TRAILR2 agonist molecules. TRAILR2 is widely expressed in a wide range of cancers. Several TRAILR2-specific agonist antibodies (including lexalimumab) (HGS-ETR2) have been developed for the treatment of cancer. However, these agonist antibodies lack efficacy clinically. Without intending to be limited by theory, it is believed that this may be due to the lack of sufficient clustering of the TRAILR2 receptor, thereby being unable to effectively induce apoptosis in cancer cells.

[0148] In different attempts to promote TRAILR2-mediated apoptosis in cancer cells, a tetramer TRAILR2-binding nanobody TAS266 has been developed. In preclinical experiments, it shows anti-tumor efficacy superior to conventional TRAILR2-targeted antibodies. However, it is reported that hepatocytes in the liver may be sensitive to TRAILR2-mediated apoptosis, and therefore the non-targeted increase of TRAILR2 clustering promoted by TAS266 has a potential toxicity risk. In fact, the Phase I clinical trial of TAS266 has been terminated.

[0149] Therefore, if a molecule that activates this pathway too effectively is used, severe side effects may be induced by inducing apoptosis in non-cancerous cells. On the other hand, if only weakly activating molecules are used, these molecules have been shown to have poor anticancer activity, although they are well tolerated.

[0150] One approach is the combined targeting of TRAIL receptors and cancer cell-specific markers (i.e., proteins that are not expressed or rarely expressed by non-cancer cells, also referred to as "anchor proteins") (see, e.g., WO 2018 / 115231A2, in which CDH17 is used as an anchor protein). However, such tumor-specific target proteins are rare, and this scarcity remains a major disadvantage commonly faced in the development of cancer-specific therapies. Although attempts have been made to target more widely expressed lineage antigens, the value of these therapies is limited by the toxicity caused by the expression of these antigens in certain normal tissues, such as by the toxicity caused by the expression of Epcam in the gastrointestinal tract (Kebenko et al., Oncoimmunology 2018, Vol. 7, No. 8). Although various methods are currently being sought to reduce the toxicity associated with off-target antigen expression, toxicity is still dose-limiting for many compounds.

[0151] The present inventors have identified anchoring proteins that are not present in significant amounts in serum and that localize in cancer cells co-expressing TRAILR2. Importantly, the anchoring proteins selected are not expressed in the liver, which is expected to be advantageous due to the potential hepatotoxicity described above.

[0152] The present inventors identified Cadherin-3 as a suitable anchoring target that can be used in combination with TRAIL receptor binding molecules.

[0153] Cadherin-3 (CDH3) is a member of the cadherin superfamily of genes that encode calcium-dependent, membrane-associated glycoproteins. The encoded protein is cadherin-like and consists of an extracellular region containing five cadherin domains, a transmembrane region, and an intracellular domain.

[0154] The present inventors analyzed the expression of CDH3 in tumor tissues and found that TRAILR2 and CDH3 were 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, skin melanoma, and in bladder urothelium and cervical cancer), and rarely or not co-expressed in non-cancerous cells. Notably, CDH3 was not detected in normal liver tissue or hepatocytes, which have been reported to be sensitive to TRAILR2 activation.

[0155] Thus, provided herein are binding molecules comprising at least one antigen binding site that specifically binds to TRAILR2 and at least one antigen binding site that specifically binds to CDH3.

[0156] In the accompanying experimental data, it can be seen that such molecules are able to induce apoptosis in vitro and in vivo in cells in which both CDH3 and TRAILR2 are expressed simultaneously. Surprisingly, bispecific binding molecules that bind to the EC1 domain of TRAILR2 and CDH3 are significantly more effective in inducing apoptosis than such bispecific binding molecules that specifically target the EC2 domain of CDH3, as strongly demonstrated by the in vivo data shown in Example 6.

[0157] Importantly, as also shown in the Examples, the same molecules do not substantially induce apoptosis in cells expressing TRAILR2 but not CDH3. Therefore, the binding molecules of the present invention are effective for treating 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.

[0158] Furthermore, the binding molecules of the invention are highly stable under low pH conditions, which enables efficient large-scale production, making them excellent candidates for therapeutic proteins.

[0159] Notably, the binding molecules of the invention show essentially no internalization activity in representative assays. As shown in Example 7, there is no measurable internalization of the exemplary CDH3 / TRAILR2 EC1 specific binding molecules. For the bispecific binding molecules of the invention, minimal or very slow internalization is beneficial to support sustained and efficient activation of TRAILR2 at the cell surface.

[0160] Thus, the CDH3 / TRAILR2 binding molecules of the present invention have significant advantages over molecules known in the art and provide utility for treating cancers including pancreatic cancer, head and neck cancer, and lung cancer. Some of these advantages include improved in vivo efficacy, fewer side effects, greater stability, and improved ease of production; especially when compared to molecules known in the art.

[0161] The 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 TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and at least one antigen binding site (second antigen binding site) that specifically binds to E-cadherin-3 (CDH3) (preferably to the EC1 domain of CDH3).

[0162] Until the present invention, the preparation of binding molecules capable of specifically binding to TRAILR2 and CDH3, especially the EC1 domain of CDH3, has never been disclosed or even slightly contemplated. Nevertheless, individually, each protein and its associated genes are known in the art and are well represented in biological databases.

[0163] For the avoidance of doubt, "TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2)" refers to the human protein provided as UniProtO14763 http: / / www.uniprot.org / uniprot / O14763, and the nucleic acid sequence encoding the protein.

[0164] For the avoidance of doubt, "Cadherin-3 (CDH3)" refers to the human protein provided as UniProt P22223 http: / / www.uniprot.org / uniprot / P22223, and the nucleic acid sequence encoding the protein.

[0165] The present invention relates to binding molecules having binding specificity for at least two different targets. With regard to the present invention, the binding molecules are derived from antibodies. Techniques for preparing binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-mortar" engineering (see, e.g., U.S. Pat. No. 5,731,168). The binding molecules of the present invention can also be prepared by engineering electrostatic guidance effects to prepare antibody Fc-heterodimer molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, for example, U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., Immunol., 148(5):1547-1553 (1 992)); using "diabody" technology for preparing bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., Immunol. 147:60 (1991).

[0166] Terms not specifically defined herein should be understood according to the meanings given to them by those skilled in the art based on the disclosure and context. However, as used in the specification, unless otherwise specified to the contrary, the following terms have the specified meanings and follow the following conventions.

[0167] Terms and definitions used

[0168] Molecules (such as the binding molecules of the invention or fragments thereof) that can "bind", "bind to", "specifically bind to", or "specifically bind to", "have affinity for" and / or "have specificity for" an epitope, antigen or protein (or at least a portion, fragment or epitope thereof) are said to be "against" or "directed against" that epitope, antigen or protein, or to be "binding" molecules with respect to such epitopes, antigens or proteins. As used herein, when referring to binding molecules, the term "bispecific" refers to molecules (such as the binding molecules of the invention) that can specifically bind to or have specificity for at least two different antigens or proteins (or at least a portion, fragment or epitope thereof).

[0169] As used herein, the term "antigen binding site" refers to a domain in a binding molecule that confers binding to a specific antigen. Antigen binding sites were originally derived from antibodies, but developments in this field have brought additional possibilities, making it possible to design and / or obtain antigen binding sites without having to produce naturally occurring antibodies for a target of interest. Regardless of its source, the "antigen binding site" according to the present invention at least includes the minimum structural elements that allow binding to its specific target antigen, i.e., necessary and sufficient structural elements. Therefore, the "antigen binding site" according to the present invention includes at least three heavy chain CDR sequences (in the case of a single domain antibody), more preferably at least three light chains and three heavy chain CDR sequences. As discussed below, these CDRs are generally present in the so-called variable domains or variable regions (Fv) of antibodies. It should be understood that although the antigen binding site at least includes the minimum structural elements, it generally encompasses additional elements (such as framework regions). Therefore, as used according to the present invention, the antigen binding site can also be defined via the sequence of each combination 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 comprised in a polypeptide and / or each of said CDRs or said variable domains is one or more polypeptides or one or more peptides.

[0170] "Antibodies" or "immunoglobulin molecules" (also called immunoglobulins, abbreviated Ig) are gamma globulins that can be found in the blood or other body fluids of vertebrates and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are usually composed of basic structural units, each with two large heavy chains and two small light chains, to form, 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 called antigens through non-covalent interactions. This binding is specific in the sense that antibodies will only bind to specific structures with high affinity. The unique portion of an antigen recognized by an antibody is called an epitope or antigenic determinant. The portion of an antibody that binds to an epitope is sometimes called a paratope and is present in the so-called variable domain or variable region (Fv) of an antibody. The variable domain contains three so-called complementary determining regions (CDRs) separated by framework regions (FRs).

[0171] In the context of the present invention, reference to CDRs is based on the definition of Kabat (EA Kabat, TT 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 (Chemical Computing Group, as described by 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).

[0172] As used herein, the expressions "variable domain" or "variable region" or Fv refer to the parts of the light and heavy chain pairs that are directly involved in the binding of the antibody to the 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 light and heavy chain domains have the same general structure, and each domain comprises four framework (FR) regions connected by three HVRs (or CDRs), the sequences of which are widely conserved. The framework region presents a β-sheet conformation, and the CDRs can form loops connecting the β-β sheet structures. The CDRs in each chain maintain their three-dimensional structure through the framework region and form an antigen binding site together with the CDRs from the other chain. The heavy and light chain CDR regions of the antibody play a particularly important role in the binding specificity / affinity of the antibodies according to the present invention and therefore provide another object of the present invention.

[0173] An immunoglobulin domain is essentially composed of four "framework regions", referred to in the art and hereinafter 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 separated by three "complementarity determining regions" or "CDRs", referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by carrying an antigen binding site.

[0174] The term "constant domain" or "constant region" as used in this application refers to the sum of the domains of an antibody excluding the variable region. Such constant domains and constant regions are well known in the prior art and are described, for example, by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91).

[0175] The "Fc portion" of an antibody is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. "Fc portion of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. According to the amino acid sequence of the constant region of the heavy chain, antibodies or immunoglobulins are divided into the following categories: IgA, IgD, IgE, IgG and IgM. According to the heavy chain constant region, different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. Based on complement activation, C1q binding and Fc receptor binding, the Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. While the effect of an antibody on the complement system depends on certain conditions, binding to CIq is caused by a defined binding site in the Fc part. Such binding sites are known in the prior art and are described, for example, 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, EP 0307434. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbered according to the Kabat EU index, see below). Among these residues, the most critical residues for mediating C1q and Fcγ receptor binding in IgG1 are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168). Antibodies of subclass IgG1 and IgG3 generally show complement activation and C1q and C3 binding, while IgG2 and IgG4 do not activate the complement system and do not bind C1q and C3.

[0176] Antibodies have been further developed in the art and have become a versatile tool in medicine and technology. Therefore, in the context of the present invention, the terms "binding molecule", "antibody molecule" or "antibody" include not only antibodies as found in nature comprising, for example, two light chains and two heavy chains or as in camelid species comprising only two heavy chains, but further encompass all molecules comprising at least one paratope having binding specificity for an antigen and having structural similarity to the variable domains of immunoglobulins.

[0177] The terms "antibody" and "antibody molecule" are used interchangeably herein.

[0178] Thus, antibodies may include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, antibody fragments, in particular Fv, Fab, Fab' or F(ab')2 fragments, single chain antibodies, in particular single chain variable fragments (scFv), small modular immunopharmaceuticals (SMIPs), domain antibodies, nanobodies and / or diabodies. Antibodies may have effector functions, such as ADCC or CDC, which are usually mediated by the Fc portion (antibody constant region) of the antibody, or they may not have effector functions, for example by lacking an Fc portion or having a blocking, shielded Fc portion, essentially an Fc portion that is not recognized or not fully recognized by immune cells or immune system components (such as the complement system). Monoclonal antibodies (mAbs) are monospecific antibodies with identical amino acid sequences. They can be prepared from hybrid cell lines (called hybridomas) by fusion tumor technology, which represent clones of B cells that produce specific antibodies fused to 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 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). "Recombinant antibodies" or "recombinant binding molecules" are antibodies or binding molecules that have been produced by recombinant engineered host cells. It is optionally isolated or purified.

[0179] 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 methods well known in the art.

[0180] Monoclonal antibodies (mAbs) are monospecific antibodies with identical amino acid sequences. They can be produced by hybridoma technology from hybrid cell lines (called hybridomas), which represent clones of B cells that produce specific antibodies and myeloma (B cell cancer) cells fused (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-7). Alternatively, monoclonal antibodies can 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).

[0181] For use in humans, it is often necessary to reduce the immunogenicity of antibodies originally derived from other species such as mice. This can be done by constructing chimeric antibodies, or by a method called "humanization". In this context, "chimeric antibodies" should be understood as antibodies comprising a sequence portion (e.g., variable domain) derived from a species (e.g., mouse) fused to a sequence portion (e.g., constant domain) derived from a different species (e.g., human). "Humanized antibodies" are antibodies comprising variable domains originally derived from non-human species, in which certain amino acid mutations have been made so that the total sequence of the variable domains is more similar to the sequence of human variable domains. Methods for antibody chimerization and humanization 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 antibodies based on sequences derived from the human genome, such as antibodies established by phage display or using transgenic animals (see, e.g., WO 90 / 05144). As used herein, the term "antibody" explicitly includes such humanized antibodies, chimeric antibodies, and human antibodies.

[0182] A "humanized" antibody refers to an antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody substantially comprises at least one (usually two) variable regions, wherein all or substantially all hypervariable regions (e.g., complementarity determining regions (CDRs)) correspond to hypervariable regions of non-human antibodies, and all or substantially the entire framework regions (FRs) correspond to framework regions of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0183] In addition, techniques for producing antibodies based on sequences derived from the human genome have been developed, for example by phage display or the use of transgenic animals (WO 90 / 05144; D. Marks, HR Hoogenboom, TP Bonnert, J. McCafferty, AD 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 20021(2): 189-203; Lonberg N, Huszar D. "Human antibodies from transgenic mice". Int Rev. Immunol. 1995; 13(1): 65-93.; Brüggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice". Curr Opin Biotechnol. 1997 August; 8(4): 455-8.). In the context of the present invention, such antibodies are "human antibodies".

[0184] The term "antibody", in particular the antibody molecules described herein, may also include fragments of immunoglobulins that retain antigen binding properties, such as Fab, Fab' or F(ab')2 fragments. Such fragments can be obtained by fragmentation of immunoglobulins, for example by proteolytic digestion, or by recombinant expression of such fragments. For example, immunoglobulin digestion can be achieved by conventional techniques, for example using papain or pepsin (WO 94 / 29348). Papain digestion of antibodies generally produces two identical antigen binding fragments, so-called Fab fragments, each having a single antigen binding site, and a residual Fc fragment. Pepsin treatment produces F(ab')2. In Fab molecules, the variable domains are each fused to an immunoglobulin constant domain, which is preferably of human origin. Therefore, 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 recombinantly expressing the corresponding nucleic acid in a host cell, see below.

[0185] A variety of techniques have been developed for replacing the variable domains of immunoglobulins or molecules derived from such variable domains under different molecular environments. These should also be considered as "antibodies" according to the present invention. Generally, these antibody molecules are smaller in size than immunoglobulins and may contain a single 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, the variable regions being linked together with a short linker that is usually serine (S) or glycine (G) (WO 88 / 01649; WO 91 / 17271; Huston et al.; International Reviews of Immunology, Vol. 10, 1993, 195-217). "Single domain antibodies" or "nanoantibodies" have an antigen binding site in a single Ig-like domain (WO 94 / 04678; WO 03 / 050531, Ward et al., Nature. Oct. 12, 1989; 341(6242):544-6; Revets et al., Expert Opin Biol Ther. 5(1):111-24, 2005). One or more single domain antibodies with binding specificity for the same or different antigens can be linked together. Diabodies are bivalent antibody molecules composed of two amino acid chains containing two variable domains (WO 94 / 13804, Holliger et al., Proc Natl Acad Sci US A. Jul. 15, 1993; 90(14):6444-8). Other examples of antibody-like molecules are immunoglobulin superfamily antibodies (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). A different concept leads to so-called small modular immunopharmaceuticals (SMIPs), which comprise an Fv domain connected to a single-chain hinge and effector domains, lacking the constant domain CH1 (WO 02 / 056910).

[0186] Antibody molecules can be fused (as fusion proteins) or otherwise connected (by covalent or non-covalent bonds) with other molecular entities (such as cytotoxic drugs) or entities having a desired effect on the characteristics of antibody molecules. For example, it may be necessary to improve the pharmacokinetic properties of antibody molecules, such as stability in body fluids such as blood, especially in the case of single-chain antibodies or domain antibodies. In this regard, in particular, to extend the circulation half-life of such antibody molecules, a large number of technologies have been developed, such as pegylation (WO 98 / 25971; WO 98 / 48837; WO 2004081026), antibody molecules are fused or covalently linked to another antibody molecule with affinity for serum proteins (such as albumin) (WO 2004041865; WO2004003019), or all or part of an antibody molecule and a serum protein (such as albumin or transferrin) are expressed as a fusion protein (WO 01 / 79258). Means and methods for lead compound identification and lead compound optimization in antibody design are well known in the art and have been reviewed, for example, in Goulet, DR and Atkins, WMJ Pharm Sci 2020; 109(1):74-103, or Tiller, KE and Tessier, PM (2015). Annual review of biomedical engineering, 17, 191-216.

[0187] In terms of the present invention, the 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). It is worth noting that the CDH3 antigen binding site of the binding molecule of the present invention specifically binds to the EC1 domain of CDH3.

[0188] "Binding specificity" means that the antibody molecule has a significantly higher binding affinity for the TRAILR2 or CDH3 target antigen than for a structurally unrelated molecule.

[0189] "Specific" or "selective" binding, when referring to ligand / receptor, antibody / antigen or other binding pairs, indicates a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, a given ligand binds to a specific receptor and does not bind in significant amounts to other proteins present in the sample.

[0190] An epitope is a region of an antigen that is bound by an antibody or antigen binding portion. The term "epitope" includes any polypeptide determinant that is capable of specific binding to an antibody or antigen binding portion. In certain embodiments, epitope determinants include chemically active surface groups of molecules (such as amino acids, glycan side chains, phosphoryl or sulfonyl groups), and in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not lost. As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen binding portion to an antigenic epitope in an in vitro assay, preferably a plasma resonance assay ( GE-Healthcare Uppsala, Sweden).

[0191] The antibodies or binding molecules described herein bind to their antigen with an affinity that is at least two times greater than the affinity for an unrelated antigen, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater. In a preferred embodiment, the antibody will have an affinity greater than about 109 L / mol, as determined, for example, by Scatchard analysis (Munsen et al. (1980) Analyt. Biochem. 107: 220-239).

[0192] Affinity is the interaction between a single antigen binding site on an antibody molecule and a single epitope. It is expressed by the association constant KA = kass / kdiss or the dissociation constant KD = kdiss / kass.

[0193] As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope of an antigen, for example in an in vitro assay, preferably in a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden), for example, measured at room temperature. Binding affinity is defined by the terms kass (rate constant for the association of the antibody from the antibody / antigen complex), kdiss (dissociation constant), and KD (kdiss / kass). 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 generally bind to their target antigen with an affinity of 10-9 M or less.

[0194] In one aspect, the antibody binds to the TRAILR2 or CDH3 target antigen with an affinity, for example, as determined by surface plasmon resonance analysis (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 April; 5(2): 282-6.), wherein the KD value is in the range of 1 pM to 100 μM, preferably in the range of 1 pM to 1 μM. Antibody affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, RJ and Brault P-A., "Kinetic exclusion assay technology: Characterization of Molecular Interactions." ASSAY and Drug Development Technologies. 2004 December 2(6): 647-657).

[0195] The binding affinity of an antibody molecule can 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 in the present invention.

[0196] In one embodiment, the bispecific binding molecules of the invention can induce TRAILR2-mediated apoptosis in one or more cancer cell types (e.g., colon adenocarcinoma cell line GP2d or lung cancer cell line NCI-H358), inhibiting cell growth by more than 50% at a concentration of 1 nM or lower, even more preferably lower than 0.01 nM.

[0197] In another embodiment, the binding molecules of the invention are unable to induce TRAILR2-mediated apoptosis in CDH3-negative cells, inhibiting cell growth by less than 50% at concentrations up to 1 nM, or more preferably up to 10 nM, and even more preferably up to 100 nM.

[0198] As is generally known and agreed in the art, amino acid residues will be represented according to the standard three-letter or single-letter amino acid code. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion or substitution of a specified number of amino acid residues at a certain position of the reference sequence compared to the second sequence. In the case of one or more substitutions, such one or more substitutions will preferably be one or more conservative amino acid substitutions, which means that the amino acid residue is substituted by another amino acid residue with a similar chemical structure and has little or 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, wherein conservative amino acid substitutions are preferably those in which one amino acid within the following groups (i)-(v) is substituted with another amino acid residue within the same group: (i) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and GIy; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, Ile, VaI and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows:

[0199] Ala is substituted with Gly or with Ser;

[0200] Arg was replaced by Lys;

[0201] Asn is substituted with Gln or with His;

[0202] Asp is replaced by Glu;

[0203] Cys was replaced by Ser;

[0204] Gln is replaced by Asn;

[0205] Glu is replaced by Asp;

[0206] Gly is substituted with Ala or with Pro;

[0207] His is replaced by Asn or by Gln;

[0208] Ile is substituted with Leu or with VaI;

[0209] Leu is substituted with Ile or with VaI;

[0210] Lys is substituted with Arg, Gln or Glu;

[0211] Met is substituted with Leu, Tyr or Ile;

[0212] Phe is substituted with Met, Leu or Tyr;

[0213] Ser is replaced by Thr;

[0214] Thr is replaced by Ser;

[0215] Trp is replaced by Tyr;

[0216] Tyr is substituted with Trp or with Phe;

[0217] VaI is substituted with Ile or with Leu.

[0218] As used herein, the term "isolated" refers to material that is removed from its original or natural environment (e.g., if it is a naturally occurring substance, its natural environment). For example, a naturally occurring nucleic acid molecule or polypeptide present in a living animal is not isolated, but the same nucleic acid molecule or polypeptide is isolated by human intervention to separate it from some or all of the coexisting materials in the natural system. 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, but they are still isolated because the vector or composition is not part of the environment in which the nucleic acid molecule or polypeptide is found in nature. For example, a nucleic acid molecule or polypeptide is considered to be "in substantially isolated (form)" when it has been separated from at least one other component with which it is normally associated in the source or medium (e.g., another nucleic acid molecule, another polypeptide, another biological component or macromolecule or at least one contaminant, impurity or trace component) as compared to the natural biological source and / or reaction medium or culture medium from which the nucleic acid molecule or polypeptide was obtained. In particular, a nucleic acid molecule or polypeptide is considered to be "substantially isolated" when it has been purified at least 2 times, particularly at least 10 times, more particularly at least 100 times, or even 1000 times or more. A nucleic acid molecule or polypeptide in "substantially isolated form" is preferably substantially homogeneous, as determined by using a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis). The binding molecules and nucleic acids of the present invention are preferably isolated.

[0219] Unless otherwise indicated, as used herein, the term "sequence" (e.g., in terms such as "immunoglobulin sequence," "binding molecule sequence," or "polypeptide sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the same, unless the context requires a more restrictive interpretation.

[0220] As used herein, in the case of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" means that two or more sequences or subsequences are identical, or have a specific percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine the percentage of identity, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the amino acid residue or nucleotide occupied by a position in the first sequence is the same as that of the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the 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, after introducing gaps in the sequence as needed, the two sequences being compared are of the same length (e.g., excluding additional sequences beyond the range of the compared sequences). For example, when comparing variable region sequences, leader sequences and / or constant domain sequences are not considered. For a sequence comparison between two sequences, a "corresponding" CDR refers to a CDR that is at the same position in both sequences (eg, CDR-H1 in each sequence).

[0221] Determining the percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm 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 an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program with score = 100 and wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to a protein of interest. To obtain a gapped alignment for comparison purposes, a gapped BLAST as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402 can be used. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules (ibid.). When using 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 for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is a part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a 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. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are obtained by observing aligned residue pairs; if ktup=1, single aligned amino acids are examined. For protein sequences, ktup can be set to 2 or 1, or to 6 for DNA sequences. If ktup is not specified, the default value is 2 for proteins and 6 for DNA.Alternatively, the CLUSTAL W algorithm may be used for protein sequence alignment as described by Higgins et al., 1996, Methods Enzymol. 266:383-402. Preferably, the CLUSTAL W algorithm described above is used.

[0222] As used herein, the term "comprising" means that other components and / or steps may be included in addition to the components and / or steps specifically described. However, this term also encompasses that the claimed subject matter is entirely composed of the components and / or steps described.

[0223] As used herein, the term "at least" refers to any number including a specific number of narrations and any number higher than it. For example, "at least one" encompasses exactly one, and more than one, including but not limited to two, such as three or four. Further including, for example, five, six, seven, eight, nine, 10, 15, such as 20, 30, 40, 50, 75, 100, 150, 200, 300, 400 or 500, and any integer between or higher than the numbers of these specific narrations. With regard to the term "at least one antigen binding site", it is particularly preferred that the term encompasses one, two, three or four antigen binding sites. Most preferably, the term relates to exactly one antigen binding site. In these cases where one or more antigen binding sites for a target are selected, these multiple antigen binding sites can be selected independently, i.e., they can be the same or they can be different from each other.

[0224] As used herein, the term "polypeptide" describes a linear amino acid molecule chain 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 describes a linear amino acid chain containing up to 30 amino acids. The term "(poly)peptide" as used according to the present invention refers to a group of molecules comprising a peptide group consisting of up to 30 amino acids and a polypeptide group consisting of more than 30 amino acids.

[0225] As used herein, the term "linker" encompasses both peptide linkers (i.e., amino acid sequences) and non-peptide linkers (separate parts of its covalent or non-covalently linked molecules). As used herein, the term "non-peptide linker" refers to a linking group having two or more reactive groups but excluding peptide linkers as defined below. For example, a non-peptide linker can be a polymer having reactive groups at both ends, which are individually combined with reactive groups (e.g., amino termini, lysine residues, histidine residues, or cysteine ​​residues) of the binding portion of the molecule of the present invention. The reactive groups of the polymer include aldehyde, propionaldehyde, butyraldehyde, maleimide, ketone, vinyl sulfone, thiol, hydrazide, carbonyl imidazole (CDI) group, nitrophenyl carbonate (NPC) group, thiol, isocyanate, and succinimide derivatives. Examples of succinimide derivatives include succinimidyl propionate (SPA), succinimidyl butyric acid (SBA), succinimidyl carboxymethyl ester (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.

[0226] As contemplated herein, a peptide linker is a (poly)peptide linker of 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 15 to 30 amino acids in length. Non-limiting examples of commonly used small linkers include sequences of glycine and serine amino acids, referred to as GS mini-linkers. Preferred examples of linker sequences are Gly / Ser linkers of varying lengths, such as (glyxsery)z linkers, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3 and (gly3ser2)3. The number of amino acids in these linkers can vary, for example they can 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 multiples thereof, such as three or more repeats of these four / six amino acids. Most preferably, such GS mini-linkers have 20 amino acids and the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232). Other examples of linkers include the following:

[0227]

[0228] 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.

[0229] It is known in the art that the properties of the linker, i.e., length and / or composition, such as amino acid sequence, can modulate or enhance the stability and / or solubility of the molecule containing the linker. Typically, the length and sequence of the linker are selected depending on the composition of the respective molecule of interest. Methods for designing and testing the suitability of different linkers are well known to those skilled in the art, see, e.g. T. et al. Protein Engineering, Design and Selection, Vol. 14, No. 10, 2001, pp. 815-823. For example, the properties of the molecule can be easily tested by comparing the binding affinity of the binding parts of the molecules of the present invention. In the case of the trispecific molecules of the present invention, the corresponding measurements of each binding part can be performed separately. The stability of the resulting molecule can be measured by measuring the residual binding capacity of the molecule after incubation for several time periods at 37° C. in human serum using an ELISA-based method. Other suitable tests can be found in, for example, Brian R. Miller, BR et al. Protein Engineering, Design and Selection, Vol. 23, No. 7, 2010, pp. 549-557 or Kügler, M. et al. Protein Engineering, Design and Selection, Vol. 22, No. 3, 2009, pp. 135-147.

[0230] According to the present invention, the term "nucleic acid molecule" can be used interchangeably with the term "polynucleotide" in this article, including DNA, such as cDNA or genomic DNA, and RNA, such as mRNA. Further include nucleic acid simulation molecules known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid simulation molecules or nucleic acid derivatives according to 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 a kind of RNA derivative, and its ribose ring is constrained by the methylene bond between 2'-oxygen atom and 4'-carbon atom. They can contain additional non-natural or derived nucleotide bases, as those skilled in the art will readily appreciate.

[0231] Binding molecules of the present invention

[0232] 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 site that specifically binds to cadherin-3 (CDH3). Preferably, the at least one antigen binding site that binds to CDH3 specifically binds to the EC1 domain of CDH3.

[0233] Therefore, the binding molecules of the present invention (also referred to herein as "proteins of the present invention" or "binding agents of the present invention") at least comprise two different antigen binding sites specifically described, i.e., at least one binding site for TRAILR2 and at least one binding site for CDH3. Because of at least two specificities, the binding molecules of the present invention are also referred to herein as "bispecific binding molecules" of the present invention.

[0234] As used herein, the term CDH3 refers to "cadherin-3", 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 region. Human CDH3 is represented by SEQ ID NO: 98 and the database accession number UniProtP22223, which can be obtained at http: / / www.uniprot.org / uniprot / P22223.

[0235] Cadherins are a group of transmembrane proteins that serve as the main adhesion molecules located in adherens junctions. They can regulate cell-cell adhesion through their extracellular domains, and their cytoplasmic domains are connected to the actin cytoskeleton by binding to catenin. Structurally, cadherins contain multiple domains: classically, these include a signal sequence; a propeptide of about 130 residues; a single transmembrane domain and five tandemly repeated extracellular cadherin domains, four of which are cadherin repeats, and the fifth usually contains four conserved cysteines and a C-terminal cytoplasmic domain. However, if a protein has one or more cadherin repeats, it is designated as a member of the broadly defined cadherin family. Cadherin repeats are independently folded sequences of approximately 110 amino acids, which usually contain motifs with the conserved sequences DRE, DXNDNAPXF, and DXD.

[0236] As used herein, the term "EC1 domain of CDH3" refers to the first extracellular cadherin repeat of CDH3. Specifically, the EC1 domain of CDH3 is represented by the amino acid sequence of SEQ ID NO:99.

[0237] Thus, the term "EC2 domain of CDH3" refers to the second extracellular cadherin repeat of CDH3, represented by SEQ ID NO:100.

[0238] With regard to its form, the binding molecules of the present invention are not particularly limited, provided that they comprise at least two different antigen binding sites (TRAILR2 and CDH3) specifically described and are capable of binding to these two targets. Therefore, the form may be based on the form of naturally occurring antibodies or antibody derivatives or fragments of such antibodies and antibody mimetics. Such forms may be modified as needed to accommodate all three antigen binding sites, for example by additionally comprising other antibody fragments (particularly Fv, Fab, Fab' or F(ab')2 fragments), single-chain antibodies (particularly single-chain variable fragments (scFv)), small modular immunopharmaceuticals (SmallModularImmunopharmaceutical; SMIP), domain antibodies or nanobodies. Other non-limiting examples of suitable forms of individual or all antigen binding sites that may be employed include antibody mimetics as defined above.

[0239] 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 the conventional Y-shaped structure of a full-length antibody comprising 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) comprises 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) comprises one or more scFv. Even more preferably, the binding molecule described herein comprises two antigen binding sites that specifically bind to TRAILR2, each comprising an scFv, i.e., such binding molecules comprise two scFvs that are target-specific for TRAILR2.

[0240] A "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 domains and the linker have one of the following orders from N-terminal to C-terminal direction: a) VH-linker-VL, b) VL-linker-VH, and wherein the linker is a polypeptide of 15 to 25 amino acids in length, preferably 20 amino acids.

[0241] In addition, these single-chain Fv fragments can be further stabilized by incorporating disulfide bonds between the VH and VL domains, within the VH domain, or within the VL domain by incorporating cysteine ​​residues. The term N-terminus refers to the first amino acid of a polypeptide chain, while the term C-terminus refers to the last amino acid at the C-terminus of a polypeptide chain. Thus, embodiments of the present invention are those wherein the one or more scFvs comprise additional cysteine ​​residues to form disulfide bonds.

[0242] In an embodiment of the invention, the stability of the scFv portion can be increased by incorporating two cysteine ​​residues in close 3-dimensional proximity to form disulfide bonds within the scFv (referred to herein as scFvss). In the case where the scFv is derived from the V region sequence of TRv1 (as discussed below), example potential sites for such stabilizing disulfide bonds that can 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 positions 4 and 100 of VL, and (d) between positions 6 and 112 of VH. To achieve stabilization via engineered disulfide bonds, the residues at these positions are preferably substituted with cysteine ​​residues.

[0243] Preferably, the antigen binding site for TRAILR2 is a scFv fused to the C-terminus of the heavy chain of an immunoglobulin molecule; for example, a scFv fused to a heavy chain of an Ig molecule; or two scFvs, one scFv fused to one of the two heavy chains and one scFv fused to the other heavy chain, thereby forming a modified heavy chain.

[0244] In a preferred embodiment, the bispecific binding molecule of the present invention comprises a modified immunoglobulin molecule, wherein (i) the immunoglobulin heavy chain comprises the amino acid sequence of the heavy chain variable domain that specifically binds to CDH3, the immunoglobulin heavy chain constant domain, and scFv, the scFv specifically binds to TRAILR2, comprises the amino acid sequence of the light chain and heavy chain variable domains, and the scFv is connected to the C-terminus of the Ig constant domain; and (ii) the immunoglobulin light chain comprises the amino acid sequence of the light chain variable domain that specifically binds to CDH3 and the light chain constant domain. Preferably, the modified immunoglobulin molecule comprises two immunoglobulin heavy chains (e.g., modified heavy chains) and two immunoglobulin light chains.

[0245] The manner in which the various components are fused to each other is well known in the art. The fusion may be carried out, for example, via a peptide linker, or via a non-peptide linker. Preferably, the fusion is carried out via (one or more) peptide linkers.

[0246] In a specific aspect of the invention, one or more scFvs that specifically bind to TRAILR2 are fused to an Ig molecule that specifically binds to CDH3 via a peptide linker, preferably a peptide linker of about 4 to 20 amino acids in length (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 Ig molecule.

[0247] Methods for linking scFv molecules to the C-terminus of the heavy chain of an IgG molecule or linking variable domains within a scFv molecule are well known in the art. Typically, a small linker sequence of glycine and serine amino acids (also called a GS mini-linker) is used.

[0248] The number of amino acids in the linker can vary between 4 and 10 or more, as described herein. In practice, the linker is typically formed by combining a nucleic acid molecule encoding an IgG of interest (in the case of the present invention, it should include nucleic acids encoding the variable domains of the heavy chain for the CDH3 binding site of the IgG type and the constant domains) with a nucleic acid encoding the desired scFv (in the case of the present invention, it should include nucleic acids encoding the variable domains of the heavy and light chains, in VL-VH or VH-VL orientation for the TRAILR2 binding site), separated by a nucleic acid molecule encoding a linker sequence (e.g., a GS mini-linker of any one of 5, 10, 15 or 20 amino acids, preferably a linker such as GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232)). Subsequently, as further explained below, this nucleic acid molecule encoding a complete modified heavy chain is placed in an expression vector and introduced into an appropriate host cell, such that a complete IgG heavy chain-scFv single polypeptide is formed.

[0249] 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.

[0250] Preferably, the immunoglobulin molecule is a monoclonal molecule, a chimeric molecule, a humanized molecule or a human immunoglobulin (e.g., an antibody) molecule. Further preferably, the heavy chain constant region of the immunoglobulin molecule is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions. In another preferred embodiment, the light chain constant region of the immunoglobulin molecule is κ or λ. Preferably, the Ig molecule is IgG.

[0251] In one embodiment, the present invention provides a binding molecule as a multispecific binding protein, comprising (i) an Ig molecule having two heavy chains and two light chains that specifically binds to CDH3, and (ii) two scFv molecules (scFv), each specifically binding to TRAILR2. Preferably, each heavy chain of the Ig molecule has an scFv fused to its C-terminus, thereby forming a bispecific tetravalent binding protein.

[0252] In one embodiment, the invention provides a binding molecule (also referred to herein as a multispecific binding protein or a modified Ig molecule) having:

[0253] (i) two heavy chains (e.g., modified heavy chains), each comprising, from N-terminus to C-terminus:

[0254] - a heavy chain variable domain (eg, a murine, humanized or human VH domain) that is specific for CDH3, preferably specific for the EC1 domain of CDH3,

[0255] - a constant domain of IgG (e.g. human IgG1 or IgG4),

[0256] - a peptide linker (e.g. a GS minilinker), and

[0257] - a scFv specific for TRAILR2 (e.g., a scFv comprising, from N-terminus to C-terminus, a VH domain (e.g., a murine, humanized or human VH domain), a linker and a VL domain (e.g., a murine, humanized or human VL domain), or vice versa a VL domain, a linker and a VH domain); and

[0258] (ii) two light chains, each comprising, from N-terminus to C-terminus:

[0259] - a light chain variable domain (eg, a murine, humanized or human VL domain) that is specific for CDH3, preferably specific for the EC1 domain of CDH3,

[0260] - a light chain constant domain (eg a human kappa chain).

[0261] Since the Fc region of an antibody interacts with many Fc receptors, which produce a variety of important functional capabilities (which are called "effector functions"), in certain embodiments, the antibody is a full-length antibody or an antibody containing a portion of the Fc region, as long as the antibody exhibits specific binding to the relevant portion of the antigen and to both Fc receptors and complement. The choice of the type and length of the constant region depends on whether effector functions (such as complement fixation or antibody-dependent cell-mediated cytotoxicity) are desired features, and on the desired pharmacological properties of the antibody protein.

[0262] In an embodiment of the present invention, the binding molecules of the present invention may have an Fc region or its related portion that has been engineered to avoid unintended cross-linking by soluble Fcγ receptors or complement C1q. In one embodiment, such binding molecules or antibody variants have much lower affinity for Fcγ receptors and complement C1q than the parent antibody. (Hereinafter, unless otherwise indicated, in the context of antibody molecules, or in the context of IgG or Fc regions, the term "parent" refers to unengineered antibody molecules, Fc regions or IgG, respectively, and the mutated (engineered) molecules are derived from them.). Therefore, one embodiment of the present invention is an Fc variant in which the Ig molecule comprises an affinity for Fcγ receptors or complement receptors or both compared to the wild-type Fc region. Such Ig molecules are referred to herein as IgG1 (KO).

[0263] Another embodiment of the present invention is that the binding molecule of the present invention comprises an Fc region or its relevant part that has been engineered to modulate serum levels (half-life) by optimizing its interaction with the neonatal Fc receptor (FcRn), for example, by a point mutation at position H310A in the CH2 domain. Such Ig molecules are referred to herein as IgG1FcRnmut.

[0264] Another embodiment of the invention is an Ig molecule wherein the binding molecule comprises a hinge region variant containing IgG4 that removes the exchange of heavy chains with other IgG4 molecules. Such Ig molecules are referred to herein as IgG4Pro.

[0265] The present invention provides a bispecific binding molecule, which has 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).

[0266] Methods for preparing binding sites that bind to specific target antigens are well known in the art. Those skilled in the art can readily use these methods to design antigen binding sites with the necessary specificity for TRAILR2 or CDH3 target antigens.

[0267] Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies can be produced by any of the following methods: inducing the in vivo production of antibody molecules, screening immunoglobulin libraries (Orlandi et al., 1989. Proc. Natl. Acad. Sci. USA 86: 3833-3837; Winter et al. 1991, Nature 349: 293-299) or producing 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 viru (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).

[0268] In some embodiments, the antigen binding site (for CDH3 and / or TrailR2) is a "humanized" antigen binding site (e.g., comprising humanized VH / VL domains) comprising amino acid residues from non-human hypervariable regions (HVRs; e.g., complementarity determining regions (CDRs)) and amino acid residues from human framework sequences. In some embodiments, the antigen binding site (for CDH3 and / or TRAILR2) is a human antigen binding site (e.g., comprising human VH / VL domains) comprising CDR and FR sequences both derived from sequences of the human genome.

[0269] The amino acid sequences of specific antigen binding sites are provided in the specification and sequence listing.

[0270] Details of preferred embodiments of the invention comprising specific antigen binding sites for TRAILR2 and / or CDH3 are provided below.

[0271] In a preferred embodiment, at least one antigen binding site that specifically binds to CDH3 comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3).

[0272] In another preferred embodiment, at least one antigen binding site that specifically binds to CDH3 comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3).

[0273] In another preferred embodiment, at least one antigen binding site that specifically binds to CDH3 comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3).

[0274] In another preferred embodiment, at least one antigen binding site that specifically binds to CDH3 comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

[0275] 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 sequence 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 sequence of SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2) and SEQ ID NO:79 (CDR3).

[0276] Thus, in a specific aspect, the binding molecules described herein comprise: one antigen binding site that specifically binds to CDH3, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence 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, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2) and SEQ ID NO: 79 (LCDR3).

[0277] In another specific aspect, the binding molecules described herein comprise: one antigen binding site that specifically binds to CDH3, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (LCDR1), SEQ ID NO: 18 (LCDR2) and SEQ ID NO: 19 (LCDR3); and at least one, preferably two, antigen binding sites that specifically bind to TRAILR2, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 77 (LCDR1), SEQ ID NO: 78 (LCDR2) and SEQ ID NO: 79 (LCDR3).

[0278] In another specific aspect, the binding molecules described herein comprise: one antigen binding site that specifically binds to CDH3, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO:27 (LCDR1), SEQ ID NO:28 (LCDR2) and SEQ ID NO:29 (LCDR3); and at least one, preferably two, antigen binding sites that specifically bind to TRAILR2, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO:77 (LCDR1), SEQ ID NO:78 (LCDR2) and SEQ ID NO:79 (LCDR3).

[0279] In yet another specific aspect, the binding molecules described herein comprise: one antigen binding site that specifically binds to CDH3, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO:37 (LCDR1), SEQ ID NO:38 (LCDR2) and SEQ ID NO:39 (LCDR3); and at least one, preferably two, antigen binding sites that specifically bind to TRAILR2, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO:77 (LCDR1), SEQ ID NO:78 (LCDR2) and SEQ ID NO:79 (LCDR3).

[0280] The CDRs disclosed herein and depicted in SEQ ID No above are presented according to Kabat nomenclature and are shown below in Table 1. As used herein, HCDR stands for heavy chain CDR and LCDR stands for light chain CDR.

[0281] Since additional nomenclature is known in the art, the most commonly used CDR sequences based on these nomenclatures are also shown in Table 1 below. These numbering systems are based on

[0282] (i) CCG (Chemical Computing Group, as described by Almagro et al., Proteins 2011; 79: 3050-3066 and Maier et al., Proteins 2014; 82: 1599-1610),

[0283] (ii) Chothia (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917),

[0284] (iii) IMGT (Lefranc MP, Dev Comp Immunol. 2003Jan; 27(1):55-77) and

[0285] (iv) North (North B, J Mol Biol. (2011) 406:228-56).

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] The amino acid positions of the CDRs herein (see Table 1) are linear according to Kabat, CCG, Chothia, IMGT and North position designations, i.e., the amino acids of each full-length molecule chain are numbered consecutively starting with number 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 in length would start with number 1 at the N-terminus and would end with number 118 at the most C-terminal amino acid. Thus, any reference to, for example, position 25 means the 25th amino acid counted from the N-terminus of the molecule being referred to.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] According to the present invention, the terms "immunoglobulin heavy chain variable domain" and "immunoglobulin light chain variable domain" are used according to the definitions in the art.

[0299] Specifically, in the binding molecules of the present invention, at least one antigen binding site against CDH3 is selected from the antigen binding sites described above, while the antigen binding site against TRAILR2 is selected by a person skilled in the art from those TRAILR2-specific antigen binding sites available in the art or from those disclosed herein.

[0300] Preferably, the antigen binding site that specifically binds to TRAILR2 is a single chain variable fragment (scFv). More preferably, the scFv is configured so that the heavy chain variable domain is at its N-terminus and the light chain variable domain is at its C-terminus.

[0301] Methods for linking a polypeptide of interest (including scFv molecules) to the C-terminus of a heavy chain of, for example, an IgG molecule are well known in the art. It will be appreciated that the fusion of the scFv with the Ig molecule may be a direct fusion or may be via a linker as described above, preferably a peptide linker. In practice, the linkage is typically achieved by combining a nucleic acid molecule encoding an IgG of interest with a nucleic acid encoding a desired polypeptide (e.g., scFv), interspersed with a nucleic acid molecule encoding a linker sequence, if necessary, to form a single nucleic acid molecule comprising all three elements. This complete HC-scFv encoding nucleic acid molecule is then placed in an expression vector and introduced into an appropriate host cell so that a complete IgG heavy chain-scFv single polypeptide is formed, and optionally the same operation is performed for the IgG light chain-scFv counterpart.

[0302] 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.

[0303] Therefore, in a specific aspect, the binding molecules described herein comprise 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:2 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO:6; 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.

[0304] In another specific aspect, 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: 12 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; and at least one, preferably two, antigen binding sites that specifically bind to TRAILR2, which comprises 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.

[0305] In another specific aspect, 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:22 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO:26; 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.

[0306] In yet another specific aspect, 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.

[0307] In a preferred embodiment of the present invention, the binding molecule comprises:

[0308] 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;

[0309] 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;

[0310] 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

[0311] 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.

[0312] In specific embodiments, the binding molecules provided herein comprise:

[0313] i. a heavy chain comprising an 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 an 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;

[0314] ii. a heavy chain comprising an 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 an 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;

[0315] iii. a heavy chain comprising an 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 an 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

[0316] 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.

[0317] In a specific aspect, the binding molecules described herein therefore comprise: a heavy chain comprising an 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 an 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, which are target specific for TRAILR2 and comprise or consist of: an immunoglobulin heavy chain variable domain having an 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 an 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: NO:76 is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO.71.

[0318] In another specific aspect, the binding molecules described herein therefore comprise: a heavy chain comprising an 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 an 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, which are target specific for TRAILR2 and comprise or consist of: an immunoglobulin heavy chain variable domain having an 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 an 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: NO:76 is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO.71.

[0319] In another specific aspect, the binding molecules described herein therefore comprise: a heavy chain comprising an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence 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 an amino acid sequence of SEQ ID NO: 85, or an amino acid sequence 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 or consist of: an immunoglobulin heavy chain variable domain having an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence 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 an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: NO:76 is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO.71.

[0320] In yet another specific aspect, the binding molecules described herein therefore comprise: a heavy chain comprising an 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 an 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, which have target specificity for TRAILR2 and comprise or consist of: an immunoglobulin heavy chain variable domain having an 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 an 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: NO:76 is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to an amino acid sequence. In a preferred embodiment, at least one scFv that specifically binds to TRAILR2 comprises the amino acid sequence of SEQ ID NO.71.

[0321] In a specific embodiment, the binding molecule of the present invention comprises a modified heavy chain, preferably wherein a TRAILR2-specific scFv is fused to the C-terminus of an Ig 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.

[0322] Specifically, the binding molecule of the present invention comprises:

[0323] 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;

[0324] 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;

[0325] 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

[0326] 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.

[0327] In preferred embodiments, the binding molecules described herein therefore comprise: 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.

[0328] In another preferred embodiment, the binding molecules described herein therefore comprise: 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.

[0329] In another preferred embodiment, the binding molecules described herein therefore comprise: 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.

[0330] In another preferred embodiment, the binding molecules described herein therefore comprise: 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.

[0331] The present invention further describes 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 described in detail above, and at least one antigen binding site (second antigen binding site) that specifically binds to the extracellular domain 2 (EC2 domain) of E-cadherin-3 (CDH3).

[0332] In one aspect, the binding molecules provided herein thus bind to an antigen comprising or consisting 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.

[0333] Preferably, the at least one antigen binding site that specifically binds to the EC2 domain of CDH3 is selected from the group consisting of:

[0334] i. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2) and SEQ ID NO: 49 (CDR3);

[0335] ii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2) and SEQ ID NO: 59 (CDR3); and

[0336] iii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3).

[0337] Even more preferably, the antigen binding site that specifically binds to the EC2 domain of CDH3 is selected from the group consisting of:

[0338] i. 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;

[0339] ii. 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; and

[0340] iii. 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.

[0341] Preferably, the binding molecule described herein and having target specificity for the EC2 domain of CDH3 comprises:

[0342] 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;

[0343] 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;

[0344] 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.

[0345] In a particular aspect, the binding molecule targeting the EC2 domain of TRAILR2 and CDH3 comprises a modified heavy chain, preferably wherein a TRAILR2-specific scFv is fused to the C-terminus of the Ig 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.

[0346] Specifically, the binding molecule comprises:

[0347] 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;

[0348] 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

[0349] 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.

[0350] Monospecific antibody molecules of the invention

[0351] Further provided herein is an antibody molecule that specifically binds to CDH3 (e.g., a full-length antibody / immunoglobulin molecule containing a Y-shaped structure having two heavy chains and two light chains, or a fragment thereof (such as an Fv, Fab, Fab' or F(ab')2 fragment), a single-chain antibody, a single-chain variable fragment (scFv)). Specifically, provided herein is an antibody molecule that specifically binds to the EC1 or EC2 domain of CDH3. In some embodiments, the antibody molecule that is specific for CDH3 is a recombinant monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody molecule.

[0352] In some embodiments, an antibody molecule specific for CDH3 comprises any one of the CDR combinations shown in (i) to (vii) below:

[0353] i. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3);

[0354] ii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3);

[0355] iii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3);

[0356] iv. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3);

[0357] v. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2) and SEQ ID NO: 49 (CDR3);

[0358] vi. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2) and SEQ ID NO: 59 (CDR3); or

[0359] vii. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3).

[0360] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3).

[0361] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3).

[0362] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3).

[0363] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

[0364] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2) and SEQ ID NO: 49 (CDR3).

[0365] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2) and SEQ ID NO: 59 (CDR3).

[0366] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3).

[0367] In some embodiments, the antibody molecule specific for CDH3 comprises:

[0368] i. a VH comprising the amino acid sequence of SEQ ID NO: 2 and a VL comprising the amino acid sequence of SEQ ID NO: 6;

[0369] 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;

[0370] 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;

[0371] 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;

[0372] 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;

[0373] 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

[0374] 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.

[0375] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0376] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0377] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0378] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0379] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0380] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0381] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, comprising 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.

[0382] In some embodiments, the CDH3-specific antibody as defined above further comprises a human heavy chain constant domain (eg, an IgG constant domain) and a human light chain constant domain (eg, a kappa or lambda light chain constant domain).

[0383] In a specific embodiment of an antibody molecule specific for CDH3, the antibody molecule comprises:

[0384] 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;

[0385] 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;

[0386] 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;

[0387] 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;

[0388] 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;

[0389] 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;

[0390] 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.

[0391] 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: 80 and a light chain comprising the amino acid sequence of SEQ ID NO: 81.

[0392] 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: 82 and a light chain comprising the amino acid sequence of SEQ ID NO: 83.

[0393] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which 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.

[0394] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which 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.

[0395] 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.

[0396] Specifically, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 91.

[0397] Specifically, provided herein is an antibody or an antigen-binding fragment thereof that specifically binds to CDH3, which comprises 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.

[0398] The CDH3-specific antibodies provided herein can be used to label, locate, identify or target cells expressing CDH3 in vitro, in vivo or ex vivo (e.g., in ELISA assays, FACS assays, immunohistology or the like) by attaching dyes, drugs or another molecule with binding specificity to different antigens. In some embodiments, CDH3-specific antibodies specifically bind to the surface of CDH3-expressing cells and are used to locate and / or identify such cells. In some embodiments, the CDH3 antibodies provided herein are used to identify cells expressing CDH3 (e.g., tumor cells). In some embodiments, the CDH3 antibodies provided herein are used to deliver drugs or cytotoxic agents to target cells (e.g., tumor cells expressing CDH3) by attaching such drugs or cytotoxic agents to the CDH3 antibodies, thereby, for example, killing the target cells.

[0399] The present invention also provides a method for detecting E-cadherin-3 (CDH3) in a sample, the method comprising the following steps:

[0400] (a) contacting the sample with an anti-CDH3 antibody molecule as defined above;

[0401] (b) allowing an antibody-antigen complex to form in the sample; and

[0402] (c) Detecting the anti-CDH3 antibody.

[0403] Means and methods for detecting antibodies are well known in the art and include, for example, immunohistochemistry, immunoblotting, and ELISA.

[0404] Methods for identifying whether a particular tumor expresses TRAILR2 and / or CDH3 are well known in the art. For example, immunohistochemistry can be used to determine whether tumor tissue expresses TRAILR2 and / or CDH3 (eg, using the TRAILR2 and / or CDH3 antibody molecules described herein).

[0405] The present invention further provides a kit for detecting E-cadherin-3 (CDH3), wherein the kit comprises an anti-CDH3 antibody molecule as defined above and instructions for use.

[0406] Further provided herein is a kit for detecting E-cadherin-3 (CDH3) and TRAILR2, wherein the kit comprises an anti-CDH3 antibody molecule, preferably as defined above, and an anti-TRAILR2 antibody molecule, and instructions for use.

[0407] Nucleic acid molecules, expression vectors and host cells of the present invention

[0408] The present invention further relates to a nucleic acid molecule encoding a bispecific binding molecule or antibody molecule described herein or a portion thereof. The present invention further encompasses a set of nucleic acid molecules encoding a bispecific binding molecule or antibody molecule described herein.

[0409] According to the invention, the nucleic acid molecule "encodes" a binding molecule of the invention or a part thereof, which means that the nucleic acid molecule is provided in an expressible form, i.e. in a form that ensures that the binding molecule of the invention (or a respective part thereof) can be expressed thereby.

[0410] In some embodiments, the binding molecules of the present invention or the antibody molecules of the present invention comprise antibody heavy chain and / or light chain polypeptides. As those skilled in the art will appreciate, nucleic acid molecules encoding heavy chain polypeptides, light chain polypeptides or heavy chain polypeptides and light chain polypeptides can be easily prepared.

[0411] The term "a part thereof" reflects the following fact: as understood by those skilled in the art, not all elements of the binding molecule of the present invention need to be encoded on a single nucleic acid molecule. On the contrary, two or more nucleic acid molecules can be relied on to encode certain parts of the binding molecule of the present invention respectively. Therefore, the present invention also encompasses a group of isolated nucleic acid molecules, wherein the group encodes all parts of the binding molecule of the present invention together, so that the expression of this group of isolated nucleic acid molecules leads to the production of the complete binding molecule of the present invention. In other words, one or more nucleic acid molecules are provided herein, which encode the individual polypeptide chains of the binding molecule of the present invention on individual nucleic acid molecules respectively, including heavy chain, light chain, scFv and combinations thereof, or are combined in one nucleic acid molecule.

[0412] Preferably, the nucleic acid molecule is a DNA molecule comprising a coding sequence. More preferably, the DNA molecule further comprises a regulatory sequence, and optionally a natural or artificial intron (such as a β-globin intron from Homo sapiens with an embedded miRNA-557 expression cassette). It may have its original codon or may have an optimized codon usage, which is particularly suitable for expression in an intended host cell or host organism. Such nucleic acid molecules of the present invention can be easily prepared or obtained by a person skilled in the art by methods known per se, such as by automated DNA synthesis, separation from natural sources and / or recombinant DNA technology, based on the information given herein about the amino acid sequence of the binding molecule of the present invention.

[0413] Nucleic acid molecules of the present invention include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the sequence table. In addition, the present invention encompasses nucleic acid molecules complementary to the DNA molecules defined above, and nucleic acid molecules hybridized thereto under highly stringent binding and washing conditions as defined in WO 2007 / 042309. Preferred molecules (from the mRNA point of view) are those molecules having at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein. For example, if the purpose is to express the bispecific binding molecules or antibody molecules of the present invention in eukaryotic cells, the DNA sequence will have to be designed to match the codon usage in eukaryotic cells. If antibodies need to be expressed in Escherichia coli (E. coli) or other prokaryotic systems, these sequences will have to be designed to match the codon usage in Escherichia coli or the respective prokaryotic systems. Variants of the DNA molecules of the present invention can be constructed in several different ways, as described in, for example, WO 2007 / 042309.

[0414] Preferably, the one or more nucleic acids are isolated, the term "isolated" being further defined above.

[0415] The present invention further relates to an expression vector comprising one or more nucleic acid molecules of the present invention.

[0416] To produce a binding molecule or antibody of the invention, a DNA molecule encoding a binding molecule or antibody molecule described herein, or a portion thereof, is inserted into an expression vector such that the sequence is operably linked to transcriptional and translational control sequences.

[0417] To produce the binding molecules or antibodies of the invention, one skilled in the art may choose from a wide 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.

[0418] According to the present invention, the vector is an expression vector, i.e. a vector that can express 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). Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes and the like. Expression vectors and expression control sequences that are compatible with the host cell are usually selected. The expression vector generally comprises at least one nucleic acid molecule of the present invention operably linked to one or more suitable regulatory components (such as one or more promoters, one or more enhancers, one or more terminators and the like). Specific examples of such regulatory components and other components suitable for expressing the polypeptide of the present invention or required for expressing the polypeptide of the present invention, such as integration factors, selection markers, signals or leader sequences, reporter genes and the like are disclosed, for example, on pages 131 to 133 of WO2006 / 040153.

[0419] Non-limiting examples of promoter sequences (exemplified for expression in mammalian cells) are promoters and / or enhancers derived from CMV (such as the CMV promoter / enhancer of human cytomegalovirus or the CMV simian virus 40 (SV40) promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma virus, and strong mammalian promoters, such as the natural immunoglobulin and actin promoters. Examples of polyadenylation signals are hamster growth hormone or bovine growth hormone poly A, SV40 late or early poly A; alternatively, the 3′UTR of an immunoglobulin gene may be used, etc.

[0420] The recombinant expression vector may also carry 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 (such as a β-lactamase gene that confers ampicillin resistance to amplify plasmids in E. coli). The recombinant expression vector may also encode a signal peptide that promotes secretion of the resulting polypeptide. The nucleic acid molecules encoding the respective polypeptide chains may be cloned into the vector so that the signal peptide is linked 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 from a non-immunoglobulin. Alternatively, the DNA sequence encoding the full-length chain of the protein of the present invention may already contain a signal peptide sequence.

[0421] As noted above, the coding sequence inserted into the vector can be synthesized, for example, by standard methods, or isolated from natural sources, or produced in a semi-synthetic manner, i.e., by combinatorial chemical synthesis and recombinant technology. The coding sequence can be joined to the transcriptional regulatory element and / or other amino acid coding sequence using established methods. A commonly used approach is, for example, to use a vector encoding a functionally complete human CH (constant weight) immunoglobulin sequence, wherein appropriate restriction sites are engineered to make it easy to insert and express any antigen binding site, such as a single-chain Fab sequence or any heavy chain / light chain variable domain. For the antibody heavy chain, it can be, but is not limited to, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.

[0422] In those cases where more than one nucleic acid molecule is needed to constitute the binding molecule of the present invention, these more than one nucleic acid molecule can be inserted into different expression vectors or inserted into the same expression vector. In the latter case, they can be under the control of the same regulatory element (such as a promoter, enhancer, terminator and the like), or they can each have their own set of regulatory elements. According to the present invention, it is particularly preferred that in those cases where more than one nucleic acid molecule encodes the individual elements of the binding molecule of the present invention, all individual nucleic acid molecules required to form the binding molecule of the present invention are present on a single expression vector, and preferably, each nucleic acid molecule has its own set of regulatory elements.

[0423] 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 transfer by viral vectors.

[0424] Therefore, the present invention also relates to a host cell transfected with the expression vector of the present invention.

[0425] Host cells can be any suitable cells 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, humans, mice, rats, monkeys, and rodent cell lines. Specific mammalian cell lines that can be used as host cells for expression are well known in the art and particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., Hep G2 and A-549 cells), 3T3 cells, or derivatives / progeny of any such cell lines. Suitable culture media and conditions for the host cells described above are known in the art.

[0426] Methods of manufacturing and preparation

[0427] To produce the antibodies of the present invention, those skilled in the art can select from various methods well known in the art.

[0428] For the production of antibodies comprising two complete heavy chains and two complete light chains, such as those of the IgG1 or IgG4 class, see Norderhaug et al., J Immunol Methods 1997, 204(1):77-87; Kipriyanow and LeGall, Molecular Biotechnology 26:39-60, 2004; Shukla et al., 2007, J. Chromatography B, 848(1):28-39.

[0429] Fab molecules can be produced by expressing nucleic acids encoding such constructs in host cells such as E. coli, Pichia pastoris, or mammalian cell lines (e.g., CHO or NSO). Methods that allow these chains to correctly fold, associate, and disulfide bond to synthesize functional Fab molecules comprising a Fd fragment and a 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).

[0430] Also known are methods for making scFv antibodies by recombinantly expressing a nucleic acid encoding a scFv construct in a host cell such as E. coli, Pichia pastoris, or a mammalian cell line such as CHO or NS0, thereby producing a functional scFv molecule (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).

[0431] Specifically, the present invention provides a method for producing a binding molecule or antibody molecule described herein, the method comprising the following steps:

[0432] (a) culturing a host cell of the invention under conditions that allow expression of a binding molecule of the invention;

[0433] (b) optionally recovering the molecule; and optionally,

[0434] (c) further purifying and / or modifying and / or formulating the binding molecule.

[0435] The protein of the present invention is produced by culturing host cells for a period of time sufficient to allow the host cells to express the protein.

[0436] Conditions suitable for cultivating prokaryotic or eukaryotic hosts are well known to those skilled in the art. In order to improve the productivity and solubility of the expression product, the culture medium may be buffered or supplemented with a suitable additive known to enhance or promote both. Generally speaking, those skilled in the art also know that these conditions may have to be suitable for the needs of the host and the requirements of the expressed molecule. In the case of nucleic acid molecules of the present invention in a vector present in an inducible promoter-controlled host cell, the expression of the molecule of interest can be induced by adding a suitable inducing agent. Suitable expression schemes and strategies are known to those skilled in the art.

[0437] Subsequently, the binding molecules of the present invention are recovered and further purified if necessary. Preferably, they are recovered from the culture medium as secretory molecules. However, if, for example, expressed in the absence of a secretory signal, they can also be recovered from the host cell lysate. It should be understood that the term "recovering the molecule" refers to separating the binding molecules of the present invention encoded by the nucleic acid molecules of the present invention, i.e., the binding molecules present in the host cell of the present invention due to the host cell being transformed or transfected by the nucleic acid molecules of the present invention or the vector.

[0438] The optionally selected steps of purifying the binding molecules of the present invention further help to obtain substantially homogeneous preparations of the molecules. Means and methods for purifying molecules of interest are well known, and those skilled in the art can, for example, use standard protein purification methods for recombinant proteins and host cell proteins and adjust them in a manner suitable for the respective molecules. For example, current state-of-the-art purification methods suitable for obtaining the binding molecules of the present invention include removing cells and / or granular cell fragments from culture medium or lysate as a first step, followed by purification from contaminating soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, Sephadex chromatography, silica or cation exchange resins.

[0439] As a final optional step in the method of obtaining the binding molecules of the present invention, the purified protein molecules can be dried, for example lyophilized, as described below for therapeutic applications, or formulated in other ways as desired. In addition, the resulting binding molecules of the present invention can be subjected to further modification, for example to remove undesirable post-translational modifications and the like.

[0440] Pharmaceutical composition and medical use of the binding molecule or pharmaceutical composition of the present invention

[0441] The present invention further relates to a pharmaceutical composition comprising or consisting of one or more binding molecules or antibody molecules described herein. In one embodiment, the one or more binding molecules are the only one or more pharmaceutically active agents. In an alternative embodiment, in addition to the one or more binding molecules, the composition comprises one or more other pharmaceutically active agents, such as further defined below.

[0442] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention comprises the compounds described above, alone or in combination. It may optionally comprise other molecules capable of altering the characteristics of the compounds of the present invention, for example stabilizing, regulating and / or activating their function. The composition may be in solid, liquid or gaseous form, and in particular may be in powder form, such as a lyophilized powder, one or more solutions, one or more tablets or an aerosol. Preferably, the composition is a lyophilized powder or a solution.

[0443] For use in therapy, the bispecific binding molecules described herein are formulated into a pharmaceutical composition form suitable for promoting administration to animals or humans. Therefore, the pharmaceutical composition of the present invention preferably also comprises a pharmaceutically acceptable carrier. Compositions comprising such carriers can be prepared by well-known conventional methods. Generally, pharmaceutical compositions comprising the binding molecules of the present invention can be prepared by mixing the binding molecules with such pharmaceutically acceptable carriers and (optionally) excipients or stabilizers. "Pharmaceutically acceptable carrier" means any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulation material or formulation aid. In addition, other excipients, regulators or stabilizers are non-toxic at the dosage and concentration used. Pharmaceutically acceptable carriers, excipients, modifiers and stabilizers include, but are not limited to: buffer systems such as phosphate, citrate, acetate and other inorganic or organic acids and salts thereof; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; alkyldimethylbenzyl ammonium chloride, benzathonine chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, oligosaccharides or polysaccharides and other carbohydrates, including glucose, mannose, sucrose, trehalose, dextrin or polydextrose; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or ionic or non-ionic surfactants such as TWEEN TM (Polysorbate), PLURONICS TM or fatty acid esters, fatty acid ethers or sugar esters. In addition, organic solvents may be contained in the formulation, such as ethanol or isopropanol. Excipients may also have the function of modifying release or modifying absorption.

[0444] Typically, aqueous solutions or suspensions will be preferred. In general, suitable formulations for therapeutic proteins (such as binding molecules of the present invention) are buffered protein solutions, such as solutions of proteins comprising a suitable concentration (such as 0.001 to 400 mg / ml, preferably 0.005 to 200 mg / ml, more preferably 0.01 to 200 mg / ml, more preferably 1.0-100 mg / ml, such as 1.0 to 40.0 mg / ml, preferably 10.0 to 30 mg / ml, most preferably 30 mg / ml (iv administration) or 100 mg / ml (sc administration)), and aqueous buffers such as the following:

[0445] - Phosphate buffered saline, pH 7.4,

[0446] -Other phosphate buffers, pH 6.2 to 8.2,

[0447] - Acetate buffer, pH 3.2 to 7.5, preferably pH 4.8 to 5.5

[0448] - Histidine buffer, pH 5.0 to 7.0, preferably pH 5.3 to 5.5

[0449] - Succinate buffer, pH 3.2 to 6.6, or

[0450] - Citrate buffer, pH 2.1 to 6.2,

[0451] and optionally, salts (e.g. NaCl) and / or stabilizers (such as sucrose, trehalose, lysine) and / or other polyols (such as mannitol and glycerol) for providing isotonicity of the solution, and optionally detergents, for example to prevent aggregation (e.g. 0.02% Tween-20 or Tween-80).

[0452] A preferred buffered protein solution for intravenous administration is one comprising about 10 mg / ml of a binding molecule of the invention dissolved in 10 mM citrate buffer pH 5.5, 207 mM sucrose, 25 mM lysine HCl and 0.02% polysorbate 20.

[0453] A particularly preferred buffered protein solution for intravenous administration is one comprising about 30 mg / ml of the binding molecule of the invention dissolved in about 20 mM His / His-HCl buffer pH 5.3 to 5.5 (preferably pH 5.3), 220 mM sucrose and 0.02% polysorbate 20 and water for injection (WFI).

[0454] Formulations for subcutaneous application may include significantly higher concentrations, such as up to 100 mg / ml or even greater than 100 mg / ml of the antibodies of the invention. However, it will be clear to those skilled in the art that the ingredients and amounts thereof as given above represent only one preferred choice. Alternatives and variants thereof will be directly apparent to those skilled in the art, or can be easily conceived starting from the above disclosure.

[0455] The pharmaceutical compositions of the present invention can be administered to an individual using any suitable mode of administration, including, for example, parenteral administration by infusion or injection (intravenous, intraarticular, intramuscular, subcutaneous, intrasternal, intraperitoneal, intradermal), as well as transdermal, intranasal, buccal or oral administration or administration by inhalation. For administration of solutions or reconstituted lyophilized powders, parenteral modes of administration are preferred.

[0456] In general, in order to treat, prevent and / or alleviate the diseases, disorders and conditions mentioned herein and depending on the specific disease, disorder or condition to be treated, the effectiveness of the specific binding molecules of the present invention to be used, the specific route of administration and the specific drug formulation or composition used have an impact on the actual dose to be administered. In addition, the actual pharmaceutically effective amount or therapeutic dose will also depend on factors known to those skilled in the art, such as the age and weight of the patient. In any case, the binding molecules of the present invention or the pharmaceutical composition of the present invention will be based on the unique condition of the patient to allow the delivery of the dosage and mode of the pharmaceutically effective amount. Preferably, the binding molecules of the present invention or the pharmaceutical composition of the present invention will be used with an amount between 0.005 and 20.0 mg per kilogram of body weight and dosage, preferably between 0.05 and 10.0 mg / kg / dosage, and more preferably between 0.5 and 5 mg / kg / dosage, continuously (e.g., by infusion) or more preferably as a single dose. The interval for administration can be, for example, twice a week, once a week or once a month, but can vary significantly, especially depending on the parameters mentioned above. Therefore, in some cases, it may be sufficient to use less than the minimum dose given above, while in other cases the upper limit may have to be exceeded. When a larger amount is administered, it may be desirable to divide it into multiple smaller doses in one day. Preferably, once a week, with a dosage range between 0.005 and 20.0 mg per kilogram of body weight and dosage, preferably between 0.05 and 10.0 mg / kg / dose, and more preferably between 0.5 and 5 mg / kg / dose.

[0457] The efficacy of the binding molecules of the present invention and compositions comprising them can be tested using any suitable in vitro assay, cell-based assay, in vivo assay known per se and / or animal model, or any combination thereof, depending on the specific disease involved. Suitable assays and animal models will be clear to those skilled in the art, and include, for example, the assays and animal models used in the examples below.

[0458] The binding molecules of the invention or the pharmaceutical compositions of the invention may be used independently or in combination with other pharmacologically active ingredients, such as current state-of-the-art or standard of care compounds, such as cytostatic or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immunomodulators / checkpoint inhibitors and the like.

[0459] Therefore, another aspect of the present invention provides a binding molecule of the present invention, or a pharmaceutical composition comprising a binding molecule of the present invention and one or more other active ingredients and optionally a pharmaceutically acceptable carrier.

[0460] Without wishing to be limited thereto, cytostatic and / or cytotoxic active substances which can be administered as combination partners according to the invention include hormones, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors (growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (fFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (eEGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, e.g. HER2, HER3, HER4) and hepatocyte growth factor (HGF)), inhibitors such as (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 (e.g., antifolates (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 (arabinoside) C), fludarabine); antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustin, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas (such as carmustin and lomustin), thiotepa); antimitotic agents (e.g., vinca alkaloids, such as vinblastine, vindesin, vinorelbin, and vincristine);and taxanes, such as paclitaxel and docetaxel); angiogenesis inhibitors, including bevacizumab, ramucirumab and aflibercept, tubulin inhibitors;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors), inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (such as volasertib), CDK inhibitors (including CDK9 inhibitors), Aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors, MCL-1 inhibitors, c-FLIP inhibitors, or KRAS inhibitors (such as KRAS G12c, KRAS G12D, or KRAS G13d)), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-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 inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals (including but not limited to systemic targeted biological agents), immunotherapeutics (such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3 and TIM3 binding molecules / immunoglobulins, such as ipilimumab, nivolumab, pembrolizumab), interferon, interferon α or rituximab; oncolytic viruses;Anticancer vaccines and various chemotherapeutic agents (such as amifostin, anagrelid, clodronat, filgrastin, leucovorin, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer); proteasome inhibitors (such as bortezomib); Smac and BH3 mimetics; agents that restore p53 functionality (including mdm2-p53 antagonists); inhibitors of the Wnt / β-catenin signaling pathway; matrix modulators (such as (preferably bispecific) molecules targeting CD137 and FAP); and / or cyclin-dependent kinase 9 inhibitors.;

[0461] According to the present invention, treatment with a binding molecule of the present invention or a pharmaceutical composition of the present invention in combination with a drug selected from the group consisting of:

[0462] (i) anti-VEGF antibodies (bevacizumab and other anti-angiogenic substances), e.g., with or without chemotherapy combinations (including doxorubicin / cyclophosphamide combinations and / or capecitabine / docetaxel combinations in the neoadjuvant setting; taxane / platinum regimens for first-line and subsequent lines of treatment), especially in breast cancer patients;

[0463] (ii) chemotherapeutic agents (including 5-fluorouracil (5-FU), irinotecan, oxaliplatin and TAS-102);

[0464] (iii) anti-EGFR antibodies (cetuximab and panitumumab in KRAS wild-type tumors), for example, with or without chemotherapy combination (including irinotecan), anti-VEGF antibody combination (bevacizumab and other anti-angiogenic substances), or regorafenib combination for the treatment of CRC patients; and / or

[0465] (iv) EGFR inhibitors such as gefitinib, afatinib, nintedanib, lapatinib, erlotinib, asimertinib; and / or

[0466] (v) ALK inhibitors; and / or

[0467] (vi) ROS1 inhibitors; and / or

[0468] (vii) immunotherapeutic agents, including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents, such as ezabenlimab, pembrolizumab and nivolumab, and other antibodies, as disclosed in WO2017 / 198741; and / or

[0469] (viii) Matrix modulators, such as (preferably bispecific) molecules targeting CD137 and FAP.

[0470] In a particularly preferred embodiment, the binding molecule of the present invention or the pharmaceutical composition of the present invention is used in combination with an immune checkpoint inhibitor, preferably in combination with a PD-1 antagonist, such as an anti-PD-1 antibody or an anti-PDL-1 antibody for treating cancer. 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 WO2017 / 198741 (incorporated herein by reference), more preferably, the anti-PD-1 antibody is Ebenlimab. Preferably, the anti-PDL-1 antibody is selected from the group consisting of: atezolizumab, avelumab and durvalumab. Specifically, such therapies can be combined with chemotherapeutic agents such as cisplatin, carboplatin or gemcitabine.

[0471] In another preferred embodiment, the binding molecules or pharmaceutical compositions described herein are used to treat cancer in combination with a single chemotherapeutic agent such as cisplatin, carboplatin, paclitaxel, docetaxel, 5-FU, methotrexate, cetuximab, capecitabine, afatinib, irinotecan, oxaliplatin, gemcitabine, paclitaxel, docetaxel, capecitabine, pemetrexed, or cetuximab.

[0472] In particularly preferred embodiments, the binding molecules or pharmaceutical compositions described herein are used to treat cancer in combination with a combination regimen selected from the group consisting of:

[0473] - Cetuximab, platinum (cisplatin or carboplatin), and 5-FU;

[0474] - Cisplatin and cetuximab;

[0475] - Cisplatin or carboplatin in combination with docetaxel or paclitaxel;

[0476] - Cisplatin and 5-FU;

[0477] - Cisplatin or carboplatin in combination with docetaxel and cetuximab;

[0478] - Cisplatin or carboplatin in combination with paclitaxel and cetuximab;

[0479] - pembrolizumab, platinum (cisplatin or carboplatin), and paclitaxel;

[0480] - Pembrolizumab, platinum (cisplatin or carboplatin), and docetaxel;

[0481] -Cisplatin and etoposide;

[0482] - Carboplatin and etoposide; and

[0483] - Cyclophosphamide in combination with doxorubicin and vincristine.

[0484] In a preferred embodiment, the cancer to be treated with the binding molecules described herein is gastric cancer or esophageal adenocarcinoma, and the treatment method comprises a combination of the binding molecules described herein and a chemotherapeutic agent selected from the group consisting of irinotecan, oxaliplatin, paclitaxel and capecitabine.

[0485] In another preferred embodiment, the cancer to be treated with the binding molecules described herein is pancreatic ductal adenocarcinoma (PDAC), and the treatment method comprises a combination of the binding molecules described herein and a chemotherapeutic agent selected from the group consisting of irinotecan, gemcitabine, paclitaxel and capecitabine.

[0486] In another preferred embodiment, the cancer to be treated with the binding molecules described herein is lung adenocarcinoma, and the method of treatment comprises a combination of the binding molecules 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 a binding molecule of the invention, oxaliplatin and cisplatin.

[0487] In another preferred embodiment, the cancer to be treated with the binding molecules described herein is lung squamous cell carcinoma (lung SCC), and the treatment method comprises a combination of the binding molecules 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 a binding molecule of the invention, oxaliplatin and cisplatin.

[0488] In another preferred embodiment, the cancer to be treated with the binding molecules described herein is squamous cell carcinoma of the head and neck (SCCHN or HNSCC), and the treatment method comprises a combination of the binding molecules described herein and a chemotherapeutic agent selected from the group consisting of oxaliplatin, paclitaxel, docetaxel and cetuximab.

[0489] In another embodiment, the binding molecules or pharmaceutical compositions described herein are used in combination with radiation therapy to treat cancer.

[0490] The present invention further relates to a binding molecule of the present invention or a pharmaceutical composition of the present invention for use in a medical bed. The present invention further relates to a binding molecule of the present invention or a pharmaceutical composition of the present invention for use in the preparation of a medicament.

[0491] In addition, the present invention also relates to the binding molecule of the present invention or the pharmaceutical composition of the present invention, which is used in a method for treating, improving or preventing cancer. The present invention further relates to a method for treating, preventing or improving cancer, which comprises administering a therapeutically effective amount of the binding molecule of the present invention or the pharmaceutical composition of the present invention to a patient in need.

[0492] A "therapeutically effective amount" of a molecule to be administered is the minimum amount necessary to prevent, ameliorate or treat clinical symptoms of cancer, particularly the minimum amount that is effective for the particular cancer being treated.

[0493] As used herein, the term "cancer" is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs (regardless of histopathological type or stage of invasion). Thus, all cancers, tumors, neoplasms, etc. mentioned below that are characterized by a specific location / origin in the body are meant to include both the primary tumor and metastatic tumors derived therefrom.

[0494] Cancers, tumors, and other proliferative diseases whose growth can be inhibited using the multispecific binding molecules described herein are any tumor expressing TRAILR2 / CDH3, including but not limited to: head and neck cancer, preferably HNSCC; lung cancer; preferably NSCLC; pancreatic cancer; cervical cancer; ovarian cancer; endometrial cancer; breast cancer, preferably TNBC; liver cancer (hepatoblastoma or hepatocellular carcinoma); prostate cancer; gastric sarcoma; gastrointestinal stromal tumor, esophageal cancer; colon cancer; colorectal cancer; kidney cancer; skin cancer; or gastrointestinal cancer. Gastrointestinal cancer includes but is not limited to esophageal cancer (e.g., gastroesophageal junction cancer), gastric (gastric) cancer, hepatocellular carcinoma, biliary cancer (e.g., bile duct cancer), gallbladder cancer, pancreatic cancer, or colorectal cancer (CRC).

[0495] In a preferred embodiment of the binding molecule or pharmaceutical composition for use according to the present invention, or the method for treating, preventing or ameliorating cancer of the present invention, or the use of the present invention, the cancer is lung cancer, specifically lung adenocarcinoma, lung squamous cell carcinoma (lung SCC) and non-small cell lung cancer (NSCLC); head and neck cancer, specifically head and neck squamous cell carcinoma (HNSCC); pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC); breast cancer, specifically triple-negative breast cancer (TNBC); gastric cancer (GC); ovarian cancer; endometrial cancer or esophageal cancer.

[0496] In particularly preferred embodiments of the invention, the cancer is pancreatic cancer, non-small cell lung cancer (NSCLC) or head and neck squamous cell carcinoma (HNSCC).

[0497] Pancreatic cancer (PAC) is a malignant disease that causes >400,000 deaths worldwide each year. It is one of the most common causes of cancer-related deaths in industrialized countries. Despite therapeutic interventions such as surgery and chemotherapy, pancreatic cancer, which accounts for about 90% of all pancreatic cancer cases, usually has an extremely poor prognosis, with approximately 25% of people surviving one year and only 5% of patients surviving five years. Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplastic disease, accounting for more than 90% of all pancreatic malignancies.

[0498] Lung cancer is the leading cause of cancer-related death in the United States. From 2011 to 2017, the 5-year relative survival rate of lung cancer patients was 22%. NSCLC is any type of epithelial lung cancer except small cell lung cancer (SCLC). The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma and adenocarcinoma, but there are several other types that are cancers with lower frequency of occurrence. In terms of category, NSCLC is generally less sensitive to chemotherapy and radiation therapy than SCLC. Patients with resectable disease can be cured by surgery, or chemotherapy followed by surgery. However, a large number of patients suffer from unresectable disease, and radiation therapy can be used to achieve local control, but cure is only seen in a few patients. Therefore, other therapeutic agents are very much needed to treat this disease.

[0499] Most head and neck cancers originate from the mucosal epithelium in the oral cavity, pharynx, and larynx, and are collectively referred to as head and neck squamous cell carcinomas (HNSCC). Oral and laryngeal cancers are generally associated with tobacco use, alcohol abuse, or both, while pharyngeal cancer is increasingly attributed to infection with the human papillomavirus (HPV), primarily HPV-16. Head and neck cancers may also begin in the salivary glands, sinuses, or muscles or nerves in the head and neck, but these types of cancers are much less common than squamous cell carcinomas.

[0500] Oral HNSCC is generally treated with surgical resection followed by adjuvant radiation or chemotherapy plus radiation (known as chemoradiation or chemoradiotherapy (CRT)), depending on the stage of the disease. With the exception of early-stage oral cancer, which is treated with surgery alone, treatment for most HNSCC patients is complex and requires a multimodal approach, and therefore requires interdisciplinary care. Therefore, targeted therapeutics are urgently needed to treat HNSCC patients, and especially those in advanced stages.

[0501] Colorectal cancer (CRC) is a unique malignant disease listed in ICD-10 and is one of the leading causes of cancer morbidity and mortality worldwide. Approximately 25% of CRC patients present with significant cancer metastasis and metastatic disease develops in 40-50% of newly diagnosed patients. Although recent improvements in chemotherapy have extended the survival duration of metastatic CRC, most patients will die from their disease. Therefore, there is a huge demand for other therapeutic agents for the treatment of this disease.

[0502] It is known that approximately 30%-50% of colorectal cancers have a mutated (abnormal) KRAS gene. KRAS mutations frequently found in neoplasms include mutations at exon 2 (codons 12 and 13) and exon 3 (codon 61) and can be analyzed by tumor biopsy. They include activating mutations that result in sustained signal transduction, stimulating downstream signaling pathways involved in cell growth, proliferation, invasion, and cancer metastasis. Therefore, in one embodiment, the binding molecules of the present invention are used to treat KRAS mutant colorectal cancer (i.e., patients with KRAS mutant tumors). In an alternative embodiment, the binding molecules of the present invention are used to treat KRAS wild-type colorectal cancer (i.e., patients with KRAS wild-type tumors).

[0503] Esophageal cancer is one of the most commonly diagnosed cancers worldwide. Similar to pancreatic cancer, it is difficult to diagnose and is often diagnosed at an advanced stage, resulting in an extremely poor prognosis for this indication. As a result, it accounts for approximately 5% of cancer-related deaths, making it the sixth most common cause of cancer-related death.

[0504] With approximately 2.3 million new cases worldwide (GLOBOSCAN 2020), breast cancer is the most commonly diagnosed malignancy in women. Triple negative breast cancer (TNBC) constitutes 15-20% of breast cancer and occurs more frequently in women under 40 years of age. Due to its highly invasive nature, TNBC is accompanied by distant cancer metastasis in almost 50% of patients. Compared to other types of breast cancer, TNBC patients show a shorter survival time, with a 5-year mortality rate of approximately 40% and a mortality rate of up to 75% after disease recurrence. The endocrine or molecular targeted therapies currently used in other BC subtypes do not represent options for TNBC due to their molecular expression patterns. Due to the extremely poor efficacy of chemotherapy-based systemic TNBC treatments (such as adjuvant chemoradiotherapy after surgery), it is highly desirable to develop new treatment modalities for this disease.

[0505] Ovarian cancer represents the seventh most common type of cancer in women worldwide, and it has the highest incidence among malignant tumors in women. Among epithelial cancer types, serous ovarian carcinoma is the most common one. Ovarian cancer is diagnosed at a late stage in approximately 80% of cases and therefore usually has a poor prognosis. Especially for recurrent ovarian cancer, current treatment is characterized by high intensity, poor quality of life and low chance of cure. Therefore, new therapeutic strategies are urgently needed to treat this disease.

[0506] The present inventors above have identified that the binding molecules described herein have many utilities for targeting cancer cells and can therefore be used in the therapy of cancers expressing both TRAILR2 and CDH3. Methods for identifying whether a particular tumor expresses TRAILR2 and CDH3 are well known in the art. For example, immunohistochemistry can be used to determine whether tumor tissue expresses TRAILR2 and CDH3 (e.g., using TRAILR2 and / or CDH3 antibody molecules described herein), and therefore should be suitable for treatment with the binding molecules of the present invention.

[0507] The binding molecules of the invention can be used in treatment regimens in the context of first line, second line or any other line of treatment and maintenance therapy.

[0508] In another aspect, the binding molecules of the present invention are used in combination with a device suitable for administering the binding molecules, such as a syringe, an injection pen, a mini pump or another device. In another aspect, the binding molecules of the present invention are included in a kit of subpackaged parts, for example, also including a package insert with instructions for use of the binding molecules.

[0509] The present invention further comprises the following items:

[0510] 1. A binding molecule comprising

[0511] (a) at least one antigen binding site that specifically binds to TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2), and

[0512] (b) at least one antigen binding site that specifically binds to E-cadherin-3 (CDH3), wherein the at least one antigen binding site that specifically binds to CDH3 is selected from the group consisting of:

[0513] i. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3);

[0514] ii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3);

[0515] iii. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3); and

[0516] iv. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

[0517] 2. A binding molecule as described in item 1, wherein the at least one antigen binding site that specifically binds to TRAILR2 comprises: a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO:77 (CDR1), SEQ ID NO:78 (CDR2) and SEQ ID NO:79 (CDR3).

[0518] 3. The binding molecule of item 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.

[0519] 4. A binding molecule as described in item 3, wherein the one or more scFvs have a VL-VH orientation from N-terminus to C-terminus.

[0520] 5. The binding molecule of item 3 or 4, wherein the one or more scFvs are fused to the C-terminus of the Ig molecule, preferably wherein the first scFv is fused to the first heavy chain of the Ig molecule and the second scFv is fused to the second heavy chain, respectively.

[0521] 6. A binding molecule according to any one of items 3 to 5, wherein each of the one or more scFvs is fused to the Ig molecule via a peptide linker, preferably a peptide linker of about 4 to 20 amino acids in length.

[0522] 7. The binding molecule of any one of items 3 to 6, wherein the Ig molecule is IgG.

[0523] 8. The binding molecule of any one of items 1 to 7, wherein the antigen binding site that specifically binds to CDH3 is selected from the group consisting of:

[0524] 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;

[0525] 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;

[0526] 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

[0527] 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.

[0528] 9. The binding molecule according to any one of items 1 to 8, comprising:

[0529] 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;

[0530] 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;

[0531] 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

[0532] 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.

[0533] 10. A binding molecule as described in any one of items 1 to 9, wherein the at least one antigen binding site that specifically binds to TRAILR2 is an antigen binding site comprising a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 72 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 76.

[0534] 11. The binding molecule of claim 10, comprising

[0535] 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;

[0536] 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;

[0537] 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

[0538] 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.

[0539] 12. An antibody or antigen-binding fragment thereof that specifically binds to CDH3, comprising:

[0540] i. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3);

[0541] ii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 17 (CDR1), SEQ ID NO: 18 (CDR2) and SEQ ID NO: 19 (CDR3);

[0542] iii. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2) and SEQ ID NO: 29 (CDR3);

[0543] iv. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3);

[0544] v. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 47 (CDR1), SEQ ID NO: 48 (CDR2) and SEQ ID NO: 49 (CDR3);

[0545] vi. a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 57 (CDR1), SEQ ID NO: 58 (CDR2) and SEQ ID NO: 59 (CDR3); or

[0546] vii. A heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3).

[0547] 13. The antibody or antigen-binding fragment thereof of claim 12, comprising:

[0548] 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;

[0549] 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;

[0550] 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;

[0551] 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;

[0552] 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;

[0553] 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

[0554] 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.

[0555] 14. The antibody or antigen-binding fragment thereof of item 12 or 13, comprising:

[0556] 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;

[0557] 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;

[0558] 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;

[0559] 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;

[0560] 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;

[0561] 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

[0562] 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.

[0563] 15. The antibody or antigen-binding fragment thereof of any one of items 12 to 14, which is selected from the group consisting of chimeric antibodies, humanized antibodies and human antibodies or antibody fragments, and scFv, Fab fragments, monovalent antibody fragments and F(ab')2 fragments.

[0564] 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 thereof according to any one of items 12 to 15.

[0565] 17. An expression vector comprising the nucleic acid of item 16.

[0566] 18. A vector as described in item 17, wherein the vector is a plasmid.

[0567] 19. A vector as described in item 17, wherein the vector is a viral vector.

[0568] 20. A host cell comprising the expression vector of any one of items 17 to 19.

[0569] 21. A method for producing a binding molecule as described in any one of items 1 to 11 or an antibody or antigen-binding fragment thereof as described in any one of items 12 to 15, comprising

[0570] i. culturing the host cell of item 20 under conditions allowing expression of the molecule, and

[0571] ii. recovering the molecule, and optionally

[0572] iii. further purifying and / or modifying and / or formulating the molecule.

[0573] 22. A binding molecule according to any one of items 1 to 11 for use in medicine.

[0574] 23. A binding molecule as described in any one of items 1 to 11 for use in treating cancer.

[0575] 24. A binding molecule for use as described in claim 23, wherein the cancer is pancreatic cancer, lung cancer or head and neck cancer.

[0576] 25. A pharmaceutical composition comprising the binding molecule of any one of items 1 to 11 and a pharmaceutically acceptable carrier.

[0577] 26. A pharmaceutical composition according to claim 25, which is lyophilized, stabilized and / or formulated for administration by injection.

[0578] 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 E-cadherin-3 (CDH3).

[0579] Features and advantages of the present invention will become apparent from the following more detailed embodiments, which illustrate by way of example the principles of the invention. Example

[0580] Example 1: Prevalence of both CDH3 and TRAILR2 in selected tumor indications

[0581] Immunohistochemistry (IHC) of TRAILR2 and CDH3 was performed as follows:

[0582] Two μm sections of each FFPE block were prepared on a microtome, placed on glass slides and dewaxed. CDH3 sections were stained with primary rabbit monoclonal Abcam #ab242060 (1:50 in diluent with casein Roche #760-219) on an automated platform Ventana Discovery Ultra using program #183. DR5 IHC was performed using Ventana Discovery program number 148 with the following antibodies: rabbit anti-DR5 (cell signalling #69400S, batch: 1; 1:50 in discovery antibody diluent (Roche #760-108)). After the automated staining run, the slides were washed in distilled water in a mild detergent, then rinsed thoroughly in distilled water, placed in a 90% ethanol bath for 1 minute, then moved to three 100% ethanol baths for 1 minute, then moved to two xylene baths for 30 seconds, and finally covered with a coverslip with mounting agent. Slides were scanned (3DHistech) and submitted for analysis.

[0583] CDH3 expression was tested in non-tumor tissues including liver (n=40) by IHC, and no CDH3 expression was found (data not shown). Co-expression of TRAILR2 and CDH3 at the mRNA level was detected in several indications such as pancreatic cancer (PAC), head and neck cancer, lung cancer, colorectal cancer, esophageal cancer, triple-negative breast cancer, and bladder urothelial cancer, ovarian cancer, endometrial cancer and cervical cancer (TCGA), some of which are shown in Table 2 below.

[0584] Table 2: Prevalence of CDH3 and TRAILR2 expression in different indications (TCGA data)

[0585]

[0586] The total number of informative cases and the number and proportion (in %) of cases showing features of interest (mRNA expression, TPM ≥ 30) are indicated for each cancer type. TPM: transcripts per million reads, TR2 / CDH3: TRAILR2 and CDH3 double-positive cases, TCGA: The Cancer Genome Atlas. HNSCC: head and neck squamous cell carcinoma.

[0587] For example, Figure 2 A and Figure 2 B shows the protein expression and membrane localization associated with the mRNA expression of TRAILR2 and CDH3 in colorectal cancer.

[0588] Figure 2 C shows representative IHC images of CDH3 and TRAILR2 in tissue sections of head and neck squamous cell carcinoma and esophageal squamous cell carcinoma.

[0589] Example 2: Design and generation of binding molecules that recognize human cadherin 3 (CDH3) and human TRAIL receptor 2 (TRAILR2)

[0590] In this study, a binding molecule that binds CDH3 and TRAILR2 and induces apoptosis in cancer cells expressing both CDH3 and TRAILR2 was developed. The molecular design includes an IgG antibody specific for CDH3 and a scFv specific for TRAILR2 coupled to the C-terminus of the heavy chain. The bispecific molecule contains a flexible peptide sequence between the variable heavy chain (VH) and variable light chain (VL) domains of the scFv, and the scFv domain is connected to the IgG antibody via other series of linkers.

[0591] The binding molecule is bispecific and tetravalent. A schematic diagram of the design is shown in Figure 3 In. An example of a binding molecule of the present invention is depicted, which comprises (i) an Ig molecule that specifically binds to CDH3, comprising two heavy chains and two light chains, and (ii) two scFv molecules that specifically bind to TRAILR2. The N-terminus of the scFv is fused to the C-terminus of each heavy chain of the Ig molecule, thereby forming a symmetrical, bispecific and tetravalent antibody-like molecule.

[0592] The following examples illustrate methods for generating bispecific molecules that bind CDH3 and TRAILR2 and the biological activities of these molecules.

[0593] 2.1 Preparation of binding domains that recognize CDH3 and TRAILR2 using high-throughput V genes recovered from hybridomas and cultured single B cells

[0594] In order to prepare bispecific molecules that bind to human CDH3 and TRAILR2, it is necessary to obtain variable domains that bind to individual target antigens (anti-CDH3 and anti-TRAILR2). To achieve this goal, cloned hybridomas or single B cells from AlivaMab humanized mice (Ablexis, San Francisco, Calif., USA: AlivaMab transgenic mouse platform with human immunoglobulin loci) immunized with CDH3 or TRAILR2 were cultured in vitro. Supernatants were screened for reactivity against recombinant human CDH3 or TRAILR2 by AlphaLISA (PerkinElmer, Waltham, Mass., USA) and for reactivity against GP2d cells by flow cytometry.

[0595] Immunoglobulin (Ig) VH and VL genes were then amplified from the identified positive clones. To isolate RNA from hybridomas, approximately 2×10 6 Cells from a single clone were precipitated and used as source material. For a single B cell, 100 to 500 cells amplified from a separate B cell were used as source material. RNA was isolated using RNeasy Plus (Qiagen, Hilden, Germany). cDNA was then synthesized using Smarter cDNA Synthesis Kit (Clontech, Mount View, CA) according to the manufacturer's instructions.

[0596] To facilitate cDNA synthesis, oligodT was used to prime reverse transcription of all messenger RNAs, followed by "5' capping" with Smarter IIA oligonucleotides. Subsequent amplification of VH and VL fragments was performed using 2-step PCR amplification using a 5' primer targeting the Smarter IIA cap and a 3' primer targeting the common region in CH1. Briefly, each 50 μl PCR reaction consisted of: 20 μM forward and reverse primer mixture, 25 μl PrimeStar Max DNA polymerase premix (Clontech), 2 μl unpurified cDNA, and 21 μl double distilled H2O. The cycling program started at 94°C for 3 min, followed by 35 cycles (94°C for 30 seconds, 50°C for 1 min, 68°C for 1 min), and ended at 72°C for 7 min. A second round of PCR was performed with VL and VH second round primers containing 15 bp complementary extensions of the respective regions "overlapping" in their respective pTT5 parent vectors (VH and VL). The second round of PCR was performed with the following program: 94°C for 3 min; 35 cycles (94°C for 30 sec, 50°C for 1 min, 68°C for 1 min), and ended with 72°C for 7 min.

[0597] use The VL gene was directionally cloned into the pTT5huIgK vector using the HD cloning kit (Clontech, USA) and the VH gene was directionally cloned into the pTT5 huIgG1KO vector. HD cloning, PCR products were purified and treated with cloning enhancers before 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 the complete V gene fragment was obtained.

[0598] Using this method, large numbers of Ig VH and VL gene pairs encoding binding domains specific for CDH3 were generated.

[0599] 2.2 Confirmation and screening of recombinant antibodies

[0600] Recombinant antibodies are prepared by transient transfection of CHO-E37 cells with corresponding heavy and light chain encoding plasmids. The supernatant containing the expressed recombinant antibodies is determined by flow cytometry for the binding assay of cell lines expressing human or cynomolgus macaque CDH3. In brief, cells are incubated with recombinant supernatants, washed, and the mAb bound from the supernatant is detected with anti-human-IgG-APC (Jackson ImmunoResearch 109-136-098). The signal / background ratio (S / B) (variable region to irrelevant protein and different constant region skeletons) is calculated by dividing the median fluorescence intensity (MFI) of the sample by the median fluorescence intensity of the same heavy control. Interested clones are selected for multi-specific formatting. Multi-specific binding proteins are produced and further evaluated in mechanism and functional screening (such as cell binding, cytotoxicity and apoptotic protease activation analysis).

[0601] 2.3 Construction of bispecific molecules binding CDH3 and TRAILR2

[0602] To construct a gene segment encoding TRAILR2 scFv, the VL and VH gene pairs encoding the TRAILR2 binding variable domain (SEQ ID NO: 76 and SEQ ID NO: 72, respectively) were joined by a gene segment encoding a flexible linker having the peptide sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 232). The resulting scFv encoding gene segment was in turn cloned in frame to the 3' end of the gene encoding the heavy chain of the human IgG antibody. These coding segments were synthesized by overlapping PCR methods and cloned into the expression vector pTT5. Then, the VL and VH gene pairs encoding the CDH3 binding variable domain prepared in Example 2.1 were formatted into Figure 3 The bispecific format shown in . The VH gene was cloned as an in-frame fusion to the 5' end of the gene encoding human Igγ in the pTT5 expression vector. The gene encoding the TRAILR2 binding scFv was cloned in-frame at the 3' end of the same Igγ coding segment. 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.

[0603] Using methods known in the art, the VL and VH gene pairs encoding CDH3 binding variable domains prepared in Example 2.1 were further used to prepare antibody molecules (full-length antibody molecules comprising two light chains and two heavy chains) that specifically bind to CDH3 (and referred to as CDH3v1, CDH3v2, CDH3v3, CDH3v4, CDH3v5, CDH3v6 or CDH3v7, see Tables 11-17), and used to detect / label cells or as control antibodies in the examples described below. Subsequently, anti-TRAILR2 scFv (Table 10) was linked to the C-terminus of each heavy chain of the anti-CDH3 antibody via the linker described above, thereby generating bispecific and tetravalent CDH3 / TRAILR2 binding molecules (Tables 3-9).

[0604] Each expression vector contains a chain encoding gene (encoding signal sequence and heavy or light chain gene) eukaryotic promoter element, a prokaryotic selection marker gene (such as ampicillin) expression cassette and replication origin. These DNA plasmids are propagated in ampicillin-resistant E. coli colonies and purified.

[0605] 2.4 Expression and purification of bispecific and tetravalent molecules recognizing human TRAILR2 and human CDH3

[0606] The bispecific molecules binding to CDH3 and TRAILR2 were produced by transient transfection of CHO-E cells with pTT5 vector carrying the CDH3 / TRAILR2 chain encoding genes. Transfected CHO-E cells grown in suspension in serum-free medium were cultured in shake flasks at 37°C and 5% CO2 with agitation at 140 rpm and maintained under exponential growth conditions. On the day of transfection, cells were chemically transfected with 1 mg of light chain plasmid and 0.5 mg of heavy chain plasmid (1:3 mass ratio). Then, 1 to 2 × 10 6 CHO-E cells were inoculated into 1L FreeStyle TM CHO expression medium (Life Technologies, NY, US). The cells were then incubated for 10 to 12 days under orbital shaking, with 200 ml of commercial feed solution fed once to allow protein expression. The antibody titer in the cell culture supernatant was determined using a Pall 30 ForteBio instrument (Pall 30 ForteBio, CA, US) and protA biosensor tips.

[0607] Recombinant CDH3 / TRAILR2 binding molecules or antibodies were purified from culture supernatants in a two-step process: in the first purification step, protein A affinity chromatography was used with MabSelect TMColumn (GE Healthcare); followed by a second purification step by cation exchange chromatography using a Poros 50HS column (Applied Biosystems, Carlsbad, Calif., USA). The two-step purified material was stored in a final buffer of 50 mM sodium acetate and 100 mM NaCl, pH 5.0. The purity and degree of heterogeneity of the samples were assessed by mass spectrometry, analytical size exclusion chromatography, and analytical ultracentrifugation. The samples were confirmed to have a monomer content of ≥90% and to contain <10% impurities before functional testing.

[0608] Table 3. Amino acid sequence of the bispecific binding molecule CDH3 / TRAILR2v7

[0609]

[0610]

[0611]

[0612] Table 4. Amino acid sequences of the bispecific binding molecule CDH3 / TRAILR2v1

[0613]

[0614]

[0615] Table 5. Amino acid sequence of the bispecific binding molecule CDH3 / TRAILR2v2

[0616]

[0617]

[0618]

[0619] Table 6. Amino acid sequence of the bispecific binding molecule CDH3 / TRAILR2v3

[0620]

[0621]

[0622] Table 7. Amino acid sequence of the bispecific binding molecule CDH3 / TRAILR2v4

[0623]

[0624]

[0625]

[0626] Table 9. Amino acid sequence of the bispecific binding molecule CDH3 / TRAILR2v6

[0627]

[0628]

[0629] Table 10. Amino acid sequences of TRAILR2 scFv used in the bispecific binding molecules generated in Example 2.

[0630]

[0631] Table 11. Amino acid sequence of anti-CDH3 antibody CDH3v7

[0632]

[0633]

[0634] Table 12. Amino acid sequence of anti-CDH3 antibody CDH3v1

[0635]

[0636]

[0637] Table 13. Amino acid sequence of anti-CDH3 antibody CDH3v2

[0638]

[0639]

[0640] Table 14. Amino acid sequence of anti-CDH3 antibody CDH3v3

[0641]

[0642]

[0643] Table 15. Amino acid sequence of anti-CDH3 antibody CDH3v4

[0644]

[0645]

[0646] Table 16. Amino acid sequence of anti-CDH3 antibody CDH3v5

[0647]

[0648]

[0649] Table 17. Amino acid sequence of anti-CDH3 antibody CDH3v6

[0650]

[0651]

[0652] 2.5: Determination of the binding affinity of bispecific molecules for recombinant CDH3 based on SPR

[0653] The binding affinity of the purified CDH3 / TRAILR2 bispecific constructs for recombinant human CDH3 (full length, EC1-EC2 or EC2-EC3) was determined by surface plasmon resonance (SPR) using a ProteOn XPR36 instrument (Bio-Rad). Run washes and all dilutions (unless otherwise specified) were performed in PBS-TEDTA. GLM sensor chips (Bio-Rad) were standardized and preconditioned according to the manufacturer's recommendations. The sensor chip was activated with an equal mixture of EDC / s-NHS at a flow rate of 30 μl / min in the horizontal direction for 300 seconds and fixed with protein A / G (60 μg / ml in 10 mM acetate pH 4.5) at a flow rate of 30 μl / min in the horizontal direction for 300 seconds, generating 5000 RU of protein A / G on the surface. The sensor chip was deactivated with 1M ethanolamine hydrochloride at a flow rate of 30 μl / min in the horizontal direction for 300 seconds. The sensor chip was stabilized with 0.85% phosphoric acid at a flow rate of 100 μl / min for 18 seconds, three times in the horizontal direction and three times in the vertical direction.

[0654] To determine the binding kinetics for CDH3, the bispecific molecules were captured on the protein A / G surface for 150 seconds vertically at a flow rate of 30 μl / min. The baseline was stabilized by injecting PBS-T-EDTA horizontally for 60 seconds at a flow rate of 100 μl / min. The analyte (human full-length CDH3, CDH3[EC1-EC2] or CDH3[EC2-EC3]) was injected horizontally over the capture 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 for 18 seconds at a flow rate of 100 μl / min, once horizontally and once vertically. The data were processed by subtracting the interspot (interaction with the sensor surface) and then fitted to a 1:1 Langmuir kinetic model to obtain rate constants and affinities.

[0655] The affinities for CDH3 (full length or EC1-EC2) were in the low nM range. The binding affinities for the EC2-EC3 peptides were in the low nM range (for the EC2 mapped epitope) or below the detection level (for the EC1 mapped epitope).

[0656] The results are shown in Table 18 below, which classifies the resulting binding molecules comprising CDH3v1, CDH3v2, CDH3v3, or CDH3v7 as EC1 binders, and binding molecules comprising CDH3v4, CDHv5, or CDHv6 as EC2 binders.

[0657] Table 18: Affinity (KD) of CDH3 / TRAILR2 binding proteins for human CDH3 as determined by SPR analysis

[0658]

[0659] BDL: Below Detection Limit

[0660] Example 3: In vitro cell killing assay

[0661] The examples described above show the preparation of bispecific molecules that recognize TRAILR2 and human CDH3. Figure 3 A number of different bispecific molecules of the format depicted in order to examine whether these molecules could cause a reduction in cell viability and whether any such reduction was due to apoptosis.

[0662] 3.1 Characterization of target expression in the GP2d cell line model.

[0663] The cell line GP2d derived from human colon adenocarcinoma was selected for cell killing assay. TRAILR2 and CDH3 protein surface expression on GP2d cells was confirmed by flow cytometry as follows. Cells were detached using Versene solution (Gibco15040066) and washed twice with FACS buffer (PBS, Gibco 14190; 3% FCS, Gibco 26140; and 0.09% NaN3, Sigma Aldrich S2002). Cells were counted using ViCell (Beckman Coulter Life Sciences) and the cell number was adjusted to 2×10 6Cells / ml. After being inoculated in a 96-well round-bottom culture plate with 100 μl / well cell suspension, the culture plate was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Then, the cells were resuspended in a primary antibody diluent (1 μg / ml) and incubated at 4 ° C for 60 min. The cells were washed twice with FACS buffer, and 100 μl / well of the appropriate dilution of the secondary antibody / binding antibody was added, and incubated in the dark at 4 ° C for 45 min. After being washed twice with FACS buffer, the cells were resuspended in 100 μl FACS buffer per well and analyzed on FACS Canto (BD Biosciences). For TRAILR2 detection, a combined anti-human CD262 (DR5) PE (eBioscience, 12-9908-42) was used. For CDH3 detection, an anti-P-cadherin antibody (GeneTex GTX52961) was used, followed by a secondary anti-mouse IgG FITC. As a control, a mouse IgG isotype control was used. Figure 4 In the figure, the protein surface expression of TRAILR2 and CDH3 in GP2d cells is shown, in which both proteins show significant expression.

[0664] 3.2 Effects of bispecific CDH3 / TRAILR2 molecules on GP2d cells (in vitro 2D)

[0665] GP2d cells have been identified as cancer cell lines suitable for assessing the function of bispecific CDH3 / TRAILR2 binding molecules, and the following assays were designed. GP2d cells were plated in culture medium (RPMI1640 / Glutamax, Gibco 61870-010; plus 10% FCS, Gibco 26140). After standing overnight at 37°C and 5% CO2, the cells were incubated with 50 μl of different antibodies or binding molecule dilutions at the desired concentration during 24 hours. Then, cell viability was assessed using CellTiter-Glo luminescent cell viability from the assay (Promega G7571) according to the instructions provided by the manufacturer. Finally, luminescence was recorded using a VICTOR X4 2030 multi-label plate reader from Perkin Elmer. Two TRAILR2 binders: anti-TRAILR2 IgG, commonly known as lexatumumab (HGS-ETR2, WO2003054216A3), and anti-TRAILR2 nanobody (WO2011098520A1), were used as controls (both were synthesized in-house). The sequence source of lexatumumab was obtained from the World Health Organization, as listed in Recommended international nonproprietary names (World Health Organization. Recommended international nonproprietary names, List 57. WHO Drug Information 2007; 21: 53-83). The expression vector of the anti-TRAILR2 nanobody produced as described in WO 2011 / 098520, SEQ ID No: 032.

[0666] GP2d cells were incubated for 24 h with: (i) the bispecific molecule (CDH3 / TR2v1), (ii) anti-TRAILR2 antibody (lexatumumab) alone, (iii) anti-CDH3 antibody (anti-CDH3v1 or anti-CDH3v2) alone, or (iv) an equal combination of anti-TRAILR2 and CDH3 (CDH3v1 or CDH3v2) antibodies.

[0667] Figure 5The effects of these molecules on cell viability are shown. Two single CDH3 molecules (anti-CDH3v1 or anti-CDH3v2) have no effect on cell viability. A single TRAILR2 antibody (lexatumumab) can significantly reduce cell viability (EC50: 5.802nM), and the addition of a free combination of anti-CDH3 (anti-CDH3v1 or anti-CDH3v2) does not change the effects observed under a single anti-TRAILR2 antibody. The bispecific CDH3 / TRAILR2 binding molecule CDH3 / TRAILR2v1 described above causes a 17-fold increase in potent killing of GP2d cells (EC50: 0.337nM) compared to a single anti-TRAILR2 antibody (lexatumumab) or a combination of an anti-TRAILR2 antibody and an anti-CDH3 antibody (anti-CDH3v1 or anti-CDH3v2).

[0668] In the same assay, using GP2d cells, a number of different bispecific molecules were compared with the tetravalent anti-TRAILR2 Nanobody.

[0669] Figure 6 The potency of the four bispecific molecules (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, CDH3 / TRAILR2v7) that showed binding to TRAILR2 and the EC1 domain of CDH3 was approximately 55-fold, 11-fold, or 7-fold lower than that of the anti-TRAILR2 nanobody.

[0670] Figure 7 The potency of three bispecific molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) that bind to TRAILR2 and the EC2 domain of CDH3 was approximately 15-fold, 327-fold, or 8-fold lower than that of the anti-TRAILR2 nanobody.

[0671] 3.3 Effects of bispecific CDH3 / TRAILR2 molecules on CDH3-deficient GP2d cells (in vitro 2D)

[0672] 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. Figure 8As shown in , flow cytometry confirmed the absence of CDH3 protein on GP2d-CDH3 knockout (CDH3-KO) cells, which retained TRAILR2 protein on the surface similar to isogenic GP2d (CDH3 wild-type) cells. The same bispecific molecules previously tested on GP2d cells were tested on GP2d CDH3 KO cells.

[0673] Fig. 9 It was shown that TRAILR2 antibody alone (Lesatumumab) significantly reduced cell viability of GP2d CDH3 knockout cells (EC50: 7.192 nM), similar to the effect observed on GP2d wild-type cells (EC50: 5.802 nM; Figure 5 ), and the addition of free combination anti-CDH3 (anti-CDH3v1 or anti-CDH3v2) did not change the effect observed with anti-TRAILR2 antibody alone. In contrast, the bispecific CDH3 / TRAILR2 binding molecule CDH3 / TRAILR2v1 had only a slight effect on cell viability of GP2d CDH3 knockout cells and did not achieve 50% cell killing at the highest tested concentration (200nM).

[0674] Fig.10 The potency of anti-TRAILR2 nanobodies on GP2d CDH3 KO cells was shown to be similar to that previously detected on GP2d WT 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 significant lack of potency, and their potency was approximately 41666-fold, 12602-fold, or 21504-fold lower than that of anti-TRAILR2 nanobodies.

[0675] Fig.11 The potency of three bispecific molecules (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) that bind to TRAILR2 and the EC2 domain of CDH3 was shown to be more than 30,000-fold lower than that of the anti-TRAILR2 nanobody.

[0676] This confirms that the bispecific molecules generated from the present study induce apoptosis only in the presence of CDH3; whereas the two anti-TRAILR2 antibodies lesatumumab and the anti-TRAILR2 nanobody induce apoptosis regardless of the presence of CDH3.

[0677] Example 4: Effects of bispecific CDH3 / TRAILR2 molecules on NCI-H358 cells (in vitro 3D)

[0678] To extend the in vitro analysis, the bispecific CDH3 / TRAILR2 molecules were tested on cell lines grown as spheroids (3D culture models) to mimic the growth conditions of tumors in vivo. NCI-H358 cells derived from lung adenocarcinoma were plated in 2% Cultrex 3DRGF BME (R+D Systems 3445-005-01) in 96-well clear round-bottom ultra-low attachment microplates (Corning, 7007) at 2,000 cells / well. Cells were treated with serial dilutions of the same molecules tested on GP2d cells in Example 3 for 48 h. 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 or anti-CDH3v2), or (iv) a combination of anti-TRAILR2 (lexatumumab) and anti-CDH3 (CDH3v1 or CDH3v2) antibodies.

[0679] Fig.12 The effects of these molecules on cell viability are shown in the NCI-H358 3D model. No effect on cell viability was detected with anti-CDH3 molecules alone (anti-CDH3v1 or anti-CDH3v2), lexatumumab alone, or a combination of anti-CDH3 and lexatumumab. Similar efficacy was detected only with CDH3 / TRAILR2v1 and anti-TRAILR2 nanobodies. Fig.13 Figure 2 shows testing of CDH3 / TRAILR2 molecules that bind to EC1 (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, CDH3 / TRAILR2v3, and CDH3 / TRAILR2v7), all of which showed similar efficacy to TAS266. In contrast, CDH3 / TRAILR2 molecules that bind to EC2 (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, CDH3 / TRAILR2v6) showed reduced efficacy compared to the EC1 binder and TAS266 ( Fig.14 ).

[0680] This unexpectedly shows that bispecific molecules that bind only EC1 can induce apoptosis with similar efficacy to anti-TRAILR2 nanobodies, although with significantly increased accuracy, because only CDH3 expressing cells are targeted. In contrast, bispecific molecules CDH3 / TRAILR2v4, CDH3 / TRAILR2v5 and CDH3 / TRAILR2v6 that bind EC2 do not reach the efficacy level of anti-TRAILR2 nanobodies.

[0681] Example 5: CDH3 / TRAILR2 molecules induce caspase-8 activation in GP2d cells

[0682] Next, it was studied whether the reduction of cell viability was caused by the induction of TRAIL-induced apoptosis (which is mediated by the recruitment and activation of apoptosis protease-8). In order to determine whether the antibodies and binding molecules prepared herein can specifically activate apoptosis pathways, the inventors measured the activity of apoptosis protease-8 in the GP2d cell line. In order to set up the experiment, GP2d cells were left to stand overnight at 37°C and 5% CO2, and incubated with different antibody or binding molecule dilutions in the range of 0.001 to 10 nM on the second day. At 7h after treatment, Promega apoptosis protease-Glo 8 was used to measure apoptosis protease-8 activity from the collected cell extracts, respectively. Then, the luminescence of each sample was measured using a VICTOR X4 2030 multi-label plate reader from Perkin Elmer.

[0683] Fig.15 Shown with the bispecific CDH3 / TRAILR2 binding molecules of the present invention, anti-TRAILR2 nano antibodies or lesamumab treatment 7h, relative to the untreated control of apoptosis protease activity. Lesamumab increased activated apoptosis protease 8 only about 2 times at the highest concentration (10nM). In contrast, an increase in activated apoptosis protease 8 was detected under 0.1nM (or higher) concentrations of CDH3 / TRAILR2v1 and anti-TRAILR2 nano antibodies. The content of activated apoptosis protease 8 was similarly increased under higher concentrations of bispecific CDH3 / TRAILR2 binding molecules and anti-TRAILR2 nano antibodies, and was about 15 times higher than the background under 10nM treatment. This data confirms that the reduction in cell viability observed in the cell killing assay is not due to a nonspecific mechanism, and that the bispecific CDH3 / TRAILR2 molecules can effectively 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 caspase-8 activation compared to the anti-TRAILR2 antibody lesatumumab.

[0684] Example 6: In vivo anti-tumor activity of CDH3 / TRAILR2 molecules in the GP2d xenograft model

[0685] The in vivo efficacy of CDH3 / TRAILR2 molecules binding to three EC1 domains and binding to three EC2 domains was investigated. For this purpose, GP2d cancer cells were implanted into immunodeficient mice and the effect of administration of the molecules of the invention on tumor volume was measured. Female BomTac:NMRI-Foxn1nu mice were implanted subcutaneously with 5.0×10 6 Gp2d cells (0.1 mL 5% FBS in PBS:Matrigel at a ratio of 1:1 (v / v)) and tumor growth was monitored until it reached approximately 200 mm 3 Mice were randomly divided into seven groups and administered a single injection of vehicle control, EC1 binder (CDH3 / TRAILR2v1, CDH3 / TRAILR2v2, or CDH3 / TRAILR2v3), or EC2 binder (CDH3 / TRAILR2v4, CDH3 / TRAILR2v5, or CDH3 / TRAILR2v6) at 1 mg / kg intravenously (iv). Tumor growth was subsequently monitored for 34 days, with the exception of the vehicle-treated cohort, which was followed after 24 days when tumors reached 1000 mm 3 The animals were euthanized when the tumor volume reached the upper limit.

[0686] Fig.16 The data presented in demonstrate that the binding molecules of the invention are able to induce a significant and prolonged reduction in tumor volume when compared to a control group. Unexpectedly, the EC1 binding molecules showed superior efficacy compared to the EC2 binding molecules. The EC1 binding agent caused a durable regression with 132%, 125% and 125% tumor growth inhibition, respectively. In contrast, although single treatment with the EC2 binding molecule caused initial regression, tumor regrowth was observed from day 10 onwards, resulting in only 70%, 61% and 102% tumor growth inhibition, respectively.

[0687] Example 7: Internalization ability of EC1-binding bispecific CDH3 / TRAILR2 molecules

[0688] The rate of endocytosis-mediated internalization and lysosomal degradation of surface receptor-bound biologics affects their stability and therefore their plasma half-life and duration of response. Compounds previously developed by others that bind CDH3 EC1 show high internalization capacity (WO12057328A1) and therefore may be less likely to support persistent and potent activation of TRAILR2 at the cell surface if used in a bispecific compound setting.

[0689] Compared with the commercially available anti-CDH3 antibody (#MAB861, R&D systems) to analyze the ability of exemplary EC1-binding bispecific CDH3 / TRAILR2 compounds, TRAILR2-binding antibody lesatumumab, or anti-CDH3 EC1-specific antibodies to internalize. To achieve this goal, GP2d colorectal cancer cells were seeded in 96-well culture plates (#3340, Corning, NY, USA) one day before compound exposure. Before adding compounds, antibodies were incubated with Next, Fabfluor-pH antibody labeling reagent (#4722, Sartorius, Goettingen, Germany) was added to the cells at a concentration of 1 nM in a total volume of 100 μl. The cells were analyzed for red Fabfluor signal as a surrogate for internalization in an S3 live cell analyzer (Sartorius) at 10× magnification, four images / well every 1 h for 24 h. Fig.17 The data presented in demonstrate that the bispecific CDH3 / TRAILR2 compounds, the anti-TRAILR2 antibody lesatumumab, and the CDH3 compounds binding to EC1 lack measurable internalization (no detectable Fabfluor red signal) at the applied concentrations compared to the moderately internalizing anti-CDH3 antibody MAB861. The absence of measurable internalization of the exemplary bispecific CDH3 / TRAILR2 antibodies binding to EC1 prepared herein is based on the durable tumor regression observed in the GP2d xenograft model, as shown in FIG. Fig.16 Confirmed in.

[0690] Example 8: Stability of bispecific CDH3 / TRAILR2 binding molecules under low pH conditions

[0691] The stability of the bispecific CDH3 / TRAILR2 molecules was tested under low pH conditions. During manufacturing, the biological product is exposed to low pH conditions in order to achieve self-affinity column elution ("capture step"). Low pH conditions are also required to inactivate potential contaminants (such as bacteria, fungi and viruses). Effective removal of potential contaminants is an important step to ensure drug safety during the manufacture of biological products using mammalian cell lines. However, for many recombinant proteins, incubation at low pH induces aggregation and reduces the yield of recovered protein. Therefore, the stability of proteins at low pH is an important factor for the successful and effective manufacture of therapeutic proteins.

[0692] To test the stability of the bispecific CDH3 / TRAILR2 binding molecules at low pH, the pH was adjusted to pH 3.5 with 1 M acetic acid, followed by incubation at room temperature for 90 minutes and then neutralized to pH 5.0 with 1 M Tris buffer (pH 9.0). The sample concentration was measured and the potential formation of aggregates (high molecular weight species, HMWS) or fragments (low molecular weight species, LMWS) was determined by analytical size exclusion chromatography (aSEC).

[0693] The results are summarized in Table 19 below, which demonstrates 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 (<1.06%) or LMWS (0%), or any significant decrease in the main peak (<1.06%) compared to the starting material, which demonstrated good stability after 90 minutes of incubation at pH 3.5.

[0694] Table 19: Affinity (KD) of CDH3 / TRAILR2 binding proteins for human CDH3, determined by SPR analysis

[0695]

Claims

1. 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 E-cadherin-3 (CDH3), wherein the at least one antigen binding site that specifically binds to CDH3 is selected from the group consisting of: i. an antigen binding site comprising a heavy chain CDR comprising the amino acid sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2) and SEQ ID NO: 39 (CDR3).

2. The binding molecule of claim 1, wherein the at least one antigen binding site that specifically binds to TRAILR2 comprises a heavy chain CDR comprising the amino acid sequence 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 sequence of SEQ ID NO: 77 (CDR1), SEQ ID NO: 78 (CDR2) and SEQ ID NO: 79 (CDR3).

3. The binding molecule of 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.

4. The binding molecule of claim 3, wherein the one or more scFvs are fused to the C-terminus of the Ig molecule, preferably, wherein a first scFv is fused to a first heavy chain and a second scFv is fused to a second heavy chain of the Ig molecule, respectively.

5. The binding molecule of any one of claims 1 to 4, wherein the at least one antigen binding site that specifically binds to CDH3 is selected from the group consisting of: 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.

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

76.

7. The binding molecule of any one of claims 1 to 6, wherein the at least one antigen binding site that binds to CDH3 comprises: 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.

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

71.

9. The binding molecule according to any one of claims 1 to 8, comprising 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.

10. An isolated nucleic acid encoding the binding molecule of any one of claims 1 to 9. An expression vector comprising the nucleic acid according to claim 10 . A host cell comprising the expression vector according to claim 11 .

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

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

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

16. A pharmaceutical composition comprising the binding molecule of 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 E-cadherin-3 (CDH3).

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