Novel trispecific binding molecules

Abstract

The present invention relates to novel trispecific binding molecules. The present invention also relates to nucleic acids encoding such binding molecules, to methods for preparing such binding molecules, to host cells expressing or capable of expressing such binding molecules, to compositions comprising such binding molecules, and to the use of such binding molecules or such compositions for therapeutic purposes, in particular in the field of cancer diseases.

Description

[Technical field]

[0001] The present invention relates to novel trispecific binding molecules. The present invention also relates to nucleic acids encoding such binding molecules, to methods for preparing such binding molecules, to host cells expressing or capable of expressing such binding molecules, to compositions comprising such binding molecules, and to the use of such binding molecules or such compositions for therapeutic purposes, in particular in the field of cancer diseases.

[0002] 2. Background of the Invention Cancer is a group of diseases generally based on abnormal cell proliferation and the potential of cancerous cells to invade or spread throughout the body. It is a serious disease and a leading cause of death worldwide.

[0003] Various methods of treatment have been used in an attempt to manage and in some cases treat cancer, including surgery, chemotherapy, radiation therapy, and hormone therapy. Antibodies also offer the potential to be powerful therapeutic agents for the treatment of cancer. Antibodies are designed to recognize and bind to specific proteins on the surface of cells, their target antigens. This binding can trigger a number of different biological responses, depending, for example, on the cell type expressing the target antigen, the function of the target antigen protein, or the structure of the antibody itself.

[0004] Some antibodies can, for example, directly bind to cancer cells and stop or reduce cell division of these cells, for example by interfering with cellular pathways, thereby slowing or preventing abnormal cell growth. Alternatively, such cancer cell targeting antibodies can also be attached with drugs or radioactive particles, thereby delivering these therapeutic agents to cancer cells and acting independently of the cells' intrinsic pathways. A different approach to these cancer cell targeting methods is based on antibodies that can induce the immune system to attack and kill cancer cells. This can be achieved by redirecting immune cells, such as cytotoxic T cells, to the cancer cells, or by directly affecting the activity of the immune system itself.

[0005] Although both cancer cell targeting approaches as well as immune cell redirection approaches offer powerful mechanisms of action (MoA) for the treatment of cancer, they often rely on the expression of cancer cell-specific markers, i.e., target proteins that are not or barely expressed by non-cancer cells. Such tumor-specific target proteins are rare, and this scarcity remains a major drawback typically faced in the development of cancer-specific therapeutics.

[0006] While there have been attempts to redirect immune cells to more broadly expressed lineage antigens, the value of these therapies has been limited by the toxicity caused by the expression of these antigens in certain normal tissues, such as Epcam expression in the gastrointestinal tract (Kebenko et al., Oncoimmunology 2018, Vol. 7, No. 8). While various approaches to reduce the toxicity associated with exosite antigen expression are currently being pursued, toxicity remains dose-limiting for many compounds.

[0007] Thus, despite the fact that there have been advances in the treatment of certain cancers in recent years and that a number of different approaches are currently being pursued, there remains a need to provide new therapeutically suitable compounds for the treatment of cancer. Thus, it is an object of the present invention to provide such pharmacologically active agents that can be used in the treatment of various cancer diseases.

[0008] In particular, it is an object of the present invention to provide such pharmacologically active agents, compositions, and / or methods of treatment that offer certain advantages over agents, compositions, and / or methods currently in use and / or known in the art. These advantages include in vivo efficacy, improved therapeutic and pharmacological properties, fewer side effects, and other advantageous properties, such as improved ease of preparation or reduced cost of manufacture, especially when compared to candidate drugs already known in the art.

[0009] This need is addressed by the embodiments provided herein.

[0010] BRIEF SUMMARY OF THE INVETION The present invention is based on the concept of combining three antigen-binding sites within a single binding molecule: a first antigen-binding site that specifically binds to trophoblast cell surface antigen 2 (TROP2), a second antigen-binding site that specifically binds to cadherin 17 (CDH17), and a third antigen-binding site that specifically binds to cluster of differentiation 3 (CD3). As discussed in more detail below, one advantage of the molecules of the invention is their low affinity, high avidity design, which provides excellent specificity for target cells.

[0011] Therefore, a first aspect of the present invention provides a binding molecule comprising: (a) at least one antigen binding site that specifically binds to trophoblast cell surface antigen 2 (TROP2) with a Kd≧1 nM; (b) at least one antigen binding site that specifically binds to cadherin-17 (CDH17); wherein the at least one antigen binding site that specifically binds to CDH17 is selected from the group consisting of antigen binding sites (i) to (ii): (i) comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 34 (CDR3). (ii) an antigen-binding site comprising a heavy chain CDR and a light chain CDR comprising the amino acid sequences of SEQ ID NO:32 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:34 (CDR3) and a light chain comprising the amino acid sequences of SEQ ID NO:39 (CDR1), SEQ ID NO:40 (CDR2), and SEQ ID NO:37 (CDR3); and (c) at least one antigen-binding site that specifically binds to cluster of differentiation 3 (CD3).

[0012] In a preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to TROP2 is selected from the group consisting of antigen-binding sites (i) to (vi): (i) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); (ii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3); (iii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (iv) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:19 (CDR2), and SEQ ID NO:15 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:20 (CDR1), SEQ ID NO:17 (CDR2), and SEQ ID NO:18 (CDR3); (v) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2), and SEQ ID NO:23 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:24 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:26 (CDR3); and (vi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2), and SEQ ID NO:29 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:30 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:31 (CDR3).

[0013] In a preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to TROP2 is selected from the group consisting of antigen-binding sites (i) to (xii): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 83 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (iii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 87 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 88; (iv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 90; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 91 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 92; (vi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (vii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (viii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96; (ix) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (x) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (xi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99; or (xii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96.

[0014] In a preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CDH17 is selected from the group consisting of antigen-binding sites (i) to (ii): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 100 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 101; and (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 102 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103.

[0015] In a preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CD3 is selected from the group consisting of antigen-binding sites (i) to (xxxi): (i) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (ii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 46 (CDR3); (iii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (iv) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 50 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (v) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (viii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 55 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (ix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (x) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 60 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xiii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 62 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xiv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xvi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xvii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xviii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 72 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xx) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxiii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 75 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxiv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 76 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxvi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxvii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxviii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxx) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); and (xxxi) An antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3).

[0016] In a preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CD3 is selected from the group consisting of antigen-binding sites (i) to (xvi): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:104 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:123, and SEQ ID NO:129; (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 110 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 111; (iii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 112 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 113; (iv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 118 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 119 and SEQ ID NO: 122; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 124 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 130, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 127; (vi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 131 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 132; (vii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 133 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; (viii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 136 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 137; (ix) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and SEQ ID NO: 156, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 139; (x) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 161, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 141; (xi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 144 and SEQ ID NO: 146, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 145; (xii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, and SEQ ID NO: 162, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 148; (xiii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 149 and SEQ ID NO: 152, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 150; (xiv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (xv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 158; and (xvi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 160.

[0017] In a preferred embodiment of the binding molecule of the invention, the binding molecule is a modified immunoglobulin (Ig) molecule, preferably a modified IgG molecule, in which the at least one antigen binding site that specifically binds TROP2 and the at least one antigen binding site that specifically binds CDH17 are present in the variable region of the Ig molecule, and in which the at least one antigen binding site that specifically binds CD3 is an scFv fused to the TROP2-CDH17-specific Ig molecule.

[0018] In a preferred embodiment of the binding molecule of the invention, the scFv is fused to the C-terminus of a heavy chain of an Ig molecule, preferably to a portion of the heavy chain of an Ig molecule that contains at least one antigen binding site that specifically binds to CDH17.

[0019] In a preferred embodiment of the binding molecule of the invention, the binding molecule comprises a combination of a first immunoglobulin light chain and an immunoglobulin heavy chain selected from: (a)(ai) to (a-xii), preferably linked together by a peptide linker: (ai) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 169 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 170; (a-ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 171 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 172; (a-iii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 173 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 174; (a-iv) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 175 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 176; (av) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 177 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 178; (a-vi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 179 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 180; (a-vii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 181 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 182; (a-viii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 183 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 184; (a-ix) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 185 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 186; (ax) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 187 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188; (a-xi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 189 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190; or (a-xii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 191 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192; and a second immunoglobulin heavy chain and immunoglobulin light chain combination selected from (b)(bi) to (b-ii), preferably linked together by a peptide linker: (bi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 196 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 197; or (b-ii) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 198 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 199; (c) a single-chain variable fragment (scFv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, preferably linked to the C-terminus of the immunoglobulin heavy chain of the second combination of immunoglobulin heavy chain and immunoglobulin light chain.

[0020] In a preferred embodiment of the binding molecule of the invention, the binding molecule comprises or consists of: (ai) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 267; (a-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 267; (a-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 267; (a-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 267; (av) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 267; (a-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 267; (a-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 267; (a-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 267; (a-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 267; (ax) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 267; (a-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 267; (a-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 267; (bi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 268; (b-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 268; (b-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 268; (b-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 268; (bv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 268; (b-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 268; (b-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 268; (b-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 268; (b-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 268; (bx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 268; (b-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 268; (b-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 268; (ci) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 269; (c-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 269; (c-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 269; (c-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 269; (cv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 269; (c-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 269; (c-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 269; (c-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 269; (c-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 269; (cx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 269; (c-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 269; (c-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 269; (di) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 270; (d-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 270; (d-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 270; (d-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 270; (dv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 270; (d-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 270; (d-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 270; (d-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 270; (d-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 270; (dx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 270; (d-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 270; (d-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 270; (ei) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 271; (e-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 271; (e-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 271; (e-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 271; (ev) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 271; (e-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 271; (e-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 271; (e-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 271; (e-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 271; (ex) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 271; (e-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 271; (e-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 271; (fi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 272; (f-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 272; (f-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 272; (f-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 272; (fv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 272; (f-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 272; (f-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 272; (f-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 272; (f-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 272; (fx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 272; (f-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 272; (f-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 272; (gi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 424; (g-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 424; (g-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 424; (g-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 424; (gv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 424; (g-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 424; (g-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 424; (g-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 424; (g-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 424; (gx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 424; (g-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 424; (g-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 424; (hi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 425; (h-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 425; (h-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 425; (h-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 425; (hv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 425; (h-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 425; (h-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 425; (h-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 425; (h-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 425; (hx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 425; (h-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 425; (h-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 425; (ii) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 426; (i-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 426; (i-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 426; (i-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 426; (iv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 426; (i-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 426; (i-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 426; (i-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 426; (i-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 426; (ix) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 426; (i-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 426; (i-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 426; (ji) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 427; (j-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 427; (j-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 427; (j-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 427; (jv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 427; (j-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 427; (j-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 427; (j-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 427; (j-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 427; (jx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 427; (j-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 427; (j-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 427; or (k) the amino acid sequence of SEQ ID NO: 436 and the amino acid sequence of SEQ ID NO: 437.

[0021] A further aspect of the invention provides a binding molecule comprising or consisting of: (ai) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 267; (a-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 267; (a-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 267; (a-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 267; (av) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 267; (a-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 267; (a-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 267; (a-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 267; (a-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 267; (ax) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 267; (a-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 267; (a-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 267; (bi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 268; (b-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 268; (b-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 268; (b-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 268; (bv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 268; (b-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 268; (b-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 268; (b-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 268; (b-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 268; (bx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 268; (b-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 268; (b-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 268; (ci) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 269; (c-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 269; (c-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 269; (c-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 269; (cv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 269; (c-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 269; (c-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 269; (c-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 269; (c-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 269; (cx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 269; (c-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 269; (c-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 269; (di) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 270; (d-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 270; (d-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 270; (d-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 270; (dv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 270; (d-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 270; (d-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 270; (d-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 270; (d-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 270; (dx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 270; (d-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 270; (d-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 270; (ei) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 271; (e-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 271; (e-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 271; (e-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 271; (ev) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 271; (e-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 271; (e-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 271; (e-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 271; (e-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 271; (ex) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 271; (e-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 271; (e-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 271; (fi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 272; (f-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 272; (f-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 272; (f-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 272; (fv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 272; (f-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 272; (f-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 272; (f-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 272; (f-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 272; (fx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 272; (f-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 272; (f-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 272; (gi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 424; (g-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 424; (g-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 424; (g-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 424; (gv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 424; (g-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 424; (g-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 424; (g-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 424; (g-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 424; (gx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 424; (g-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 424; (g-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 424; (hi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 425; (h-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 425; (h-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 425; (h-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 425; (hv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 425; (h-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 425; (h-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 425; (h-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 425; (h-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 425; (hx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 425; (h-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 425; (h-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 425; (ii) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 426; (i-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 426; (i-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 426; (i-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 426; (iv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 426; (i-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 426; (i-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 426; (i-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 426; (i-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 426; (ix) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 426; (i-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 426; (i-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 426; (ji) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 427; (j-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 427; (j-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 427; (j-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 427; (jv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 427; (j-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 427; (j-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 427; (j-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 427; (j-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 427; (jx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 427; (j-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 427; (j-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 427; or (k) the amino acid sequence of SEQ ID NO: 436 and the amino acid sequence of SEQ ID NO: 437.

[0022] The present invention further relates to a nucleic acid molecule encoding a binding molecule of the invention, or a part thereof.The present invention further relates to an expression vector comprising one or more nucleic acid molecules of the invention.The present invention further relates to a host cell transfected with an expression vector of the invention.

[0023] The present invention further relates to a method for producing a binding molecule of the invention, the method comprising the steps of: (a) culturing a host cell according to claim 12 under conditions allowing expression of a binding molecule according to any one of claims 1 to 11; (b) optionally recovering the molecule; and, optionally (c) further purifying and / or modifying and / or formulating the binding molecule.

[0024] The present invention further relates to pharmaceutical compositions comprising or consisting of one or more binding molecules of the invention and optionally a pharma- ceutically acceptable carrier.

[0025] The invention further relates to a binding molecule of the invention or a pharmaceutical composition of the invention for use in medicine.The invention further relates to a binding molecule of the invention or a pharmaceutical composition of the invention for use in a method for treating, ameliorating or preventing cancer.

[0026] The present invention further relates to a method for treating, preventing, or ameliorating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a binding molecule of the invention, or a pharmaceutical composition of the invention.

[0027] The present invention further relates to the use of a binding molecule of the invention for preparing a pharmaceutical composition for treating, preventing, or ameliorating cancer.

[0028] In a preferred embodiment of the binding molecule of the invention, or the pharmaceutical composition of the invention, or the method of the invention, or the use of the invention, the cancer is colorectal cancer (CRC), gastric cancer (GC) or pancreatic cancer (PAC).

[0029] In a preferred embodiment of the binding molecule of the invention, or the pharmaceutical composition of the invention, or the method of the invention, or the use of the invention, the binding molecule is used in combination with an immune checkpoint inhibitor, preferably an anti-PD-1 antibody or an anti-PD-1-L1 antibody. [Brief description of the drawings]

[0030] [Figure 1] Co-expression of CDH17 and Trop2 in GI cancer tissues but not normal tissues. Gene expression in tumors and key normal tissues. Scale is arbitrary units of log2-transformed MAS5-normalized expression values. Axis labels are gene symbols (TACSTD2.202286_s_at=Trop2, CDH17.209847_at=CDH17) and Affymetrix probe sets. [Diagram 2]Validation of target expression in GI tumors. Immunohistochemistry using antibodies binding to Trop2 (ENZ-ABS380, Enzo) and CDH17 (760-4865, Roche Ventana) in selected human cancer tissues. [Diagram 3] Schematic diagram of an exemplary trispecific binding molecule of the invention. [Figure 4] Selection of CDH17 binders with binding capacity. Binding of the four different CDH17 binding proteins indicated (in either bivalent or monovalent format) to a HEK293 cell line recombinantly expressing human CDH17, tested by flow cytometry. The anti-TNP binding arm is an irrelevant (non-binding) control directed against tetranitrophenol. [Diagram 5] Validation of binding capacity of CDH17-binders in a trispecific format. Upregulation of the T cell activation marker CD69 on CD8+ T cells mediated by Trop2 / CDH17 / CD3 molecules with distinct CDH17 antigen binding sites in the presence of HEK293 cells either expressing CDH17 alone (left panel) or co-expressing Trop2 / CDH17 (right panel). [Figure 6] Selection of Trop2 binders with binding capacity. Binding of the seven different Trop2 binders indicated (in either bivalent or monovalent format) to a HEK293 cell line recombinantly expressing human Trop2, tested by flow cytometry. The anti-TNP binding arm is an irrelevant (non-binding) control directed against tetranitrophenol. [Figure 7] Confirmation of binding capacity of Trop2 binders in a trispecific format. Upregulation of the T cell activation marker CD69 on CD8+ T cells mediated by Trop2 / CDH17 / CD3 molecules with distinct CDH17 antigen binding sites in the presence of HEK293 cells expressing CDH17 alone (C), Trop2 alone (B) or co-expressing Trop2 / CDH17 (A). [Figure 8]Analysis of the binding capacity of various trispecific Trop2 / CDH17 / CD3 binder combinations in a cell binding assay. Binding of different combinations of Trop2 and CDH17 binders in a trispecific format to HEK293 cell lines recombinantly expressing human Trop2 (A) or co-expressing human Trop2 and CDH17 (B) tested by flow cytometry. The anti-TNP binding arm is an irrelevant (non-binding) control directed against tetranitrophenol. [Figure 9] Potential in mediating T cell-induced lysis in cells expressing either CDH17, Trop2, or co-expressing both. Redirected T cell-mediated lysis (A) of HEK293 cells recombinantly expressing either CDH17 (left), Trop2 (middle), or both (right) was assessed using various Trop2 / CDH17 / CD3 binders (shown in A is one exemplary Trop2 / CDH17 / CD3 binder) as well as various control molecules that bind only one target, either monovalently or bivalently. Redirected T cell-mediated lysis of DLD-1-derived cell lines (B) expressing either Trop2 alone (left) or both CDH17 and Trop2 (right) was assessed using different Trop2 / CDH17 / CD3 binder combinations as well as a monovalent Trop2 binding control. [Figure 10] Confirmation of the contribution of CDH17 binding to avidity-induced increased potency. Redirected T cell-mediated lysis of SK-CO1 cells was assessed using different Trop2 / CDH17 / CD3 molecules as well as a Trop2 / TNP / CD3 control molecule. [Figure 11]Comparison of knob-in-hole formats. Redirected T cell-mediated lysis of SK-CO1 cells was assessed using the trispecific molecules in two different knob-in-hole formats. The reference "hole" or "knob" in brackets in the legend indicates the position of the CDH17 / CD3 arm, i.e., "hole" indicates that CDH17 / CD3 is on the hole arm (see SEQ ID NOs: 271, 272, and 424-427), while "knob" indicates that CDH17 / CD3 is on the knob arm (see SEQ ID NOs: 428-433). [Figure 12] Confirmation of tumor growth inhibition in vivo. Effect of different Trop2 / CDH17 / CD3 binders as well as a monovalent control binder on the growth of HPAF-II derived xenograft tumors in a humanized mouse model.

[0031] Detailed Description of the Invention Numerous documents are cited herein, including patent applications and manufacturer's manuals. The disclosures of these documents, while not believed to be relevant to the patentability of this invention, are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0032] definition The above summarized aspects of the invention, as well as other aspects and embodiments of the invention, will become apparent from the further description herein, in which: a) Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which would be apparent to one of ordinary skill in the art. In case of conflict, the patent specification, including definitions, shall prevail. Reference is made to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), as well as to the general background art cited herein. Moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, as would be apparent to one skilled in the art. Reference is again made to the standard handbooks, the general background art referred to above, and the further references cited therein. b) Unless otherwise indicated or defined, the term "antibody" is used as defined in the art. Antibodies are typically immunoglobulins (abbreviated Ig), preferably gamma globulin proteins (IgG; see further below). Natural antibodies can be found in the blood or other body fluids of vertebrates, where they are used by the immune system to identify and neutralize foreign bodies, such as bacteria and viruses, a function mediated by their ability to bind by covalent interactions with other molecules or structures known as antigens. This binding is specific in the sense that the antibody binds only to a particular structure with high affinity. The unique portion of an antigen recognized by an antibody is called the epitope, or antigenic determinant. Natural antibodies (e.g., including the classical Y-shaped immunoglobulin molecule) are typically composed of a basic structural unit: two large heavy chains and two small light chains each - or only two heavy chains (i.e., V and VH, respectively) as in camelids or cartilaginous fish. H H fragment and V NAREach heavy chain has a variable domain (VH) at the N-terminus, followed by three or four (in the case of IgE) constant domains (CH1, CH2, CH3, and CH4) and a hinge region between CH1 and CH2. Each light chain has two domains, an N-terminal variable domain (VL) and a C-terminal constant domain (CL). The part of the antibody that mediates binding to the epitope is sometimes called the paratope and is located in the variable domain, or variable region (Fv), of the antibody. The variable domain contains three so-called complementarity determining regions (CDRs) spaced apart by framework regions (FR). The framework regions adopt a beta-sheet conformation and the CDRs may form loops connecting the beta-sheet structure. The CDRs in each chain are held in a three-dimensional structure by framework regions and typically, together with the CDRs from the other chain (if present), mediate binding to the antigen. The term "constant domain" or "constant region" as used within this application denotes a summary of the domains of an antibody other than the variable region. Such constant domains and regions are well known in the state of the 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). The "Fc part" of an antibody is not directly involved in the binding of the antibody to an antigen, but exhibits various effector functions. "Fc part of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. Depending on the amino acid sequence of the constant region of the heavy chain, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG, and IgM. According to the heavy chain constant region, the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. The Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding, and Fc receptor binding. 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 antibodies on the complement system is condition-dependent, the binding to C1q is initiated by a defined binding site in the Fc portion. Such binding sites are known in the state of the art and are described, for example, by Kellner et al. Transfus Med Hemother. 44(5)(2017)327-336. Non-limiting examples of such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat). The most critical among these residues in mediating C1q and Fc gamma receptor binding in IgG1 are L234 and L235 (Hezareh et al., J. Virology 75(2001)12161- 12168). Antibodies of subclasses IgG1 and IgG3 usually exhibit complement activation and C1q and C3 binding, whereas IgG2 and IgG4 do not activate the complement system and do not bind C1q and C3.

[0033] The term "antibody" as used herein also includes fragments of immunoglobulins that retain antigen-binding properties, such as Fab, Fab', or F(ab')2 fragments. Such fragments may 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 using routine techniques, for example using papain or pepsin (WO94 / 29348). Papain digestion of antibodies typically produces two identical antigen-binding fragments, so-called Fab fragments, each with a single antigen-binding site and a remaining Fc fragment. Pepsin treatment results in F(ab')2. In Fab molecules, the variable domains are each fused to an immunoglobulin constant domain, preferably of human origin. Thus, the heavy chain variable domain may be fused to a CH1 domain (so-called Fd fragment), whereas the light chain variable domain may be fused to a CL domain. Such molecules may be produced by recombinant expression of the respective nucleic acid in a host cell, using methods known in the art.

[0034] Many approaches have been described in the art for modifying natural antibodies and for placing immunoglobulin variable domains, or molecules derived from such variable domains, in different molecular contexts, see for example Holliger, P., Hudson, P. Nat Biotechnol 23, 1126-1136(2005) or Nilvebrant, J. et al. Current pharmaceutical design vol. 22,43(2016):6527-6537. Typically, the purpose of these modifications is to make antibodies even more versatile tools in medicine and technology. These approaches lead to structurally modified molecules, also referred to herein as "antibody derivatives" or "modified immunoglobulins", which often differ from the basic organization of natural antibodies (i.e. antibodies composed of two large heavy chains and two small light chains, or only two heavy chains as in camelid species or cartilaginous fish). Often, these molecules are smaller in size and may contain a single or several amino acid chains compared to natural antibodies. For example, single chain variable fragments (scFv) are fusions of the variable regions of heavy and light immunoglobulin chains, linked together with a short linker, usually serine (S) or glycine (G) (see, e.g., WO88 / 01649; WO91 / 17271). "Single domain antibodies" or "Nanobodies" have an antigen-binding site in a single Ig-like domain (see, e.g., WO94 / 04678; WO03 / 050531). Single domain antibodies with binding specificities for the same or different antigens may be linked together. Diabodies are bivalent antibody molecules consisting of two amino acid chains containing two variable domains (WO94 / 13804). Another example of an antibody-like molecule is the immunoglobulin superfamily antibody (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6(2):185-96). A different concept has led to the so-called small modular immunopharmaceuticals (SMIPs), which contain an Fv domain linked to a single hinge and effector domain lacking the constant domain CH1 (WO02 / 056910).However, in some cases, modifications can also lead to molecules larger than natural antibodies when classical Ig formats are combined with additional antigen binding sites, for example in the form of scFvs, etc. All such antibody derivatives or modified immunoglobulins are also encompassed by the more general term "antibody" as used herein.

[0035] For applications in humans, it is often desirable to reduce the immunogenicity of antibodies originally derived from other species, such as mice, and therefore modification approaches such as the construction of chimeric antibodies, or the so-called "humanization" of antibodies, have been developed. In this context, a "chimeric antibody" is understood to be an antibody that comprises a sequence portion (e.g., a variable domain) derived from one species (e.g., mouse) fused to a sequence portion (e.g., a constant domain) derived from a different species (e.g., human). A "humanized antibody" is an antibody that comprises a variable domain originally derived from a non-human species, in which certain amino acids have been mutated so that the overall sequence of the variable domain more closely resembles the amino acids of the sequence of a human variable domain. Methods for chimerization and humanization of antibodies are well known in the art (Billetta R, Lobuglio AF. "Chimeric antibodies". Int Rev Immunol. 1993;10(2-3):165-76; Riechmann L, Clark M, Waldmann H, Winter G(1988). "Reshaping human antibodies for therapy". Nature:332:323). The term "human antibody" as used herein relates to antibodies generated based on sequences derived from the human genome, for example by the use of phage display or transgenic animals (see, e.g., WO90 / 05144). The term "antibody" as used herein explicitly includes such humanized antibodies, chimeric antibodies, as well as human antibodies.

[0036] Antibodies may further be fused (as fusion proteins) or otherwise linked (covalently or non-covalently) to other molecular entities having the desired effect on the properties of the antibody. For example, it may be desirable to improve the pharmacokinetic properties of antibodies, such as their stability in body fluids, such as blood, especially in the case of single chain or domain antibodies. In this regard, a number of techniques have been developed, particularly for extending the half-life of such antibodies in circulation, such as pegylation (WO98 / 25971; WO98 / 48837; WO2004081026), fusion or otherwise covalent attachment of the antibody to another antibody having affinity for a serum protein such as albumin (WO2004041865; WO2004003019), or expression of the antibody as a fusion protein with all or part of a serum protein such as albumin or transferrin (WO01 / 79258). Means and methods for lead identification and lead optimization in antibody design are well known in the art and have been reviewed, for example, in Goulet, DR and Atkins, WM J Pharm Sci 2020;109(1):74-103 or Tiller, KE, & Tessier, PM(2015). Annual review of biomedical engineering, 17, 191-216. c) "Antibody mimetics" have also been developed, which typically bear only a distant structural relationship to immunoglobulin variable domains, or no such relationship at all, but which exhibit the same particular binding specificity and affinity as immunoglobulin variable domains. Such non-immunoglobulin "antibody mimetics" are sometimes called "scaffold proteins" and can be based, for example, on genes for protein A, lipocalin, fibronectin domains, ankyrin consensus repeat domains, and thioredoxin (Skerra, Current Opinion in Biotechnology 2007, 18(4):295-304). d) The term "immunoglobulin variable domain" as used herein means an immunoglobulin domain consisting essentially of four "framework regions", which are 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; which are interrupted by three "complementarity determining regions" or "CDRs", which are 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", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be depicted as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain that carries the antigen binding site and thereby confers specificity to the antibody for the antigen. e) A molecule (such as a binding molecule of the invention, or a fragment thereof) that is capable of "binding," "binding to," "specifically binding," or "specifically binding to," "has affinity for" and / or "has specificity for" a particular epitope, antigen, or protein (or at least one portion, fragment, or epitope thereof) is said to be "for" or "directed against" that epitope, antigen, or protein, or is a "binding" molecule with respect to such epitope, antigen, or protein. f) The term "antigen-binding site", as used herein, relates to a domain of a binding molecule that confers binding to a specific antigen. Although antigen-binding sites are originally derived from antibodies, advances in the field have led to additional possibilities to design and / or obtain antigen-binding sites without the need to generate natural antibodies against the target of interest. Regardless of its origin, an "antigen-binding site" according to the invention comprises at least the minimal structural elements, i.e. the necessary and sufficient structural elements, that allow binding to its specific target antigen. Thus, an "antigen-binding site" according to the invention comprises at least three heavy chain CDR sequences (in the case of a single domain antibody), more preferably at least three light chain and three heavy chain CDR sequences. As discussed above, these CDRs are typically present in the so-called variable domains, or variable regions (Fv) of an antibody. It will be understood that an antigen-binding site comprises at least the minimal structural elements, but typically also encompasses additional elements, such as framework regions. Thus, as used according to the present invention, an antigen-binding site can also be defined via the sequence of the respective combination of heavy chain variable domains and light chain variable domains. It is particularly preferred according to the invention that the "antigen binding site" is comprised in a polypeptide and / or that said CDR or each of said variable domains is a polypeptide or peptide. g) The term "specific binding" of a binding molecule or antigen-binding site may, for example, be described in terms of their cross-reactivity. Preferably, the antigen-binding site of the binding molecule of the invention does not cross-react or does not essentially cross-react with epitopes that are distinct from, but have a similar structure to, the target antigen. Preferably, "specifically binds to" refers to an antigen-binding site that does not bind to a target with less than 65%, preferably less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, most preferably less than 98% identity (calculated using methods known in the art) to the specifically listed targets, i.e., antigens TROP2, CDH17, and CD3, respectively. The cross-reactivity of a panel of molecules under study may, for example, be tested by evaluating the binding of said panel of molecules under conventional conditions to the epitope of interest as well as to a number of more or less closely related (structurally and / or functionally) epitopes. Only molecules that bind to the epitope of interest in the relevant context (e.g., a particular motif in the structure of a protein) but do not or essentially do not bind to any other epitopes are considered to specifically bind. Corresponding methods are described in the textbook literature as well as, for example, Brooks BD. Curr Drug Discov Technol. 2014 Jun;11(2):109-12 or Abdiche YN, et al. PLoS One. 2014 Mar 20;9(3):e92451.

[0037] "Specificity", however, can also be described or specified in terms of their affinity and / or avidity.

[0038] Affinity is a measure of the binding strength between an epitope and an antigen-binding site on a binding molecule, represented by the equilibrium constant (Kd) for the dissociation of the binding molecule and the antigen: the smaller the value of Kd, the stronger the binding strength between the epitope and the binding molecule (alternatively, affinity can also be expressed as an affinity constant (Ka), which is 1 / Kd).

[0039] It is particularly preferred that an antigen-binding site that specifically binds to its target has a significantly higher binding affinity for the respective target, i.e., antigen TROP2, CDH17, or CD3, than structurally unrelated molecules. Thus, an antigen-binding site for TROP2 has a significantly higher binding affinity for TROP2 than any other structurally unrelated molecule; an antigen-binding site for CDH17 has a significantly higher binding affinity for CDH17 than any other structurally unrelated molecule, and an antigen-binding site for CD3 has a significantly higher binding affinity for CD3 than any other structurally unrelated molecule. In accordance with the present invention, an antigen-binding site is considered to have a significantly higher binding affinity for a target antigen if it binds to said target antigen with an affinity that is at least 2-fold, preferably at least 10-fold greater, preferably at least 20-fold greater, and most preferably at least 100-fold greater than its affinity for an unrelated antigen under the same conditions.

[0040] Preferred binding affinities vary depending on the binding molecule, but include those with a dissociation constant (Kd) of at least 10E-4 moles / liter or less, as measured, for example, via any standard methodology known in the art, preferably via a Biacore assay as described below and in the accompanying examples. Any Kd value above 10E-4M is generally considered to indicate non-specific binding. Preferably, the antigen-binding site specifically binds to the desired antigen with a Kd of less than 500nM, preferably less than 200nM, more preferably less than 100nM, and most preferably less than 20nM. For a particular binding agent, such as the antigen-binding site for CD3 described herein, unless otherwise defined herein, it is preferred that the dissociation constant (Kd) is less than 10nM, more preferably less than 5nM, and even more preferably less than 2nM.

[0041] Avidity is a measure of the strength of binding between a binding molecule (such as a binding molecule of the invention, or a fragment thereof) and the relevant antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the binding molecule, and the number of relevant binding sites present on the binding molecule.

[0042] As will be clear to the skilled artisan (e.g., based on the further disclosure herein), specific binding can be determined using means and methods known per se in the art, depending on the particular antigen of interest. Such means and methods include, but are not limited to, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competitive assays, as well as plasmon resonance assays (Malmqvist M., Curr Opin Immunol. 1993 Apr;5(2):282-6.), using purified wild-type antigens. Antibody affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, RJ, and Brault PA., ASSAY and Drug Development Technologies. 2004, Dec 2(6):647-657). Preferably, specific binding is measured by plasmon resonance assays using purified wild-type antigens. More preferably, the method for determining the Kd value is based on a Biacore assay, for example as described in Example 3.2 below. Thus, any Kd value referred to herein is preferably a Kd value obtained via surface plasmon resonance (SPR) on a Biacore 4000, in which the binding molecule of interest is captured on the sensor surface via Protein A / G at 10 μl / min for 60 seconds, and then the respective target molecule (e.g. human Trop2, CDH17, or CD3), preferably at a concentration of 100 nM, is applied for association at 30 μl / min for 180 seconds, followed by dissociation in HBS-EP buffer for 120 seconds. h) Amino acid residues are designated according to the standard three-letter or one-letter amino acid code, which is generally known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of the indicated number of amino acid residues at the position of the reference sequence compared to the second sequence. In the case of substitutions, such substitutions are preferably conservative amino acid substitutions, which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or essentially 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 WO98 / 49185, in which conservative amino acid substitutions are preferably those in which one amino acid residue in the following groups (i) to (v) is replaced by another amino acid residue in the same group: (i) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (non-charged) amides: Asp, Asn, Glu, and Gin; (iii) polar positively charged residues: His, Arg, and Lys; (iv) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala to GIy or Ser; Arg to Lys; Asn to Gin or His; Asp to GIu; Cys to Ser; Gin to Asn; GIu to Asp; GIy to Ala or Pro; His to Asn or Gin; Ile to Leu or VaI; Leu to Ile or VaI; Lys to Arg, Gin, or GIu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; VaI to Ile or Leu. i) The term "isolated" as used herein refers to a material that has been removed from its original or natural environment (e.g., the natural environment if it occurs in nature). 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 has been separated by human intervention from some or all of the coexisting materials in a natural system and is isolated. Such a nucleic acid molecule may be part of a vector and / or such a nucleic acid molecule or polypeptide may be part of a composition, but is still isolated in that such a 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 "essentially isolated" when, compared to its natural biological source and / or the reaction or culture medium from which it was obtained, it is separated from at least one other component that is normally associated in the source or medium, such as another nucleic acid molecule, another polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or trace component. In particular, a nucleic acid molecule or polypeptide is considered to be "essentially isolated" if it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. A nucleic acid molecule or polypeptide that is in "essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, for example polyacrylamide gel electrophoresis. The binding molecules and nucleic acids of the present invention are preferably isolated. j) Unless otherwise indicated, the term "sequence" as used herein (e.g., terms such as "immunoglobulin sequence," "binding molecule sequence," or "polypeptide sequence") should generally be understood to include both related amino acid sequences as well as nucleic acid sequences or nucleotide sequences encoding same, unless a more limited interpretation is required by context. k) As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences being compared are the same length after gaps, if appropriate, are introduced into the sequences (e.g., excluding additional sequences that extend beyond the sequences being compared). For example, when comparing variable region sequences, leader and / or constant domain sequences are not considered. For sequence comparison between two sequences, a "corresponding" CDR refers to the CDR that is at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0043] The determination of percent identity or similarity between two sequences can be achieved using a mathematical algorithm. A preferred, non-limiting example of the mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as 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, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST protein searches can be performed with the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing 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, including ADVANCE and ADAM, described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA, 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. When ktup=2, similar regions in the two sequences being compared are found by examining pairs of aligned residues; when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1-6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402. Preferably, the CLUSTAL W algorithm described above is used. l) The term "comprising" as used herein means that additional ingredients and / or steps may be included in addition to the ingredients and / or steps specifically recited, but the term also encompasses the claimed subject matter consisting of exactly the ingredients and / or steps recited. m) As used herein, the term "at least" refers to any number, including the number specifically recited and any number greater than that. For example, "at least one" includes exactly 1, as well as more than 1, including but not limited to 2, for example 3 or 4. Also included are, for example, 5, 6, 7, 8, 9, 10, 15, such as 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500, as well as any integer between or above these specifically recited numbers. With regard to the term "at least one antigen-binding site", it is particularly preferred that the term encompasses 1, 2, 3, or 4 antigen-binding sites. Most preferably, the term relates to exactly one antigen-binding site. When more than one antigen-binding site is selected for a target, these multiple antigen-binding sites can be selected independently, i.e., they can be identical, or they can be different from each other. n) The term "polypeptide" as used herein describes a linear molecular chain of amino acids, including a single polypeptide or fragment thereof, containing more than 30 amino acids. On the other hand, the term "peptide" as used in the present invention describes a linear chain of amino acids containing up to 30 amino acids. The term "(poly)peptide" as used in accordance with the present invention refers to a group of molecules that includes the group of peptides consisting of up to 30 amino acids, as well as the group of polypeptides consisting of more than 30 amino acids. o) The term "linker", as used herein, includes both peptide linkers, i.e., sequences of amino acids, as well as non-peptide linkers, which connect the individual parts of the molecule covalently or non-covalently. The term "non-peptide linker", as used herein, 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 bonded to reactive groups of the binding portion of the molecule of the invention, such as the amino terminus, lysine residue, histidine residue, or cysteine ​​residue. Reactive groups of the polymer include aldehyde groups, propionic aldehyde groups, butyraldehyde groups, maleimide groups, ketone groups, vinylsulfone groups, thiol groups, hydrazide groups, carbonyldiimidazole (CDI) groups, nitrophenylcarbonate (NPC) groups, trisylate groups, isocyanate groups, and succinimide derivatives. Examples of succinimide derivatives include succinimidyl propionate (SPA), succinimidyl butanoate (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA), succinimidyl succinate (SS), succinimidyl carbonate, and N-hydroxysuccinimide (NHS). The reactive groups at both ends of the non-peptide polymer can be the same or different. For example, the non-peptide polymer can have a maleimide group at one end and an aldehyde group at the other end.

[0044] A peptide linker, as envisaged herein, is a (poly)peptide linker of at least one amino acid in length. Preferably, the linker is 1-100 amino acids in length. More preferably, the linker is 5-50 amino acids in length, more preferably 10-40 amino acids in length, even more preferably, the linker is 15-30 amino acids in length. A non-limiting example of a small linker that is often used comprises a sequence of glycine and serine amino acids, called GS mini-linkers. The number of amino acids in these linkers can vary, for example, they can be 4 (GGGS) (SEQ ID NO: 273), or 6 (GGSGGS) (SEQ ID NO: 274), or multiples thereof, such as 2 or 3 or more repeats of these 4 / 6 amino acids. Most preferably, such a GS mini-linker has 20 amino acids and the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 275).

[0045] 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.

[0046] It is known in the art that the properties, i.e., length and / or composition, such as amino acid sequence, of the linker can modify or enhance the stability and / or solubility of the molecule comprising the linker. Typically, the length and sequence of the linker are chosen depending on the composition of the respective molecule of interest. The skilled artisan is well aware how to design and test the suitability of different linkers, see for example Volkel, T. et al. Protein Engineering, Design and Selection, Volume 14, Issue 10, 2001, Pages 815-823. For example, the properties of the molecule can be easily tested by comparing the binding affinity of the binding moieties of the molecules of the invention. In the case of the trispecific molecules of the invention, each measurement for each binding moiety may be performed separately. The stability of the resulting molecules can be measured using an ELISA-based method to determine the residual binding capacity of the molecules after incubation in human serum at 37° C. for several hours. Other suitable tests can be found, for example, in Brian R. Miller, BR et al. Protein Engineering, Design and Selection, Volume 23, Issue 7, 2010, Pages 549-557 or Kugler, M. et al. Protein Engineering, Design and Selection, Volume 22, Issue 3, 2009, Pages 135-147. (p) The term "nucleic acid molecule" is used herein interchangeably with the term "polynucleotide" in accordance with the present invention and includes DNA, such as cDNA or genomic DNA, and RNA, such as mRNA. Also included are nucleic acid mimicking molecules known in the art, such as synthetic or semi-synthetic derivatives and mixed polymers of DNA or RNA. Such nucleic acid mimicking molecules or nucleic acid derivatives in accordance with the present invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA) and locked nucleic acid (LNA). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2' oxygen and the 4' carbon. They may contain additional non-natural or derivative nucleotide bases, as will be readily understood by those skilled in the art.

[0047] Trispecific Binding Molecules of the Invention The present invention relates to a binding molecule comprising: (a) at least one antigen-binding site that specifically binds to trophoblast cell surface antigen 2 (TROP2), preferably human TROP2, with a Kd≧1 nM, (b) at least one antigen-binding site that specifically binds to cadherin 17 (CDH17), preferably human CDH17, with a Kd≧10 nM, preferably with a Kd≧100 nM, and (c) at least one antigen-binding site that specifically binds to cluster of differentiation 3 (CD3), preferably human CD3.

[0048] Thus, a binding molecule of the invention (also referred to herein as a "protein of the invention" or a "binding agent of the invention") comprises at least the three specifically recited different antigen binding sites, i.e., at least one binding site for TROP2, at least one binding site for CDH17, and at least one binding site for CD3. Because of these three specificities, a binding molecule of the invention is also referred to herein as a "trispecific binding molecule" of the invention.

[0049] The term TROP2, as used herein, refers to "trophoblast cell surface antigen 2." TROP2 belongs to the family of tumor-associated calcium signal transducers (TACSTDs) and is required for the stability of claudin-7 and claudin-1. Human TROP2 is represented by SEQ ID NO: 419 and database accession number UniProt P09758.

[0050] The term CDH17, as used herein, refers to "cadherin 17". CDH17 is a member of the cadherin superfamily of calcium-dependent membrane-associated glycoproteins. The encoded protein is cadherin-like, consisting of an extracellular region containing seven cadherin domains, and a transmembrane region, but lacks the conserved cytoplasmic domain. Expression of the protein in the gastrointestinal and pancreatic ducts has been reported. Human CDH17 is represented by SEQ ID NO: 420 and in the database accession number UniProt Q12864.

[0051] The term CD3, as used herein, refers to "cluster of differentiation 3". CD3 is a protein complex and is a T cell coreceptor involved in activating both cytotoxic T cells, i.e., CD8+ naive T cells, and helper T cells, i.e., CD4+ naive T cells. CD3 is composed of four different chains, in which the complex includes, in mammals, the CD3γ chain, the CD3δ chain, and two CD3ε chains. Together with the T cell receptor (TCR) and the ζ chain (zeta chain), CD3 generates an activation signal in T lymphocytes. Human CD3 is represented by SEQ ID NO: 421, and for the CD3 delta chain, SEQ ID NO: 422, by database accession number UniProtKB-P04234, and for the CD3 epsilon chain, SEQ ID NO: 423, by database accession number UniProtKB-P07766, and for the CD3 gamma chain, SEQ ID NO: 424, by database accession number UniProtKB-P09693, and for the CD3 zeta chain by database accession number UniProtKB-P20963.

[0052] The general structure of an antigen-binding site is well known in the art and may be, for example, a single domain, such as an epitope-binding domain, a single chain Fv (ScFv) domain, or paired VH / VL domains, as discussed herein above. In a preferred embodiment, the antigen-binding site comprises at least a light chain variable domain and a heavy chain variable domain.

[0053] As discussed in relation to the general definition, the antigen-binding sites are required to bind specifically to their respective target antigens. The binding molecules of the present invention are further characterized in that the antigen-binding sites that specifically bind to TROP2 and CDH17, respectively, bind with a Kd of less than 1 nM for TROP2 and less than 10 nM (preferably less than 100 nM) for DH17. In other words, while the antigen-binding sites are required to be specific for their target antigens, they are not selected for strong binding affinity, but instead are required to bind with a lower affinity that allows strong binding of the binding molecules of the present invention only in the presence of both targets. As discussed in more detail below and shown in the examples (especially Examples 6-8), it has been found that the requirement that these two antigen-binding sites have reduced affinity for their respective antigens results in low monovalent binding, but enhanced binding of the resulting trispecific binding molecules of the present invention due to avidity in the presence of both targets. It is particularly preferred that the antigen-binding site for TROP2 has a Kd between 1 nM and 20 nM, more preferably between 1 nM and 10 nM, even more preferably between 1 nM and 4 nM. Most preferably, the antigen-binding site specifically binding to TROP2 has a Kd between 2 nM and 5 nM. Furthermore, it is particularly preferred that the antigen-binding site for CDH17 has a Kd between 10 nM and 200 nM, more preferably between 50 nM and 150 nM, even more preferably between 75 nM and 125 nM. Most preferably, the antigen-binding site specifically binding to CDH17 has a Kd that is between 100 nM and 120 nM. Preferably, the Kd is determined as described above for SPR on a Biacore 4000.

[0054] According to the present invention, such a requirement for reduced affinity binders is not necessary for antigen binding sites that bind to CD3. Thus, as stated herein above, for antigen binding sites that target CD3, it is particularly preferred that the binder binds with a Kd of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, and most preferably less than 2 nM. A number of antigen binding sites that specifically bind to CD3 have been described in the art, for example in WO2004106383, which describes well-known antigen binding sites derived from antibodies selected from the group consisting of X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11 D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, WT31, and F101.01.e, and SP34. In particular, WO2004106383 describes that the VH and VL regions are derived from antibodies / antibody derivatives and the like that are capable of specifically recognizing the human CD3-ε chain in the context of other TCR subunits, e.g. in mouse cells transgenic for the human CD3-ε chain.Further non-limiting examples of suitable CD3-specific antigen binding sites are described in US20180118848, W02011090762 (e.g., CRIS-7, OKT3, HU291, G19-4), US9782478, US20180118827 (SP34 modification), US20160145340 (SP34 modification), US9212225 (e.g., SP34 CDR modifications), US10174124 (CD3 hybridoma clones including clones 40G5C and 38E4.V1), US9914776 and WO2020127619 (SP34 modifications) US7,728,114, US5929212, WO2014047231 (based on OKT3 or CRIS-7), WO2004108158 (based on OKT3), WO200703320 (CD3 clones 28F11, 27H5, 23F10, 15C3), US1006 6015 (SP34 modification), WO2018201051 (SP34 modification), WO201911871, US99759662, US20180326058, WO2019131988 (including SP34 (mu, hu), OKT3, UCHT1.v9, UCHT1, v1, UCHT1.vM1, HU40G5C, 38E4.V1, anti-CD3 clones AN104, AN119, AN121, AN395), US20180161428, WO2019075378 (SP34 CDR variants), WO2016187594 (e.g., muromonab CD3 (OKT3), otelizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31), US10066016 (SP34 CDR variants), WO20180209298 (tritac), or US10544221 (SP34 CDR variants).

[0055] The antigen binding sites for the listed targets may be selected by the skilled artisan from the antigen binding sites described herein, from those described in the art (such as those known in the art for CD3, detailed above) or from newly developed antigen binding sites, provided in each case that the prerequisites for specificity described above are met (i.e. specific binding for TROP2 and CDH17 for the respective target, but with the provision that the Kd is not too low as defined above). Means and methods for testing these requirements are provided above and can be applied by the skilled artisan without further ado.

[0056] While the antigen-binding sites of the binding molecules of the invention are listed as (a), (b), and (c), this listing is not intended to dictate a particular order or configuration. Thus, the antigen-binding sites can be arranged in any order within the binding molecules of the invention, for example, (a)-(b)-(c), (b)-(c)-(a), (c)-(a)-(b), (b)-(a)-(c), etc. Preferred arrangements of the antigen-binding sites within the binding molecules of the invention are described in further detail below.

[0057] Importantly, the binding molecule of the invention is capable of (substantially) simultaneously binding all three targets (TROP2, CDH17, and CD3). Thus, provided all three targets are present, the binding molecule of the invention binds to all three of them, thereby acting as a T cell engager (TcE): it binds to CD3 on the T cell, thus bringing the T cell into proximity with tumor cells expressing both TROP2 and CDH17. Whether a binding molecule of interest is capable of binding to both Trop2 and CDH17 on the surface of a target cell and is capable of inducing T cell activity can be determined as described in the appended examples, for example using recombinant HEK293 cells or cancer cell lines (such as the colorectal cancer DLD-1 cell line) that endogenously express the antigen (both of which are described in Example 9).

[0058] In a preferred embodiment, the binding molecule of the invention does not contain any further antigen binding sites other than the three antigen binding sites specifically listed for TROP2, CDH17, and CD3. Even more preferably, the binding molecule of the invention is trispecific and trivalent, i.e. it contains one binding site for each of the three antigen targets.

[0059] The binding molecule of the present invention is not particularly limited with respect to its format, provided that it comprises at least the three specifically listed different antigen binding sites (TROP2, CDH17, and CD3) and is capable of (substantially) simultaneously binding these three targets as defined herein. As such, the format can be based on the format of natural antibodies, of antibody derivatives, or of fragments of such antibodies, as well as antibody mimetics. Such formats can be modified as necessary to accommodate all three antigen binding sites, for example by additionally comprising further 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, or nanobodies. Further non-limiting examples of suitable formats that can be used for each or all antigen binding sites include antibody mimetics as defined herein above.

[0060] Preferably, the binding molecules of the invention are modified immunoglobulins (also referred to herein as modified immunoglobulin molecules). For example, in a preferred embodiment, the antigen binding sites for TROP2 and CDH17 are present in the variable regions of a binding molecule having a (classical Y-shaped) immunoglobulin format. In such a format, a first set of immunoglobulin light and heavy chains contains an antigen binding site for TROP2 in its variable region, and a second set of immunoglobulin light and heavy chains contains an antigen binding site for CDH17 in its variable region, and both sets of chains are linked to each other, for example via disulfide bridges, to form the immunoglobulin molecule.

[0061] Techniques for making such immunoglobulin molecules are well known in the art and include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (e.g., Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)); "knobs-in-holes" engineering (e.g., U.S. Pat. No. 5,731,168); engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking of two or more immunoglobulins or fragments (e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (e.g., Kostelny et al., Immunol., 229:81 (1985)). 148(5):1547-1553(1992)). Such methods are well known to the skilled artisan and are also reviewed in the art, for example in Liu H. et al.(2017)Front. Immunol. 8:38. doi:10.3389 / fimmu.2017.00038 or in Moore, GL, Methods, Volume 154, 2019, Pages 38-50.

[0062] The immunoglobulin molecule is further modified by fusion of an additional moiety containing an antigen-binding site for CD3, resulting in a modified immunoglobulin. Preferably, the antigen-binding site for CD3 is an scFv fused to the C-terminus of one or both heavy chains of the immunoglobulin molecule. Fusion of various moieties to each other is well known in the art. The fusion can be, for example, via a peptide linker or via a non-peptide linker. Preferably, the fusion is via a peptide linker.

[0063] More preferably, the immunoglobulin molecule and / or the modified immunoglobulin molecule is a monoclonal, chimeric, humanized or human immunoglobulin (e.g., antibody) molecule. Even more 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 kappa or lambda.

[0064] According to the present invention, the individual elements constituting the binding molecule of the present invention can be connected to each other via one or more linkers.Preferably, these linkers are peptide linkers, more preferably they connect the individual elements via covalent bonds.It is particularly preferred herein that each light chain is connected to each heavy chain via a linker.

[0065] In embodiments in which a binding molecule of the invention comprises more than one linker, it is envisioned that the linkers may have the same or different lengths and may be composed of the same or different structures, e.g., comprise the same or different amino acid sequences, or the same or different non-peptide polymers. In preferred embodiments, the linkers present in a binding molecule of the invention differ from each other in length and / or structure (e.g., amino acid sequence or nature of the non-peptide polymer).

[0066] Preferably, the linker is a peptide linker. More preferably, the linker is a flexible linker, for example using the amino acids alanine and serine or glycine and serine, for example the linker shown in SEQ ID NO: 275, or one of the linkers shown in SEQ ID NOs: 265 and 266. Thus, it is particularly preferred that the binding molecule of the invention is completely composed of amino acids, i.e. it is a polypeptide.

[0067] To the best of our knowledge, no trispecific antibody or recombinant trispecific antibody derivative has been reported so far, connecting Trop2 and CDH17 on cancer cells with CD3 on immune cells, in which Trop2- and CDH17-specific binders bind to their targets with Kds above 1 nM and 10 nM, respectively. WO21113748 is a patent application describing multispecific antibodies in various formats, including a molecule called "TriAx-E" that binds Trop2, CDH17, and / or CD3, but any attempt by the inventors to reproduce said molecule has failed due to insufficient stability. Classical antibody selection campaigns are usually geared towards yielding high affinity binders. This is also the approach followed in WO21113748. However, the inventors have surprisingly found that the incorporation of antigen-binding sites with high affinity for these two targets results in a narrow therapeutic window. As shown in the examples, especially in Examples 6-8, the use of higher affinities leads to monovalent binding and activity on cells expressing only one or the other target, thus increasing the risk of unwanted side effects. The use of multivalent formats also reduced the therapeutic window. These findings show that increased affinity for either of the two targets leads to a loss of specificity, an effect that is further enhanced with increased valency. Therefore, a different selection strategy was chosen, focusing on obtaining low affinity binders. As is evident from the following examples, the trispecific binding molecules of the invention provide excellent specificity for target cells. The best results were obtained for binding molecules in which low affinity and monovalent binders are combined, as shown in the following examples.

[0068] In another preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to TROP2 is selected from the group consisting of antigen-binding sites (i) to (vi): (i) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); (ii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3); (iii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (iv) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:19 (CDR2), and SEQ ID NO:15 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:20 (CDR1), SEQ ID NO:17 (CDR2), and SEQ ID NO:18 (CDR3); (v) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2), and SEQ ID NO:23 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:24 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:26 (CDR3); And (vi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2), and SEQ ID NO:29 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO:30 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:31 (CDR3).

[0069] The CDRs disclosed herein and depicted in SEQ ID NOs: 1-31 are presented according to Kabat nomenclature and are shown below in Table 1. As used herein, HCDRs represent heavy chain CDRs and LCDRs represent light chain CDRs.

[0070] Because additional nomenclature systems are known in the art, CDR sequences based on the most commonly used of these nomenclature systems are also shown below in Table 1, but only for those instances where application of these alternative nomenclature systems resulted in different amino acid sequences. These numbering systems are based on: (i) CCG (Chemical Computing Group, as exemplified in Almagro et al., Proteins 2011;79:3050-3066 and Maier et al, Proteins 2014;82:1599-1610), (ii) Chothia (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917), (iii) IMGT (Lefranc MP, Dev Comp Immunol. 2003 Jan;27(1):55-77), and (iv) North (North B, J Mol Biol.(2011)406:228-56).

[0071] The amino acid positions indicated for the CDRs herein according to Kabat, CCG, Chothia, IMGT, and North positions (see Table 1) are linear, i.e., the amino acids of each full length chain are numbered consecutively starting from the N-terminus at number 1 and ending with the number corresponding to the total number of amino acids in the molecule. For example, a heavy chain 118 amino acids in length would start at the N-terminus at number 1 and end with the most C-terminal amino acid at number 118. Thus, for example, position 25 is meant to refer to the 25th amino acid counting from the N-terminus of the molecule.

[0072] In a more preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to TROP2 is selected from the group consisting of antigen-binding sites (i) to (xii): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 83 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (iii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 87 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 88; (iv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 90; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 91 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 92; (vi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (vii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (viii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96; (ix) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (x) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (xi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99; or (xii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96.

[0073] According to the present invention, the terms "immunoglobulin heavy chain variable domain" and "immunoglobulin light chain variable domain" are used according to their definition in the art.

[0074] In another preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CDH17 is selected from the group consisting of antigen-binding sites (i) to (ii): (i) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 34 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 35 (CDR1), SEQ ID NO: 36 (CDR2), and SEQ ID NO: 37 (CDR3); and (ii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 34 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 39 (CDR1), SEQ ID NO: 40 (CDR2), and SEQ ID NO: 37 (CDR3).

[0075] The CDRs disclosed herein and depicted in SEQ ID NOs: 32-40 are also presented according to the Kabat nomenclature and are shown below in Table 1. Again, as additional nomenclatures are known in the art, CDR sequences based on the most commonly used of these nomenclatures are also shown below in Table 1, but only for those instances where application of these alternative nomenclatures resulted in different amino acid sequences.

[0076] In a more preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CDH17 is selected from the group consisting of antigen-binding sites (i) to (ii): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 100 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 101; And (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 102 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103.

[0077] In a preferred embodiment of the binding molecule of the invention, (a) the at least one antigen-binding site that specifically binds to TROP2 is comprised in a first polypeptide comprising a first immunoglobulin heavy chain constant CH1 domain, a first immunoglobulin heavy chain variable domain, optionally a first linker, a first immunoglobulin light chain constant domain, and a first immunoglobulin light chain variable domain; and / or (b) the at least one antigen binding site that specifically binds CDH17 is contained in a second polypeptide comprising a second immunoglobulin heavy chain constant CH1 domain, a second immunoglobulin heavy chain variable domain, optionally a second linker, a second immunoglobulin light chain constant domain, and a second immunoglobulin light chain variable domain.

[0078] In accordance with the present invention, the terms "immunoglobulin heavy chain constant CH1 domain" and "immunoglobulin light chain constant domain" are used according to their definition in the art. Preferably, the order listed above is from N-terminus to C-terminus.

[0079] The terms "first" and "second", and further below the term "third", when used herein in the context of particular polypeptides and immunoglobulin chains, are intended only to indicate that these molecules are different molecules (because they bind to different target antigens). As such, these terms should not be understood as referring to the exact order or sequence of the polypeptides or immunoglobulin chains within the binding proteins of the invention. Instead, the term "first" is used when referring to the aspect relating to binding to TROP2, the term "second" is used when referring to the aspect relating to binding to CDH17, and the term "third" is used when referring to the aspect relating to binding to CD3.

[0080] The term linker is defined herein above, and suitable linkers are described in the art. In a preferred embodiment of the binding molecule of the present invention, the first linker and / or the second linker comprises any one of the amino acid sequences of SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:273, SEQ ID NO:274, or SEQ ID NO:275. More preferably, the first and / or the second linker comprises the amino acid sequence of SEQ ID NO:266.

[0081] According to this embodiment of the binding molecule of the invention, the antigen-binding site for TROP2 and / or CDH17 is selected from the antigen-binding sites described herein above, whereas the antigen-binding site for CD3 is chosen by the skilled artisan from those CD3-specific antigen-binding sites available in the art or from the CD3-specific antigen-binding sites disclosed herein. More preferably, the CD3-specific antigen-binding site is chosen from one of the scFv sequences defined herein below (SEQ ID NOs: 222 to 264, preferably SEQ ID NOs: 222 to 245, more preferably SEQ ID NOs: 222 to 224).

[0082] The binding molecules of the present invention may, and preferably do, comprise an Fc portion. The term Fc portion is known in the art and is defined herein above. The Fc region of a natural immunoglobulin molecule typically interacts with a number of Fc receptors, thereby providing a number of important functional capabilities (referred to as "effector functions"). In accordance with the present invention, it is preferred that the binding molecules of the present invention comprise an Fc region, or a portion of an Fc region, that does not interfere with the specific binding of the binding molecules of the present invention to the relevant portion of the target antigen. Furthermore, the skilled artisan is well aware that 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 characteristics, and on the desired pharmacological properties of the antibody protein.

[0083] Thus, in a preferred embodiment of the binding molecules of the invention, the binding molecules have an Fc region, or relevant portion thereof, that is engineered to avoid unintended cross-linking by soluble Fc gamma receptors and / or complement C1q. Preferably, such binding molecules have a much lower affinity for Fc gamma receptors and / or complement C1q than the non-engineered binding molecule (i.e., the non-Fc engineered binding molecule from which the mutant (engineered) molecule is derived). Such immunoglobulin molecules are often referred to as Ig(KO).

[0084] In a particularly preferred embodiment of the binding molecules of the invention, binding to the complement product C1q or Fc gamma receptor by the binding molecules of the invention is ablated by use of the IgG4 constant region or of the IgG1 constant region with directed L to A mutagenesis at positions 234 and 235 (the so-called "LA-LA mutation"; Hezareh et al., J. Virology 75(2001)12161-12168). Preferably, the binding molecules of the invention comprise said L to A mutagenesis.

[0085] In another preferred embodiment of the binding molecule of the invention, the binding molecule comprises an Fc region, or a relevant section thereof, that has been engineered to modify serum levels (half-life) by optimizing its interaction with the neonatal Fc receptor (FcRn) through a point mutation at position H310A (according to the EU numbering scheme) in the CH2 domain. Such an Ig molecule is referred to herein as IgFc Rnmut.

[0086] One challenge in the design of multimeric binding molecules is their production on an industrial scale without random chain pairing and other mismatches. Those skilled in the art are aware of numerous approaches to try to prevent the formation of heavy chain homodimers in particular, including the so-called knob-in-hole (KiH) strategy, the use of opposite charges to create an electrostatic steering effect, or hydrophobic mutations to promote heterodimerization of heavy chains, CH3 strand exchange engineering domains (SEED Technology), and fusion of heterodimerization modules, such as cleavable leucine zippers at the C-terminus of the CH3 domain (LUZ-Y Technology), as recently summarized by Amaral et al. (J Appl Bioanal 6(1), 26-51(2020)).

[0087] Thus, it is particularly preferred that the binding molecules of the invention comprise at least a mutation that results in knob-in-hole formation. Preferably, the binding molecules of the invention are based on modified immunoglobulin molecules, in which the heavy chain of the Ig molecule portion comprises the following mutation: (i) the first heavy chain comprises a tryptophan (W) at position 366 [T366W] and the second heavy chain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]; (ii) the first heavy chain contains a tyrosine (Y) at position 366 [T366Y] and the second heavy chain contains a threonine (T) at position 407 [Y407T]; (iii) the second heavy chain comprises a tryptophan (W) at position 366 [T366W] and the first heavy chain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]; or (iv) the second heavy chain comprises a tyrosine (Y) at position 366 [T366Y] and the first heavy chain comprises a threonine (T) at position 407 [Y407T]; And more preferably, the portion of the immunoglobulin molecule comprising the first heavy chain is the portion that also comprises an antigen binding site that specifically binds to TROP2 (herein also referred to as the TROP2-binding portion of the Ig molecule), and the portion of the immunoglobulin molecule comprising the second heavy chain is the portion that also comprises an antigen binding site that specifically binds to CDH17 (herein also referred to as the CDH17-binding portion of the Ig molecule).

[0088] According to this preferred embodiment, the numbering is based on the respective heavy chain, with the first amino acid at the N-terminus of the constant region of the heavy chain being at position 118 according to the EU numbering scheme. In other words, the tryptophan at position 366 is the 366th amino acid in the heavy chain constant region, starting N-terminus at position 118. This numbering is well known to those skilled in the art and is described in the literature, see for example the IMGT Scientific chart with reference to the EU numbering system for CH1. Here, for IgG1, the constant region starts at amino acid 118 "A" according to Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0089] Preferably, the binding molecules of the invention comprise the mutations cited in (i) above, i.e. one binding portion of the Ig molecule comprises a knob mutation (T366W) while the other binding portion of the Ig molecule comprises hole mutations (T366S, L368A, Y407V). More preferably, the TROP2 binding portion of the Ig molecule comprises a knob mutation (T366W) while the CDH17 binding portion of the Ig molecule comprises hole mutations (T366S, L368A, and Y407V). The skilled artisan knows how to combine the target-specific antigen binding sites described herein (preferably comprising or consisting of a single-chain Fab) with Fc domains comprising such knob or hole mutations; for example, the Fc domains depicted in SEQ ID NOs: 434 and 435 can be used to place any antigen binding site of interest, preferably a Fab, in the desired knob or hole context.

[0090] In an even more preferred embodiment of the binding molecule of the invention, the heavy chain comprising the Hole mutation additionally further comprises the following mutation: v) an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F] (leading to the elimination of the Protein A binding site); and / or vi) Alanine at position 234 [L234A] and position 235 [L235A], as discussed above. The same considerations regarding the numbering defined above apply mutatis mutandis. Further mutations that can be applied by those skilled in the art are well known in the art and are described, for example, in Saunders KO (2019). Front. Immunol. 10:1296.

[0091] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises: (ai) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 169 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 170; (a-ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 171 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 172; (a-iii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 173 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 174; (a-iv) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 175 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 176; (av) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 177 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 178; (a-vi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 179 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 180; (a-vii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 181 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 182; (a-viii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 183 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 184; (a-ix) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 185 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 186; (ax) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 187 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188; (a-xi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 189 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190; or (a-xii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 191 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192; and / or (bi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 196 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 197; or (b-ii) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 198 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 199.

[0092] According to the present invention, the terms "immunoglobulin heavy chain" and "immunoglobulin light chain" are used according to their definition in the art. Thus, these terms relate to the respective chains or domains of IgG, e.g., IgG1, IgG2, or IgG4.

[0093] In particular, an immunoglobulin heavy chain typically comprises, in the order listed, a heavy chain variable domain VH, a first heavy chain constant CH1 domain, and an Fc domain, and includes a hinge region and two constant domains (CH2 and CH3), whereas an immunoglobulin light chain typically comprises, in the order listed, a light chain variable domain VL and a first light chain constant CL1 domain.

[0094] According to this embodiment of the binding molecule of the invention, the antigen binding sites for TROP2 and / or CDH17 are comprised in the specific sequences listed above, whereas the antigen binding site for CD3 is selected by the skilled artisan from those CD3-specific antigen binding sites available in the art or from the CD3-specific antigen binding sites disclosed herein. More preferably, the CD3-specific antigen binding site is selected from one of the scFv sequences defined herein below (SEQ ID NOs: 222 to 264, preferably SEQ ID NOs: 222 to 245, more preferably SEQ ID NOs: 222 to 224).

[0095] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises an immunoglobulin molecule comprising at least a first and a second polypeptide, (a) the first polypeptide specifically binds TROP2 and comprises a combination of an immunoglobulin heavy chain and an immunoglobulin light chain as defined herein above in (ai) to (a-xii), optionally linked by a first linker; and (b) the second polypeptide specifically binds to CDH17 and comprises a combination of an immunoglobulin heavy chain and an immunoglobulin light chain as defined herein above in (bi) to (b-ii), optionally linked by a second linker.

[0096] The term linker is defined herein above, and suitable linkers are described in the art. In a preferred embodiment of the binding molecule of the present invention, it comprises any one of the amino acid sequences of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 273, SEQ ID NO: 274, or SEQ ID NO: 275. More preferably, the first and / or second linker comprises the amino acid sequence of SEQ ID NO: 266.

[0097] Also, according to this embodiment of the binding molecule of the invention, the antigen binding sites for TROP2 and CDH17 are comprised in the specific sequences listed above, whereas the antigen binding site for CD3 is selected by the skilled artisan from those CD3-specific antigen binding sites available in the art or from the CD3-specific antigen binding sites disclosed herein. More preferably, the CD3-specific antigen binding site is selected from one of the scFv sequences defined herein below (SEQ ID NOs: 222 to 264, preferably SEQ ID NOs: 222 to 245, more preferably SEQ ID NOs: 222 to 224).

[0098] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises: (ai) the amino acid sequence of SEQ ID NO: 200; (a-ii) the amino acid sequence of SEQ ID NO: 207; (a-iii) the amino acid sequence of SEQ ID NO: 208; (a-iv) the amino acid sequence of SEQ ID NO: 209; (av) the amino acid sequence of SEQ ID NO: 210; (a-vi) the amino acid sequence of SEQ ID NO: 211; (a-vii) the amino acid sequence of SEQ ID NO: 212; (a-viii) the amino acid sequence of SEQ ID NO: 213; (a-ix) the amino acid sequence of SEQ ID NO: 214; (ax) the amino acid sequence of SEQ ID NO: 215; (a-xi) the amino acid sequence of SEQ ID NO: 216; or (a-xii) the amino acid sequence of SEQ ID NO: 217; and / or (bi) the amino acid sequence of SEQ ID NO: 220; or (b-ii) Contains the amino acid sequence of SEQ ID NO: 221.

[0099] Also according to this embodiment of the binding molecule of the invention, the antigen binding sites for TROP2 and CDH17 are comprised in the specific sequences listed above, whereas the antigen binding site for CD3 is selected by the skilled artisan from those CD3 specific antigen binding sites available in the art or from the CD3 specific antigen binding sites disclosed herein. More preferably, the CD3 specific antigen binding site is selected from one of the scFv sequences (SEQ ID NOs: 222-264) defined herein below. Even more preferably, the CD3 specific antigen binding site is selected from one of the scFv sequences of SEQ ID NOs: 222-245. Even more preferably, the CD3 specific antigen binding site is selected from one of the scFv sequences of SEQ ID NOs: 222-244.

[0100] In a further preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CD3 is selected from the group consisting of antigen-binding sites (i) to (xxxi): (i) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (ii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 46 (CDR3); (iii) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (iv) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 50 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (v) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (viii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 55 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (ix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (x) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xi) an antigen-binding site comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3), and light chain CDRs comprising the amino acid sequences of SEQ ID NO: 60 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xiii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 62 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xiv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 54 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xvi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xvii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xviii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 72 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xx) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxiii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 75 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxiv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxv) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 76 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxvi) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxvii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxviii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxix) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xxx) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); and (xxxi) An antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3).

[0101] The definitions and preferred embodiments listed above in the context of the antigen-binding site for TROP2 and / or CDH17 also apply to this at least one antigen-binding site specifically binding to CD3, unless stated otherwise. In particular, the order of the specific elements within the antigen-binding site, the definitions of terms not specific to TROP2 or CDH17, as well as the general design and structural aspects of the elements of the binding molecules of the invention also apply to this CD3-specific antigen-binding site. Furthermore, it is also preferred for this CD3-specific antigen-binding site that it is completely composed of amino acids, i.e. that it is comprised in a polypeptide and / or that the CDRs are peptides or polypeptides. This will also be understood to mean that any linker, if present, is preferably a peptide linker, preferably a flexible linker as defined herein above. Preferably, any such linker comprises the amino acid sequence of any one of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 273, SEQ ID NO: 274 or SEQ ID NO: 275. More preferably, the linker comprises the amino acid sequence of SEQ ID NO: 265.

[0102] The CDRs disclosed herein and depicted in SEQ ID NOs: 41-82 are also presented according to the Kabat nomenclature and are shown below in Table 1. Again, as additional nomenclatures are known in the art, CDR sequences based on the most commonly used of these nomenclatures are also shown below in Table 1, but only for those instances where application of these alternative nomenclatures resulted in different amino acid sequences.

[0103] In an even more preferred embodiment of the binding molecule of the invention, the at least one antigen binding site that specifically binds to CD3 comprises a third immunoglobulin heavy chain variable domain and a third immunoglobulin light chain variable domain, optionally linked by a third linker.

[0104] In a more preferred embodiment of the binding molecule of the invention, the at least one antigen-binding site that specifically binds to CD3 is selected from the group consisting of antigen-binding sites (i) to (xvi): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:104 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:123, and SEQ ID NO:129; (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 110 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 111; (iii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 112 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 113; (iv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 118 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 119 and SEQ ID NO: 122; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 124 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; (v) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 130, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 127; (vi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 131 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 132; (vii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 133 and an immunoglobulin light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; (viii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 136 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 137; (ix) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and SEQ ID NO: 156, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 139; (x) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 161, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 141; (xi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 144 and SEQ ID NO: 146, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 145; (xii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, and SEQ ID NO: 162, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 148; (xiii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 149 and SEQ ID NO: 152, and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 150; (xiv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (xv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 158; and (xvi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 160.

[0105] It is also preferred that the antigen-binding site that specifically binds to CD3 is a single chain variable fragment (scFv).More preferably, the scFv is arranged such that the heavy chain variable domain is at its N-terminus and the light chain variable domain is at its C-terminus.

[0106] Methods for linking a polypeptide of interest, including scFv molecules, to, for example, the C-terminus of the heavy chain of an IgG molecule, are well known in the art. It will be appreciated that the fusion of the scFv to the Ig molecule may be a direct fusion or may be via a linker, preferably a peptide linker having a length of 5-40 amino acids. More preferably, the peptide linker comprises the amino acid sequence of any one of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 273, SEQ ID NO: 274, or SEQ ID NO: 275. More preferably, the linker comprises the amino acid sequence of SEQ ID NO: 275. In practice, this linking is usually achieved by combining a nucleic acid molecule encoding the IgG of interest with a nucleic acid encoding the desired polypeptide, e.g., an scFv, flanked, if necessary, by nucleic acid molecules encoding linker sequences, thereby forming a single nucleic acid molecule containing all three elements. This complete HC-scFv-encoding nucleic acid molecule is then placed into an expression vector and introduced into a suitable host cell such that a complete IgG heavy chain-scFv single polypeptide is formed.

[0107] In an even more preferred embodiment of the binding molecule of the invention, at least one antigen-binding site that specifically binds to CD3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264. Even more preferably, the CD3-specific antigen-binding site is selected from one of the scFv sequences of SEQ ID NOs: 222 to 245. Even more preferably, the CD3-specific antigen-binding site is selected from one of the scFv sequences of SEQ ID NOs: 222 to 224.

[0108] As set forth herein above, the numbering (a), (b), and (c) of the antigen binding sites of the binding molecules of the invention are not intended to indicate any particular order or format.

[0109] However, it is preferred that the first antigen-binding site that specifically binds TROP2 and the second antigen-binding site that specifically binds CDH17 are both comprised within one immunoglobulin (Ig) molecule, i.e. they are arranged in a conventional IgG antibody format, for example forming a Y-shaped structure, as shown in FIG. 3, where one half of the Ig molecule (e.g. comprising a first light chain linked to a first heavy chain) comprises the antigen-binding site for TROP2 and the second half of the Ig molecule (e.g. comprising a second light chain linked to a second heavy chain) comprises the antigen-binding site for CDH17. Preferably, the Ig molecule is an IgG or has a format derived from an IgG. More preferably, the immunoglobulin molecule is modified such that at least one antigen-binding site that specifically binds CD3 is fused thereto, preferably as a single chain variable fragment (scFv).

[0110] Thus, in a particularly preferred embodiment of the binding molecule of the invention, the binding molecule is a modified immunoglobulin (Ig) molecule, preferably a modified IgG molecule, in which the at least one antigen binding site that specifically binds TROP2 and the at least one antigen binding site that specifically binds CDH17 are present in the variable region of the Ig molecule and the at least one antigen binding site that specifically binds CD3 is at least one scFv fused to the TROP2-CDH17 specific Ig molecule. More preferably, the scFv is fused to the C-terminus of a heavy chain of the Ig molecule, preferably to the C-terminus of only one of the two heavy chains, more preferably to the part of the heavy chain of the Ig molecule that comprises at least one antigen binding site that specifically binds CDH17, and as depicted in FIG.

[0111] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises: (a) A first immunoglobulin light chain and immunoglobulin heavy chain combination selected from (a-xii): (ai) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 169 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 170; (a-ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 171 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 172; (a-iii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 173 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 174; (a-iv) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 175 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 176; (av) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 177 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 178; (a-vi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 179 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 180; (a-vii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 181 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 182; (a-viii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 183 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 184; (a-ix) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 185 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 186; (ax) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 187 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188; (a-xi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 189 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190; or (a-xii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 191 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192; And (b) A second combination of an immunoglobulin heavy chain and an immunoglobulin light chain selected from (bi) to (b-ii): (bi) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 196 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 197; or (b-ii) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 198 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 199; And (c) A single-chain variable fragment (scFv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 244.

[0112] Preferably, the first immunoglobulin heavy chain is fused to the first immunoglobulin light chain via a first linker, preferably a linker of SEQ ID NO: 266, and / or the second immunoglobulin heavy chain is fused to the second immunoglobulin light chain via a second linker, preferably a linker of SEQ ID NO: 266, and / or the scFv is fused to the C-terminus of the second immunoglobulin heavy chain via a third linker, preferably a linker of SEQ ID NO: 275.

[0113] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises: (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 200, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 200, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 207, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 207, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (v) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 208, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (vi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 208, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (vii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 209, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (viii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 209, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (ix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 210, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (x) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 210, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 211, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 211, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 212, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 212, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 213, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xvi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 213, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xvii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 214, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xviii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 214, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 215, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xx) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 215, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xxi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 216, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xxii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 216, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; (xxiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 217, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224; or (xxiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 217, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221, and a third polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264, more preferably from the group consisting of SEQ ID NOs: 222 to 245, and even more preferably from the group consisting of SEQ ID NOs: 222 to 224.

[0114] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises or consists of a first amino acid sequence selected from the group consisting of SEQ ID NOs: 200, 207-217, and 436 and a second amino acid sequence selected from the group consisting of SEQ ID NOs: 267-272, 424-427, and 437.

[0115] In an even more preferred embodiment of the binding molecule of the invention, the binding molecule comprises or consists of: (ai) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 267; (a-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 267; (a-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 267; (a-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 267; (av) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 267; (a-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 267; (a-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 267; (a-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 267; (a-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 267; (ax) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 267; (a-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 267; (a-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 267; (bi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 268; (b-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 268; (b-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 268; (b-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 268; (bv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 268; (b-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 268; (b-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 268; (b-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 268; (b-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 268; (bx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 268; (b-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 268; (b-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 268; (ci) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 269; (c-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 269; (c-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 269; (c-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 269; (cv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 269; (c-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 269; (c-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 269; (c-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 269; (c-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 269; (cx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 269; (c-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 269; (c-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 269; (di) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 270; (d-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 270; (d-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 270; (d-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 270; (dv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 270; (d-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 270; (d-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 270; (d-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 270; (d-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 270; (dx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 270; (d-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 270; (d-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 270; (ei) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 271; (e-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 271; (e-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 271; (e-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 271; (ev) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 271; (e-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 271; (e-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 271; (e-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 271; (e-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 271; (ex) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 271; (e-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 271; (e-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 271; (fi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 272; (f-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 272; (f-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 272; (f-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 272; (fv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 272; (f-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 272; (f-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 272; (f-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 272; (f-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 272; (fx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 272; (f-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 272; (f-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 272; (gi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 424; (g-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 424; (g-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 424; (g-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 424; (gv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 424; (g-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 424; (g-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 424; (g-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 424; (g-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 424; (gx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 424; (g-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 424; (g-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 424; (hi) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 425; (h-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 425; (h-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 425; (h-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 425; (hv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 425; (h-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 425; (h-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 425; (h-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 425; (h-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 425; (hx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 425; (h-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 425; (h-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 425; (ii) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 426; (i-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 426; (i-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 426; (i-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 426; (iv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 426; (i-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 426; (i-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 426; (i-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 426; (i-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 426; (ix) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 426; (i-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 426; (i-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 426; (ji) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 427; (j-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 427; (j-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 427; (j-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 427; (jv) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 427; (j-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 427; (j-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 427; (j-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 427; (j-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 427; (jx) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 427; (j-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 427; (j-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 427; or (k) the amino acid sequence of SEQ ID NO: 436 and the amino acid sequence of SEQ ID NO: 437.

[0116] In one particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 267. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 267. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 267. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 267. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 271. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 271. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 271. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 271. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 271. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 272. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 272. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 272. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 272. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 424. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 425. In another particularly preferred embodiment, the binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 426.In another particularly preferred embodiment, a binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 427. In another particularly preferred embodiment, a binding molecule of the invention comprises the amino acid sequence of SEQ ID NO: 436 and the amino acid sequence of SEQ ID NO: 437.

[0117] As shown in the accompanying examples, these particularly preferred binding molecules of the invention bind all three target antigens, thereby bringing T cells into contact with tumor cells bearing both Trop2 and CDH17. Surprisingly, it was found that the incorporation of antigen binding sites with high affinity leads to monovalent binding and activity on cells expressing only one of these two targets, thus increasing the risk of unwanted side effects. Thus, the invention provides trispecific binding molecules comprising low affinity binders for tumor cell antigens. As is evident from the following examples, these low affinity, high affinity trispecific binding molecules of the invention provide excellent specificity for target cells.

[0118] The present invention further relates to a trispecific binding molecule comprising or consisting of: (i) the amino acid sequence of SEQ ID NO:207 and the amino acid sequence of SEQ ID NO:267; (ii) the amino acid sequence of SEQ ID NO:208 and the amino acid sequence of SEQ ID NO:267; (iii) the amino acid sequence of SEQ ID NO:209 and the amino acid sequence of SEQ ID NO:267; (iv) the amino acid sequence of SEQ ID NO:210 and the amino acid sequence of SEQ ID NO:267; (v) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:271; (vi) the amino acid sequence of SEQ ID NO:212 and the amino acid sequence of SEQ ID NO:271; (vii) the amino acid sequence of SEQ ID NO:213 and the amino acid sequence of SEQ ID NO:271; (viii) the amino acid sequence of SEQ ID NO:214 and the amino acid sequence of SEQ ID NO:271; (ix) the amino acid sequence of SEQ ID NO:215 and the amino acid sequence of SEQ ID NO: (x) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:272; (xi) the amino acid sequence of SEQ ID NO:214 and the amino acid sequence of SEQ ID NO:272; (xii) the amino acid sequence of SEQ ID NO:216 and the amino acid sequence of SEQ ID NO:272; (xiii) the amino acid sequence of SEQ ID NO:217 and the amino acid sequence of SEQ ID NO:272; (xiv) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:424; (xv) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:425; (xvi) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:426; (xvii) the amino acid sequence of SEQ ID NO:211 and the amino acid sequence of SEQ ID NO:427; or (xviii) the amino acid sequence of SEQ ID NO:436 and the amino acid sequence of SEQ ID NO:437. Table 1 below summarizes preferred amino acid sequences of the binding molecules of the invention, or portions thereof. [Table 1] TIFF2024523033000003.tif245165 TIFF2024523033000004.tif242165 TIFF2024523033000005.tif241165 TIFF2024523033000006.tif241165 TIFF2024523033000007.tif241165 TIFF2024523033000008.tif242165 TIFF2024523033000009.tif241165 TIFF2024523033000010.tif242165 TIFF2024523033000011.tif241165 TIFF2024523033000012.tif242165 TIFF2024523033000013.tif242165 TIFF2024523033000014.tif242165 TIFF2024523033000015.tif242165 TIFF2024523033000016.tif236165 TIFF2024523033000017.tif242165 TIFF2024523033000018.tif237165 TIFF2024523033000019.tif242165 TIFF2024523033000020.tif242165 TIFF2024523033000021.tif242165 TIFF2024523033000022.tif242165 TIFF2024523033000023.tif242165 TIFF2024523033000024.tif242165 TIFF2024523033000025.tif242165 TIFF2024523033000026.tif242165 TIFF2024523033000027.tif242165 TIFF2024523033000028.tif242165 TIFF2024523033000029.tif238165 TIFF2024523033000030.tif242165 TIFF2024523033000031.tif242165 TIFF2024523033000032.tif242165 TIFF2024523033000033.tif236165 TIFF2024523033000034.tif242165 TIFF2024523033000035.tif237165 TIFF2024523033000036.tif236165 TIFF2024523033000037.tif242165 TIFF2024523033000038.tif242165 TIFF2024523033000039.tif242165 TIFF2024523033000040.tif236165 TIFF2024523033000041.tif242165 TIFF2024523033000042.tif242165 TIFF2024523033000043.tif236165 TIFF2024523033000044.tif242165 TIFF2024523033000045.tif242165 TIFF2024523033000046.tif242165 TIFF2024523033000047.tif242165 TIFF2024523033000048.tif242165 TIFF2024523033000049.tif242165 TIFF2024523033000050.tif221165

[0119] The preferred antigen binding sites disclosed herein as Trop2#1, Trop2#8 to Trop2#18, CDH17#1, CDH17#8, and CD3#1 to CD3#43 have all been identified and tested by the inventors as suitable antigen binding sites for use in the trispecific binding molecules of the invention. In particular, the antigen binding sites are connected by flexible elements of polypeptide chains or linkers, which do not interfere with the structural and functional integrity of the antigen binding sites.

[0120] A monospecific binding molecule for TROP2. Further provided herein are antibody molecules that specifically bind to TROP2 (e.g., full-length antibodies / immunoglobulin molecules having a Y-shaped structure with two heavy chains and two light chains, or fragments thereof, such as Fv, Fab, Fab', or F(ab')2 fragments, single-chain antibodies, single-chain variable fragments (scFv)). In some embodiments, the antibody molecules specific for TROP2 are recombinant monoclonal antibodies, chimeric, humanized, or human antibody molecules.

[0121] In some embodiments, the antibody molecule specific for TROP2 comprises any one of the following CDR combinations as shown in (i) to (vi): (i) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); (ii) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3); (iii) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (iv) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 15 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 20 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (v) heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:21 (CDR1), SEQ ID NO:22 (CDR2), and SEQ ID NO:23 (CDR3) and light chain CDRs comprising the amino acid sequences of SEQ ID NO:24 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:26 (CDR3); or (vi) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:27 (CDR1), SEQ ID NO:28 (CDR2), and SEQ ID NO:29 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO:30 (CDR1), SEQ ID NO:25 (CDR2), and SEQ ID NO:31 (CDR3).

[0122] In some embodiments, the antibody molecule specific for TROP2 comprises: i) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 83 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (ii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (iii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 87 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 88; (iv) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 90; (v) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 91 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 92; (vi) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (vii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (viii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96; (ix) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (x) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (xi) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99; or (xii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96.

[0123] In some embodiments, the TROP2-specific antibody defined above further comprises a human heavy chain constant domain (e.g., an IgG constant domain) and a human light chain constant domain (e.g., a kappa or lambda light chain constant domain). In some embodiments, the heavy chain constant domain is human IgG1 wild type (e.g., as provided in SEQ ID NO: 414), IgG1 KO (e.g., as provided in SEQ ID NO: 415), IgG4 Pro wild type (e.g., as provided in SEQ ID NO: 416), or IgG1 FcRnmut (e.g., as provided in SEQ ID NO: 417). In some embodiments, the human light chain constant domain is human kappa (e.g., as provided in SEQ ID NO: 418).

[0124] In some embodiments, a TROP2-specific antibody has a heavy chain comprising any one of the sequences of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, or 98 fused to any one of the sequences of SEQ ID NOs: 414, 415, 416, or 417, and a light chain comprising any one of the sequences of SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 97, or 99 fused to the sequence of SEQ ID NO: 418.

[0125] The TROP2-specific antibodies provided herein can be used to label, localize, identify, or target cells expressing TROP2 in vitro, in vivo, or ex vivo (e.g., in ELISA assays, FACS analysis, immunohistology, or the like) by attaching dyes, drugs, or other molecules with binding specificity for different antigens. The TROP2-specific antibodies described herein alone have no effect on cell viability of cells expressing TROP2. In some embodiments, the TROP2-specific antibodies specifically bind to the surface of TROP2-expressing cells and are used to localize and / or identify such cells. In some embodiments, the TROP2 antibodies provided herein are used to identify cells expressing TROP2 (e.g., tumor cells). In some embodiments, the TROP2 antibodies provided herein are used to deliver drugs or cytotoxic agents to target cells (e.g., tumor cells expressing TROP2) by attaching such drugs or cytotoxic agents to the TROP2 antibodies, thereby, for example, killing the target cells.

[0126] Also provided herein is a method for detecting trophoblast cell surface antigen 2 (TROP2) in a sample, the method comprising the steps of: (a) contacting the sample with an anti-TROP2 antibody molecule as defined herein above; (b) allowing the formation of antibody-antigen complexes in the sample; and (c) Detecting anti-TROP2 antibodies.

[0127] Means and methods for detecting antibodies are well known in the art and include, for example, immunohistochemistry, immunoblotting, and ELISA.

[0128] Further provided herein is a kit for detecting trophoblast cell surface antigen 2 (TROP2), wherein the kit comprises an anti-TROP2 antibody molecule as defined herein above and instructions for use.

[0129] A monospecific binding molecule for CDH17. Further provided herein are antibody molecules that specifically bind to CDH17 (e.g., full length antibodies / immunoglobulin molecules having a Y-shaped structure with two heavy chains and two light chains, or fragments thereof, such as Fv, Fab, Fab', or F(ab')2 fragments, single chain antibodies, single chain variable fragments (scFv)). In some embodiments, the antibody molecules specific for CDH17 are recombinant monoclonal antibodies, chimeric, humanized, or human antibody molecules.

[0130] In some embodiments, an antibody molecule specific for CDH17 comprises any one of the following CDR combinations as depicted in (i) to (ii): (i) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 34 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 35 (CDR1), SEQ ID NO: 36 (CDR2), and SEQ ID NO: 37 (CDR3); And (ii) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 34 (CDR3), and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 39 (CDR1), SEQ ID NO: 40 (CDR2), and SEQ ID NO: 37 (CDR3).

[0131] In some embodiments, the antibody molecule specific for CDH17 is (i) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 100 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 101; or (ii) an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 102 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103.

[0132] In some embodiments, the CDH17-specific antibody defined above further comprises a human heavy chain constant domain (e.g., an IgG constant domain) and a human light chain constant domain (e.g., a kappa or lambda light chain constant domain). In some embodiments, the heavy chain constant domain is human IgG1 wild type (e.g., as provided in SEQ ID NO: 414), IgG1 KO (e.g., as provided in SEQ ID NO: 415), IgG4 Pro wild type (e.g., as provided in SEQ ID NO: 416), or IgG1 FcRnmut (e.g., as provided in SEQ ID NO: 417). In some embodiments, the human light chain constant domain is human kappa (e.g., as provided in SEQ ID NO: 418).

[0133] In some embodiments, a CDH17-specific antibody has a heavy chain comprising the sequence of any one of SEQ ID NOs: 100 or 102 fused to the sequence of any one of SEQ ID NOs: 414, 415, 416, or 417, and a light chain comprising the sequence of any one of SEQ ID NOs: 101 or 103 fused to the sequence of SEQ ID NO: 418.

[0134] The CDH17-specific antibodies provided herein can be used to label, localize, identify, or target cells expressing CDH17 in vitro, in vivo, or ex vivo (e.g., in ELISA assays, FACS analysis, immunohistology, or the like) by attaching dyes, drugs, or other molecules with binding specificity for different antigens. The CDH17-specific antibodies described herein alone have no effect on cell viability of cells expressing CDH17. In some embodiments, the CDH17-specific antibodies specifically bind to the surface of CDH17-expressing cells and are used to localize and / or identify such cells. In some embodiments, the CDH17 antibodies provided herein are used to identify cells expressing CDH17 (e.g., tumor cells). In some embodiments, the CDH17 antibodies provided herein are used to deliver a drug or cytotoxic agent to a target cell (e.g., a tumor cell that expresses CDH17) by attaching such a drug or cytotoxic agent to the CDH17 antibody, thereby, for example, killing the target cell.

[0135] Also provided herein is a method for detecting cadherin-17 (CDH17) in a sample, the method comprising the steps of: (a) contacting the sample with an anti-CDH17 antibody molecule as defined herein above; (b) allowing the formation of antibody-antigen complexes in the sample; and (c) detecting anti-CDH17 antibodies;

[0136] Means and methods for detecting antibodies are well known in the art and include, for example, immunohistochemistry, immunoblot, and ELISA.

[0137] Further provided herein is a kit for detecting trophoblast cell surface antigen 2 (TROP2), wherein the kit comprises an anti-TROP2 antibody molecule as defined herein above and instructions for use.

[0138] Nucleic Acid Molecules, Expression Vectors, and Host Cells of the Invention The present invention further relates to nucleic acid molecules encoding the binding molecules of the invention, or parts thereof.The present invention further encompasses sets of nucleic acid molecules encoding the binding molecules of the invention.

[0139] According to the present 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 which ensures that the binding molecule of the invention (or the respective part thereof) can be expressed.

[0140] The term "portion thereof" reflects the fact that not all elements of a binding molecule of the invention need be encoded on a single nucleic acid molecule, as will be understood by those skilled in the art. Instead, two or more nucleic acid molecules may be relied upon to individually encode specific portions of a binding molecule of the invention. Thus, the invention also encompasses a set of isolated nucleic acid molecules, where the set together encodes all portions of a binding molecule of the invention, and expression of the set of isolated nucleic acid molecules results in the production of a complete binding molecule of the invention. In other words, one or more nucleic acid molecules are provided herein that encode the individual polypeptide chains of a binding molecule of the invention, including heavy chains, light chains, scFvs, and combinations thereof, separately on individual nucleic acid molecules or in combination in one nucleic acid molecule.

[0141] Preferably, the binding molecule of the invention is encoded by two different isolated nucleic acid molecules, where a first nucleic acid molecule encodes a full length chain comprising an antigen binding site that specifically binds TROP2 (comprising a single chain Fab and Fc domain) and a second nucleic acid molecule encodes a full length chain comprising an antigen binding site that specifically binds CDH17 (comprising a single chain Fab and Fc domain) linked to a full length chain comprising an antigen binding site that specifically binds CD3 (preferably as an scFv). Upon expression, both expressed polypeptides form the complete binding molecule of the invention, e.g. via a disulfide bond between the constant domains.

[0142] Preferably, the nucleic acid molecule is a DNA molecule comprising a coding sequence. More preferably, the DNA molecule additionally comprises regulatory sequences and, optionally, natural or artificial introns (such as, for example, the β-globin intron from Homo sapiens with an embedded miRNA-557 expression cassette). It may have its native codons or may have an optimized codon usage specifically adapted for expression in the intended host cell or host organism. Such nucleic acid molecules of the invention can be easily prepared or obtained by those skilled in the art, depending on methods known per se, for example, by automated DNA synthesis, isolation from natural sources and / or recombinant DNA techniques, based on the information on the amino acid sequences for the binding molecules of the invention provided herein.

[0143] The nucleic acid molecules of the present invention include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the sequence listing. The present invention further contemplates nucleic acid molecules complementary to the above defined DNA molecules, as well as nucleic acid molecules hybridizing thereto under high stringency binding and washing conditions, as defined in WO2007 / 042309. Preferred molecules (in terms of mRNA) are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90%, most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein. By way of example, if the aim is to express the binding molecules of the present invention in eukaryotic cells, the DNA sequences must be designed to match the codon usage in eukaryotic cells. If it is desired to express the antibodies in E. coli or other prokaryotic systems, these sequences must be designed so that the codon usage is consistent with E. coli or the respective prokaryotic system. Variants of the DNA molecules of the present invention can be constructed in several different ways, for example as described in WO2007 / 042309.

[0144] Preferably, the nucleic acid is isolated, the term "isolated" being further defined above.

[0145] The present invention further relates to an expression vector comprising the nucleic acid molecule of the invention.

[0146] According to the present invention, the vector is an expression vector, i.e. a vector capable of providing expression of the respective polypeptide from the encoding nucleic acid molecule in vitro and / or in vivo (e.g. in a suitable host cell, host organism, and expression system). Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. Expression vectors and expression control sequences are typically selected to be compatible with the host cell. Expression vectors generally comprise at least one nucleic acid molecule of the present invention operably linked to one or more suitable regulatory elements, such as promoters, enhancers, terminators, and the like. Specific examples of such regulatory elements and other elements useful or necessary for expressing the polypeptides of the present invention, such as integration factors, selection markers, signal or leader sequences, reporter genes, and the like, are disclosed, for example, in WO2006 / 040153, pp. 131-133.

[0147] Non-limiting examples of promoter sequences (illustrated 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, and strong mammalian promoters such as the native immunoglobulin and actin promoters. Examples of polyadenylation signals are hamster growth hormone or bovine growth hormone polyA, SV40 late or early polyA; alternatively, the 3'UTR of an immunoglobulin gene, and the like, can be used.

[0148] The recombinant expression vector may also carry sequences that regulate replication of the vector in a host cell (e.g., origins of replication, such as the ColE1 (pUC) origin of replication) and selectable marker genes (e.g., the β-lactamase gene that confers ampicillin resistance for amplification of the plasmid in E. coli). The recombinant expression vector may also encode a signal peptide that facilitates secretion of the resulting polypeptide. The nucleic acid molecule encoding each polypeptide chain may be cloned into the vector such 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 protein. Alternatively, the DNA sequence encoding the full-length chain of the protein of the invention may already contain a signal peptide sequence.

[0149] As indicated above, the coding sequence inserted in the vector can be, for example, synthesized by standard methods or isolated from natural sources, or produced semi-synthetically, i.e., by combining chemical synthesis and recombinant techniques. Ligation of the coding sequence to transcriptional regulatory elements and / or other amino acid coding sequences can be performed using established methods. One approach that is often used is to use, for example, a vector that encodes a functionally complete human CH (constant heavy chain) immunoglobulin sequence, with engineered suitable restriction sites, so that any antigen binding site, such as a single chain Fab sequence or any heavy / light chain variable domain, can be easily inserted and expressed. For the antibody heavy chain, it can be any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulin, including but not limited to, allelic variants.

[0150] If more than one nucleic acid molecule is required to construct the binding molecule of the present invention, these more than one nucleic acid molecule can be inserted into different or the same expression vector. In the latter case, they can be under the control of the same regulatory elements, such as promoters, enhancers, terminators and the like, or they can each have their own set of regulatory elements. In accordance with the present invention, if more than one nucleic acid molecule encodes the individual elements of the binding molecule of the present invention (i.e., a polypeptide chain comprising an antigen binding site that specifically binds to TROP2, a polypeptide chain comprising an antigen binding site that specifically binds to CHD17, and a polypeptide chain comprising an antigen binding site that specifically binds to CD3), it is particularly preferred that all the individual nucleic acid molecules required to form the binding molecule of the present invention are present on a single expression vector, preferably each nucleic acid molecule having its own set of regulatory elements.

[0151] 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.

[0152] Thus, the present invention also relates to a host cell transfected with an expression vector of the present invention.

[0153] The host cell may be any suitable cell known in the art, including prokaryotic cells, such as bacteria, and eukaryotic cells, such as yeast cells or mammalian cells. Non-limiting examples of mammalian cells include, but are not limited to, human, mouse, rat, monkey, and rodent cell lines. Specific mammalian cell lines available as host cells for expression are well known in the art, including, among others, Chinese hamster ovary (CHO) cells, NS0, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., Hep G2 cells and A-549 cells), 3T3 cells, or derivatives / progenies of any such cell lines. Suitable culture media and conditions for the above host cells are known in the art.

[0154] Methods for producing the binding molecules of the invention The present invention also relates to a method for producing a binding molecule of the invention, comprising the steps of: (a) culturing a host cell of the invention under conditions allowing expression of a binding molecule of the invention; (b) optionally recovering the molecule; and, optionally, (c) further purifying and / or modifying and / or formulating the binding molecule.

[0155] The proteins of the invention are produced by culturing the host cells for a period of time sufficient to allow expression of the protein by the host cells.

[0156] Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art. Suitable additives known to buffer the medium or to enhance or promote both can be added to increase the yield and solubility of the expression product. In general, those skilled in the art will also recognize that these conditions will have to be adapted to the needs of the host and the requirements of the molecule to be expressed. In the case where an inducible promoter controls the nucleic acid molecule of the invention in a vector present in a host cell, the expression of the molecule of interest can be induced by the addition of a suitable inducer. Suitable expression protocols and strategies are known to those skilled in the art.

[0157] The binding molecules of the invention are then recovered and, if necessary, further purified. Preferably, they are recovered from the culture medium as secreted molecules. However, they can also be recovered from host cell lysates, for example if they are expressed without a secretion signal. The term "recovering the molecule" will be understood to refer to the isolation of the binding molecules of the invention encoded by the nucleic acid molecule of the invention, i.e. the binding molecules present in the host cell of the invention due to transformation or transfection of the host cell with the nucleic acid molecule or vector of the invention.

[0158] Optional steps of purifying the binding molecules of the present invention further aid in obtaining substantially homogeneous preparations of the molecules. Means and methods for purifying molecules of interest are well known, and the skilled artisan can use standard protein purification methods, for example, used for recombinant proteins and host cell proteins, and adjust them in a suitable manner for each molecule. By way of example, state-of-the-art purification methods useful for obtaining the binding molecules of the present invention include, as a first step, the removal of cells and / or particulate cell debris from the culture medium or lysate, followed by the removal of contaminating soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on an immunoaffinity column or an ion exchange column, ethanol precipitation, reversed-phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin.

[0159] As a final optional step in the process of obtaining the binding molecules of the invention, the purified protein molecules may be dried, e.g., lyophilized as described below for therapeutic applications, or formulated if desired. Furthermore, the resulting binding molecules of the invention may be subjected to further modifications, e.g., to remove unwanted post-translational modifications and the like.

[0160] Pharmaceutical compositions and medical uses of the binding molecules or of the pharmaceutical compositions of the invention The present invention further relates to a pharmaceutical composition comprising or consisting of one or more binding molecules of the invention. In one embodiment, the binding molecule is the only pharma- ceutical active agent. In an alternative embodiment, the composition comprises, in addition to the binding molecule, one or more further pharma- ceutical active agents, e.g., as further defined below.

[0161] According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention comprises the compounds listed above, alone or in combination. It may optionally comprise additional molecules capable of changing the properties of the compounds of the present invention, thereby, for example, stabilizing, regulating and / or activating their function. The composition may be in the form of a solid, liquid or gas, in particular in the form of a powder, such as a lyophilized powder, (a) a solution, (a) a tablet, or (a) an aerosol. Preferably, the composition is a lyophilized powder or a solution.

[0162] For use in therapy, the binding molecules or antibodies of the present invention are formulated in pharmaceutical compositions suitable for facilitating administration to animals or humans. Thus, the pharmaceutical compositions of the present invention also preferably include a pharma- ceutically acceptable carrier. Compositions including such carriers can be formulated by well-known conventional methods. Typically, pharmaceutical compositions including the binding molecules of the present invention can be formulated by mixing the binding molecules with such pharma- ceutically acceptable carriers, as well as (optionally) excipients or stabilizers. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any kind. Additionally, other excipients, modifiers, or stabilizers are non-toxic at the dosages and concentrations employed. Pharmaceutically acceptable carriers, excipients, modifiers, and stabilizers include, but are not limited to, buffer systems such as phosphate, citric acid, acetic acid, other inorganic or organic acids and their salts; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; proteins such as serum albumin, gelatin, or immunoglobulins; 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 dextran; 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™ (polysorbates), PLURONICS™, or fatty acid esters, fatty acid ethers, or sugar esters.Organic solvents such as ethanol or isopropanol may also be included in the formulation. Excipients may also have release-modifying or absorption-modifying functions.

[0163] Typically, aqueous solutions or suspensions are preferred. In general, suitable formulations for therapeutic proteins, such as binding molecules of the invention, include buffered protein solutions, such as solutions containing the protein at an appropriate concentration (e.g., 0.001-400 mg / ml, preferably 0.005-200 mg / ml, more preferably 0.01-200 mg / ml, more preferably 1.0-100 mg / ml, such as 1.0-10.0 mg / ml (intravenous administration) or 100 mg / ml (subcutaneous administration)), and aqueous buffer solutions, such as: -Phosphate buffered saline, pH 7.4, -Other phosphate buffers, pH 6.2-8.2, Acetate buffer, pH 3.2-7.5, preferably pH 4.8-5.5 -Histidine buffer, pH 5.5-7.0, - succinate buffer, pH 3.2 to 6.6, or -Citrate buffer, pH 2.1 to 6.2, etc. and optionally salts (e.g., NaCl) and / or stabilizers (e.g., sucrose, trehalose, lysine, etc.) and / or other polyhydric alcohols (e.g., mannitol and glycerol) to provide isotonicity to the solution, and optionally surfactants (e.g., 0.02% Tween-20 or Tween-80) to prevent aggregation.

[0164] A preferred buffered protein solution for intravenous administration is a solution containing about 10 mg / ml of the 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. Formulations for subcutaneous application may contain significantly higher concentrations of the antibody of the invention, such as up to or even above 100 mg / ml. However, it will be clear to the skilled artisan that the above given ingredients and their amounts only represent one preferred option. Alternatives and variations thereof will be immediately clear to the skilled artisan or can be easily conceived starting from the above disclosure.

[0165] The pharmaceutical compositions of the invention can be administered to a subject 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 a solution or reconstituted lyophilized powder, parenteral modes of administration are preferred.

[0166] In general, for the treatment, prevention, and / or amelioration of the diseases, disorders, and conditions referred to herein, depending on the particular disease, disorder, or condition being treated, the potency of the specific binding molecule of the invention, the particular route of administration used, and the particular pharmaceutical formulation or composition used will have an impact on the actual dose administered. Furthermore, the actual pharmacologic effective amount or therapeutic dose will also depend on factors known by those skilled in the art, such as the age and weight of the patient. In any case, the binding molecule of the invention or the pharmaceutical composition of the invention will be administered in a dosage and manner that allows a pharmacologic effective amount to be delivered based on the unique condition of the patient. Preferably, the binding molecule of the invention or the pharmaceutical composition of the invention will be administered continuously (e.g., by infusion) or more preferably as a single dose, between 0.005-20.0 mg / kg body weight / dose, preferably between 0.05-10.0 mg / kg / dose, more preferably between 0.5-5 mg / kg / dose. The dosing interval can be, for example, twice weekly, weekly, or monthly doses, but can vary significantly depending, inter alia, on the parameters mentioned above. Thus, in some cases it may be sufficient to use less than the minimum dose given above, whereas in other cases the upper limit may have to be exceeded. When administering larger amounts, it is recommended to divide them into a number of smaller doses over the course of a day. Preferably, administration is once a week at a dose range from between 0.005-20.0 mg / kg body weight / dose, preferably between 0.05-10.0 mg / kg / dose, more preferably between 0.5-5 mg / kg / dose.

[0167] The efficacy of the binding molecules of the invention, and compositions comprising same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof, depending on the particular disease involved. Suitable assays and animal models will be apparent to those skilled in the art and include, for example, the assays and animal models used in the Examples below.

[0168] The binding molecules of the invention or the pharmaceutical compositions of the invention may be used on their own or in combination with other pharmacologically active ingredients, such as state-of-the-art or standard of care compounds, such as, for example, cytostatic or cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, steroids, immunomodulators / checkpoint inhibitors, and the like.

[0169] Thus, a further aspect of the invention provides a pharmaceutical composition comprising a binding molecule of the invention, or a binding molecule of the invention, together with one or more further active ingredients and optionally a pharma- ceutically acceptable carrier.

[0170] Cytostatic and / or cytotoxic active substances which may be administered as combination partners according to the invention include, but are not limited to, hormones, hormone analogues and antihormones, aromatase inhibitors, LHRH agonists and antagonists, inhibitors of growth factors (growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, e.g. HER2, HER3, HER4) and hepatocyte growth factor (HGF)), inhibitors such as, for example, (anti)-growth factors. 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., folate antagonists), such as methotrexate, raltitrexed, pyrimidine analogues, such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine, purine and adenosine analogues, such as mercaptopurine, thioguanine, cladribine, and pentostatin, cytarabine (arabinose), C), fludarabine, etc.); antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa, etc.); mitotic inhibitors (e.g., vinca alkaloids such as vinblastine, vindesine, vinorelbine, and vincristine, etc.; and taxanes such as paclitaxel, docetaxel, etc.); angiogenesis inhibitors, tubulin inhibitors, including bevacizumab, ramucirumab, and aflibercept;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, etc.), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, P LK1 inhibitors (e.g., volasertib), inhibitors of CDK, including CDK9 inhibitors, Aurora kinase inhibitors, tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors), 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 analogues (e.g., everolimus, tem sirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutic agents such as immune checkpoint inhibitors (CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins, such as ipilimumab, nivolumab, pembrolizumab, etc.) and various chemotherapeutic agents such as amifostine, anagrelide, clodronate, filgrastin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-39 such as feron, interferon alpha, leucovorin, rituximab, 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 / beta-catenin signaling pathway; stromal regulators, such as (preferably bispecific) molecules targeting CD137 and FAP; and / or cyclin-dependent kinase 9 inhibitors.

[0171] Preferred according to the invention is treatment with a binding molecule of the invention or a pharmaceutical composition of the invention in combination with a drug selected from: (i) anti-VEGF antibodies (bevacizumab and other antiangiogenic agents) with or without chemotherapy combinations, for example, particularly in breast cancer patients (including doxorubicin / cyclophosphamide combinations and / or capecitabine / docetaxel combinations in the neoadjuvant setting; taxane / platinum regimens in first and subsequent lines of treatment); (ii) chemotherapeutic agents used for the treatment of CRC (including 5-fluorouracil, irinotecan, doxorubicin, and TAS-102); (iii) chemotherapy combinations (including irinotecan), anti-VEGF antibody combinations (bevacizumab and other antiangiogenic agents), or anti-EGFR antibodies (cetuximab and panitumumab in KRAS wild-type tumors) with or without regorafenib combinations, for example for the treatment of CRC patients; and / or (iv) immunotherapeutic agents, including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents, such as ezabenlimab, pembrolizumab, and nivolumab, as well as other antibodies disclosed in WO2017 / 198741, for example, for the treatment of CRC patients; and / or (v) Stromal regulators, such as (preferably bispecific) molecules targeting CD137 and FAP.

[0172] In a particularly preferred embodiment, the binding molecule of the invention or the pharmaceutical composition of the invention is used for the treatment of cancer in combination with an immune checkpoint inhibitor, preferably with a PD-1 antagonist, such as an anti-PD-1 antibody or an anti-PDL-1 antibody. Preferably, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, or PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 as described in WO2017 / 198741 (herein incorporated by reference), more preferably, the anti-PD-1 antibody is ezabenlimab. Preferably, the anti-PDL-1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0173] The invention further relates to a binding molecule of the invention or a pharmaceutical composition of the invention for use in medicine.The invention further relates to a binding molecule of the invention or a pharmaceutical composition of the invention for use in the preparation of a medicament.

[0174] Furthermore, the present invention also relates to a binding molecule of the invention or a pharmaceutical composition of the invention for use in a method for treating, ameliorating, or preventing cancer.The present invention further relates to a method of treating, preventing, or ameliorating cancer comprising administering a therapeutically effective amount of a binding molecule of the invention or of a pharmaceutical composition of the invention to a patient in need thereof.

[0175] A "therapeutically effective amount" of a molecule administered is the minimum amount necessary to prevent, ameliorate, or treat clinical symptoms of cancer, particularly the minimum amount that is effective against the particular cancer being treated.

[0176] The term "cancer" as used herein is meant to include all kinds 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. referred to below, characterized by their specific location / origin in the body, are meant to be included as both primary tumors and metastatic tumors derived therefrom.

[0177] Cancers whose growth can be inhibited using the polyspecific binding molecules described herein are any TROP2 / CDH17 expressing tumors, including, but not limited to, T cell lymphoma, myeloid leukemia, breast cancer, ovarian cancer, oral squamous cell carcinoma, gastrointestinal cancer, including, but not limited to, esophageal cancer (e.g., gastroesophageal junction cancer), gastric (stomach) cancer, hepatocellular carcinoma, biliary tract cancer (e.g., cholangiocarcinoma), gallbladder cancer, pancreatic cancer, or colorectal cancer (CRC). In some embodiments, the following cancers, tumors, and other proliferative diseases may be treated with the polyspecific binding molecules of the invention: head and neck cancer, preferably HNSCC; lung cancer; preferably NSCLC; breast cancer; thyroid cancer; cervical cancer; ovarian cancer; endometrial cancer; liver cancer (hepatoblastoma or hepatocellular carcinoma); pancreatic cancer; prostate cancer; gastric sarcoma; gastrointestinal stromal tumors, esophageal cancer; colon cancer; colorectal cancer; kidney cancer; skin cancer; brain tumors; glioblastoma; non-Hodgkin's lymphoma (T or B cell lymphoma); leukemia (chronic or acute myeloid leukemia, non-lymphocytic leukemia), or multiple myeloma.

[0178] In a preferred embodiment of the binding molecule or pharmaceutical composition for use according to the invention, or the method of treating, preventing or ameliorating cancer of the invention, or the use of the invention, the cancer is colorectal cancer (CRC), including metastatic CRC (mCRC), gastric cancer (GC), pancreatic cancer (PAC) or esophageal cancer. In a particularly preferred embodiment of the invention, the cancer is mCRC.

[0179] Colorectal cancer (CRC) is a distinct 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 overt metastasis, and metastatic disease occurs in 40-50% of newly diagnosed patients. Although recent improvements in chemotherapy have extended survival in metastatic CRC, the majority of patients will succumb to their disease. Therefore, there is a great need for additional therapeutic agents to treat this disease.

[0180] Approximately 30-50% of colorectal cancers are known to have mutated (abnormal) KRAS genes. KRAS mutations frequently found in neoplasms include mutations in exon 2 (codons 12 and 13) and exon 3 (codon 61), which can be analyzed from tumor biopsies. They include activating mutations that result in continued signaling, stimulating downstream signaling pathways involved in cell growth, proliferation, invasion, and metastasis. Thus, in one embodiment, the binding molecules of the invention are for use in the treatment of KRAS mutant colorectal cancer (i.e., patients with KRAS mutant tumors). In an alternative embodiment, the binding molecules of the invention are for use in the treatment of KRAS wild-type colorectal cancer (i.e., patients with KRAS wild-type tumors).

[0181] Gastric cancer, also known as stomach cancer, is the third leading cause of cancer-related deaths. While incidence has declined since the early 20th century due to improvements in food preservation, gastric cancer remains an indication with a poor prognosis, as the majority of patients already present with advanced disease and treatment options are limited to surgery, chemotherapy, radiation therapy, and a limited amount of targeted therapy.

[0182] Pancreatic cancer (PAC) is a malignant disease that causes more than 400,000 deaths per year worldwide. It is among the most common causes of cancer-related death in developed countries. Despite therapeutic interventions such as surgery and chemotherapy, pancreatic adenocarcinoma accounts for about 90% of all pancreatic cancer cases and typically has a very poor prognosis, with about 25% of people surviving for one year and only 5% surviving for five years.

[0183] Esophageal cancer is among the most frequently diagnosed cancers worldwide. Like pancreatic cancer, diagnosis is difficult and it tends to occur already at an advanced stage, leading to a very poor prognosis for this indication. As a result, it accounts for about 5% of cancer-related deaths, thus making it the sixth most common cause of cancer-related deaths.

[0184] The incidence of the two main types of esophageal cancer, esophageal squamous cell carcinoma (ESCC, 60-70% of cases) and esophageal adenocarcinoma (EAC, 20-23% of cases), varies widely among different geographic regions, with ESCC being more common in developing countries, whereas EAC predominates in developed countries. Current treatment options are mostly limited to surgery and chemotherapy or radiotherapy. Only localized tumors are treated with curative intent, whereas treatment of metastatic disease is mostly palliative.

[0185] As mentioned above, the inventors have confirmed that the binding molecules described herein have many usefulness for targeting cancer cells and can therefore be used in the treatment of cancers that express both TROP2 and CDH17. Methods for identifying whether a particular tumor expresses both TROP2 and CDH17 are well known in the art. For example, immunohistochemistry can be used to determine whether a tumor tissue expresses TROP2 and CDH17 (e.g., using the TROP2 and / or CDH17 antibodies described herein) and is therefore suitable for treatment with the binding molecules of the present invention.

[0186] The binding molecules of the invention may be used in therapeutic regimens in the first line, second line, or any further line of treatment and in the maintenance treatment setting.

[0187] In a further aspect, the binding molecules of the invention are used in combination with a device useful for administration of the binding molecules, such as a syringe, injector pen, micropump, or other device, hi a further aspect, the binding molecules of the invention are included in a kit of parts, which also includes, for example, a package insert with instructions for use of the binding molecule.

[0188] Working Example: The invention will now be described by the following non-limiting examples.

[0189] Example 1: Identification of Trop2 and CDH17 as membrane proteins co-expressed in GI cancer tissues but not in normal tissues Gene expression of Trop2 and CDH17 in tumor and key normal tissues obtained from GeneLogic was measured using Affymetrix Chipset HGU133a and HGU133b, and co-expression was confirmed in tumor tissues of specific indications, e.g., gastric, pancreatic, esophageal, and colorectal cancer (as shown in Figure 1). No co-expression was detected in normal tissues.

[0190] CDH17 and Trop2 protein expression was further assessed in human tumor tissue sections by immunohistochemistry using antibodies that bind Trop2 (ENZ-ABS380, Enzo) and CDH17 (760-4865, Roche Ventana) in single- and double-staining protocols, which additionally confirmed the co-expression of these targets on tumor cells of gastric, pancreatic, and colorectal origin (Figure 2).

[0191] Example 2: Schematic of a trispecific binding protein of the invention To achieve T cell-mediated lysis of tumor cells co-expressing Trop2 and CDH17, a polyspecific binding protein was designed that binds Trop2, CDH17, and CD3. One exemplary molecular design used has an IgG antibody scaffold and an IgG-like structure, as shown in FIG. 3. It features a knob-in-hole technique in the Fc domain for heterodimerization known in the art, with a knob portion at the anti-Trop2 binding arm and a hole portion at the anti-CDH17 binding arm. The binding protein also has a flexible peptide sequence between the light chain and the corresponding heavy chain in each arm. A CD3-binding single chain variable region is attached to the Fc portion of the hole chain in this exemplary design. Thus, the binding protein contains three antigen binding sites, one that binds Trop2 and another that binds CDH17, in which each arm contains a single chain Fab and Fc region, and the third antigen binding site binds CD3 as a scFv. In a preferred molecular design, the binding molecule is trispecific and trivalent (ie, monovalent for each of the three targets).

[0192] Example 3: Design, construction, and validation of antigen-binding sites and trispecific binding molecules Example 3.1: Preparation of antigen-binding sites recognizing Trop2 and CDH17 using hybridomas and high-throughput V gene recovery from cultured single B cells To obtain anti-Trop2 binders, hybridomas or single B cells derived from Trop2-immunized wild-type and AlivaMab-humanized mice (Ablexis, San Francisco, CA, USA: AlivaMab transgenic mouse platform with human immunoglobulin loci) were cultured in vitro. Supernatants were screened for reactivity against recombinant human Trop2 by AlphaLISA (PerkinElmer, Waltham, MA, USA) and binding to primary cancer cell lines expressing human Trop2 was confirmed by flow cytometry.

[0193] To obtain anti-CDH17 binders, immunization of wild-type mice was performed with Abpro (Abpro SOW#4). Hybridomas or single B cells derived from immunized mice were cultured in vitro. Supernatants were screened by flow cytometry with AlphaLISA (PerkinElmer, Waltham, MA, USA) for reactivity against recombinant human CDH17 and against cells of the AsPC-1 pancreatic tumor cell line (ATCC®, CRL-1682) and / or NCI-H716 colon tumor cell line (ATCC®, CCL-251) expressing human CDH17. Lymph nodes were processed according to standard Abpro hybridoma procedures. Primary screening was performed by ELISA using hCDH17-His (prepared as described in Example 4.5 below). All ELISA-positive wells were screened by flow cytometry against the two cell lines listed above. All ELISA and cell line positive wells were expanded and retested by both methods. Affinity ranking of expanded positive hybridomas was performed using the ForteBio Octet.

[0194] For both Trop2 and CDH17, immunoglobulin (Ig) VH and VL genes were then amplified from the identified positive clones. To isolate RNA from hybridomas, approximately 2×106 Individual cells were pelleted and used as source material. For single B cells, 100–500 cells expanded from single isolated B cells were used as source material. RNA was isolated using RNeasy Plus (Qiagen, Hilden, Germany). cDNA was then synthesized using the Smarter cDNA synthesis kit (Clontech, Mountain View, CA) according to the manufacturer's instructions.

[0195] To facilitate cDNA synthesis, oligo dT 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 a two-step PCR amplification using a 5' primer targeting the Smarter IIA cap and a 3' primer targeting the consensus region in CH1. Briefly, each 50 μl PCR reaction consisted of 20 μM forward and reverse primer mix, 25 μl PrimeStar Max DNA polymerase premix (Clontech), 2 μl unpurified cDNA, and 21 μl double distilled water. The cycling program started at 94°C for 3 min, followed by 35 cycles (94°C for 30 s, 50°C for 1 min, 68°C for 1 min) and ended at 72°C for 7 min. Second round PCR was performed with VL and VH second round primers that contain 15 bp complementary extensions that "overlap" their respective regions (VH and VL) in their respective pTT5 mother vectors. Second round 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), ending at 72°C for 7 min.

[0196] The In-Fusion® HD Cloning Kit (Clontech, USA) was used for directional cloning of the VL gene into the pTT5 huIgK vector and the VH gene into the pTT5 huIgG1KO vector. To facilitate In-Fusion® HD cloning, PCR products were purified and treated with Cloning Enhancer before In-Fusion HD cloning. Cloning and transformation were performed according to the manufacturer's (Clontech, USA) protocol. Miniprep DNA was subjected to Sanger sequencing to confirm that complete V gene fragments were obtained.

[0197] Using this methodology, Ig VH and VL gene pairs were prepared that encode antigen-binding sites with specificity for Trop2 and CDH17. Recombinant antibodies were produced by transient transfection of CHO-E37 cells with plasmids encoding the corresponding heavy and light chains.

[0198] Example 3.2: Confirmatory Screening of Recombinant Antibodies Supernatants containing expressed recombinant antibodies were assayed by flow cytometry for binding to cell lines expressing human or cynomolgus Trop2 and CDH17, respectively. Briefly, cells were incubated with recombinant supernatants, washed, and bound monoclonal antibodies (mAbs) from the supernatants were detected with anti-human IgG-APC (Jackson ImmunoResearch 109-136-098). Signal-to-background ratios (S / B) were calculated by dividing the median fluorescence intensity (MFI) of the samples by the median fluorescence intensity (MFI) of the isotype controls (variable regions and different constant region backbones for an irrelevant protein).

[0199] Surface plasmon resonance (SPR) on a Biacore 4000 was performed on the recombinant supernatants. Briefly, non-optimized IgG in HTP supernatants was captured on the sensor surface via Protein A / G for 60 seconds at 10 μl / min. Binding of 100 nM human Trop2 or 100 nM human CDH17 to the captured IgG was monitored for association for 180 seconds at 30 μl / min, followed by dissociation for 120 seconds in HBS-EP buffer. Regeneration of the Protein A / G surface was performed with glycine pH 2.1 between each binding cycle.

[0200] The following materials were used in this assay: Protein Reagent: recombinantly expressed human Trop2 and CDH17. System Running Buffer: HBS-EP Buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% v / v Polysorbate P20). Capture Reagent: Protein A / GG (ThermoFisher Scientific, Waltham, MA, USA), with specificity directed against all human IgG isotypes.

[0201] Clones of interest (with Kd in the nM range) were selected for the multispecific format. Multispecific binding proteins were then generated and further evaluated in mechanistic and functional screens (e.g., cell binding, cytotoxicity, and T cell activation, etc.) as described below.

[0202] Example 3.3: Humanization and optimization of Trop2 and CDH17 binders The sequences of Trop2 and CDH17 binders described above were humanized and / or optimized. Antibody sequence optimization / humanization is a methodology for engineering antibodies (against a specific antigen / epitope) produced in non-human species for use as therapeutics similar to antibodies produced in humans, thereby preserving specificity while eliminating potential adverse effects, such as immunogenicity. The sequence optimization / humanization approach utilized herein was as described by Singh et al., 2015 (Singh S et al., mAbs 2015:7(4):778-91). Briefly, closely matching human germlines were identified in silico and optimized / humanized variants were evaluated using phage screening methods. Final lead candidate sequences were selected based on binding, percent human score, and Epivax (an in silico prediction tool for potential immunogenicity) score.

[0203] Example 3.4: scFv conversion of CD3 binding agents To construct the gene segment encoding the CD3 scFv, a pair of VL and VH genes encoding the CD3-binding variable domains were derived from humanized CD3-binding agents described in the literature (Pessano et al., EMBO J. 1985 Feb;4(2):337-44; Salmeron A et al., J Immunol. 1991 Nov 1;147(9):3047-52) and linked by a gene segment encoding a flexible linker of the peptide sequence GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 265).

[0204] Example 3.5: Construction of a trispecific protein that binds Trop2, CDH17, and CD3 and controls The variable regions of Trop2, CDH17 binder, and CD3 scFv as well as the corresponding controls were cloned into the expression vector pTT5 (National Research Council, Canada) using common molecular biology techniques to form a trispecific binding protein with one Trop2-specific binding unit, a single chain Fab that binds Trop2, and one Trop2-specific binding unit comprising an Fc region (such binding units are also referred to herein as "Trop2 arms" or "Trop2 chains"), and a single chain Fab that binds CDH17, and a CDH17-specific binding unit comprising an Fc region (such binding units are also referred to herein as "CDH17 arms" or "CDH17 chains"), with the CD3 scFv being added to the C-terminus of one of the arms, preferably the CDH17 arm. A 20 amino acid spacer was used to separate the scFv from the Fc. The Fc regions of the Trop2 and CDH17 arms contain either "knob" or "hole" mutations (Atwell et al, JMB, 1997, 270, 26-35), with each strand referred to as the knob or hole strand. For multi-fragment DNA assembly, the Gibson assembly and NEBuilder HiFi DNA assembly approaches were used according to the manufacturer's protocols (New England Biolabs, Ipswich, MA, USA). DNA minipreps were sequenced.

[0205] Each expression vector contained the chain-encoding genes (Trop2 or CDH17 arms / chains), i.e., eukaryotic promoter elements for the signal sequences and light and heavy chain-encoding genes, an expression cassette for a prokaryotic selection marker gene, such as ampicillin, and an origin of replication. These DNA plasmids were propagated and purified in ampicillin-resistant E. coli colonies and cultures.

[0206] Control molecules in various assemblies including Trop2 / Trop2 / CD3, CDH17 / CDH17 / CD3, Trop2 / TNP / CD3, TNP / CDH17 / CD3, and TNP / TNP / CD3 were cloned using the Gibson assembly and NEBuilder HiFi DNA assembly approaches described above and cloned into the pTT5 vector for transient expression.

[0207] Example 4: Expression and purification of a trispecific, trivalent binding protein that binds Trop2, CDH17, and CD3 Trispecific molecules binding Trop2, CDH17, and CD3, as well as corresponding controls, were produced by transient transfection of CHO-E cells with pTT5 vectors carrying genes encoding Trop2 / CDH17 / CD3 chains. Briefly, transfected CHO-E cells growing in suspension in serum-free medium were cultured in shake flasks under agitation at 140 rpm, 37 °C, 5% CO2 and kept in exponential growth conditions. On the day of transfection, cells were chemically transfected with knob-strand and hole-strand plasmids in a mass ratio of 1:3. They were then cultured at 1–2 × 10 6 cells / ml in 1 L of Gibco® FreeStyle™ CHO Expression Medium (LifeTechnologies, NY, USA). 6 Cells were then incubated with a single feed of 200 ml of commercial feed solution under orbital shaking for 10 days to allow protein expression. Antibody titers in cell culture supernatants were measured using an Octet® instrument (Pall ForteBio, California, USA) and a protA biosensor chip according to the manufacturer's instructions.

[0208] Recombinant Trop2 / CDH17 / CD3 binding protein was purified from culture supernatant in a two-step process: first, by Protein A affinity chromatography using a MabSelect™ column (GE Healthcare); second, by cation exchange chromatography using a Poros 50 HS column (Applied Biosystems, Carlsbad, CA, 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 analytical size exclusion chromatography, mass spectrometry, and analytical ultracentrifugation. Samples carried forward for functional testing contained two-step purified material with >98% monomer content.

[0209] Example 4.1: Framework engineering of anti-Trop2 antibodies to reduce isoelectric disparity The trispecific molecule containing anti-Trop2, anti-CDH17, and anti-CD3 was subsequently further modified to minimize the amount of aggregation observed (approximately 65% ​​monomer after single-step ProA purification). In silico analysis of the individual Fv arms showed that the anti-Trop2 antibody has a pI (pI=7.7) one unit lower than the anti-CDH17 arm (pI=8.5) and the anti-CD3 arm (pI=8.8). The hypothesis of this engineering strategy was that by increasing the pI of the anti-Trop2 arm to be more consistent with the anti-CDH17 and anti-CD3 arms, aggregation would be reduced and the percentage of monomers would be increased. Mutations were selected from a subset of residues that had either a lysine or an arginine in at least one native heavy or kappa light chain framework position, independent of family. A total of seven point mutations in the heavy chain (plus parent) and four point mutations in the light chain (plus parent) were selected. Modeling of charge variants showed that incorporation of single point mutations increased the pI of anti-Trop2 Fv to an average of 8.3, while a double mutant (one on the heavy chain, one on the light chain) increased the pI to 8.8.

[0210] The following mutations were selected as suitable for increasing the pI of the anti-Trop2 Fv: VH: S19K, T23K, G43R, S61K, T72R, S83R, T85R and VL: Q3R, S65K, S76R, Q79K. Numbering is based on the Kabat numbering scheme, with the first amino acid of the VH or VL domain, respectively, counting as number 1 (i.e. the mutation S19K is a mutation at the 19th amino acid of the VH domain, etc.).

[0211] Example 4.2: Optimization of anti-CD3 scFv for improved stability To improve the stability of anti-CD3 scFv, a structural modeling-based approach was used. A molecular model for the Fv portion of CD3#1 (SEQ ID NO: 222) was generated via high-throughput antibody modeling enabled by the antibody modeler in MOE (Chemical Computing Group, ULC). The structural model was used to calculate the stability descriptors of the Fv region and their VL and VH interfaces in solution and on the molecular surface. Positions determined in silico to affect biophysical properties were identified to generate a library of variants and screened for improved serum stability in the trispecific format. Eight mutations in the light chain (R23G, E40Q, L45A, G59W, V60T, L77I, E87D, and F89Y) and nine mutations in the heavy chain (K19R, S30N, G49A, D68G, T80S, A81L, N87S, K89R, and T90A) were identified that affected the biophysical properties either individually (see, e.g., in CD3#8, SEQ ID NO:229) or in combination (see, e.g., in CD3#2 (SEQ ID NO:223), 3 (SEQ ID NO:224), 5 (SEQ ID NO:226), 7 (SEQ ID NO:228)). Again, the numbering is based on the Kabat numbering scheme, with the first amino acid of the VH or VL domain, respectively, counting as number 1 (i.e., the mutation R23G is a mutation at the 23rd amino acid of the VL domain, etc.).

[0212] Example 4.3: Percent Monomeric Content of Trop2 / CDH17 / CD3 Binding Proteins Percent monomer was determined by analytical size exclusion chromatography (aSEC) for exemplary Trop2 / CDH17 / CD3 binding proteins as shown in Table 2. aSEC was performed on a Waters (Milford, Massachusetts, USA) Acquity UPLC system using a Protein BEH SEC column 200A, 1.7 μm, 4.6×150 mm (catalog number 186005225). Run conditions were as follows: mobile phase: 50 mM sodium phosphate, 200 mM arginine, and 0.05% sodium azide; flow rate: 0.5 ml / min; run time: 5 min; sample load: 10 μg; peak detection: A280 nM; automated method of processing chromatograms. [Table 2] Table 2: Percent monomer after first and second purification steps for trispecific molecules containing Trop2, CDH17, and CD3. The sequences of each binder are shown in Table 1.

[0213] Example 4.4: Thermal Stability Thermal stability was determined by thermal shift analysis (TSA) and the results of the first melting transition (Tm1) of a representative Trop2 / CDH17 / CD3 binding protein are shown in Table 3. Fluorescence intensity profiles as a function of temperature were acquired using a QuantStudio 6 Flex real-time PCR system (Applied Biosystems, Waltham, MA) with SYPRO Orange (Invitrogen, Carlsbad, CA) as the exogenous fluorophore. Samples were diluted to 0.4 mg / ml in 10 mM histidine, pH 6.0 with 40 mM sodium chloride and 0.02% sodium azide. Melting curves were generated by ramping from 25°C to 95°C at a rate of 2°C / min and data were collected approximately every 4°C through a "ROX" filter set (Ex: 580±10 nm, Em: 623±14 nm). Data were analyzed using Protein Thermal Shift Software version 1.3 (ThermoFisher Scientific, Waltham, MA). [Table 3] Table 3: Thermal stability measured by the first melting transition (Tm1) of representative Trop2 / CDH17 / CD3 binding proteins.

[0214] Example 4.5: Production of Recombinant Proteins Human Trop2-His and human CDH17-His Cell lines producing human Trop2-His and human CDH17-His were generated using HEK-293 cells (Thermo Fisher), the Lenti-X Lentiviral System (Clontech), and plasmids encoding Human Trop2-His (human Trop2 accession number P09758) and human CDH17-His (human CDH17 accession number Q12864). For expression, cells were cultured and grown at 37°C, 5% CO2, shaking at 140 rpm. On day 0 of expression, cells were pelleted and resuspended in Expi 293 medium. On day 3 of expression, conditioned culture supernatant was harvested by pelleting cells at 4700 rpm for 40 min. Protease inhibitors were added to the biomass before purification. Expression was confirmed by Western blot. Conditioned culture supernatants were adjusted with 0.5 mM TCEP, 0.02% CHAPS, 10 mM imidazole. Purification was performed with a HisTrap Ni excel column and Buffer A: 50 mM MES, 50 mM NaCl, 0.5 mM TCEP, 0.02% CHAPS, pH 6.5. The protein of interest was eluted in Buffer A (pH 8.5) supplemented with 0.5 M imidazole using an elution gradient from 20 mM imidazole to 500 mM imidazole. Pooled fractions were dialyzed in Buffer: 50 mM MES, 50 mM NaCl, 1 mM TCEP, 0.02% CHAPS, 0.2 M arginine, 3% glycerol, pH 6.5. Purified material was characterized by mass spectrometry and analytical ultracentrifugation.

[0215] Cyno Trop2-His and Cyno CDH17-His Cell lines producing Cyno Trop2-His and Cyno CDH17-His were generated using HEK-293 cells (Thermo Fisher), the Lenti-X lentiviral system (Clontech), and plasmids encoding Cyno Trop2-His (Cyno Trop2 accession number: Accession # XP_001114599.1 (RefSeq)) and Cyno CDH17-His (Cyno CDH17 accession number: XP_005563762.1 (RefSeq)). For expression, cells were cultured and grown at 37°C, 5% CO2, shaking at 140 rpm. On day 0 of expression, cells were pelleted and resuspended in Expi 293 medium. On day 3 of expression, conditioned culture supernatant was harvested by pelleting cells at 4700 rpm for 40 min. Protease inhibitors were added to the biomass before purification. Expression was confirmed by Western blot. Conditioned culture supernatants were adjusted with 0.5 mM TCEP, 0.02% CHAPS, 10 mM imidazole. Purification was performed with a HisTrap Ni excel column and Buffer A: 50 mM MES, 50 mM NaCl, 0.5 mM TCEP, 0.02% CHAPS, pH 6.5. The protein of interest was eluted in Buffer A (pH 8.5) supplemented with 0.5 M imidazole using an elution gradient from 20 mM imidazole to 500 mM imidazole. Pooled fractions were dialyzed in Buffer: 50 mM MES, 50 mM NaCl, 1 mM TCEP, 0.02% CHAPS, 0.2 M arginine, 3% glycerol, pH 6.5. Purified material was characterized by mass spectrometry and analytical ultracentrifugation.

[0216] Example 5: Cell line generation for binder selection For generation of stable HEK293 cells expressing full-length human CDH17 alone or in combination with Trop2, the coding sequence of CDH17 (protein accession Q12864) was cloned into pCMV6 (Origene). For generation of stable HEK293 cells expressing full-length human Trop2 alone or in combination with CDH17, pCMV6 containing the coding sequence of Trop2 with a C-terminal Myc-DDK tag was obtained from Origene (#RC202519). HEK293 cells were transfected using the Cell Line Nucleofector Kit V (Amaxa) according to the manufacturer's instructions and stable clones were established using geneticin and puromycin selection, respectively.

[0217] Expression of recombinant proteins was verified by flow cytometry using anti-Trop2 (R&D# MAB650) and anti-CDH17 (R&D# MAB1032) primary antibodies followed by a PE-labeled anti-mouse secondary antibody (Dako# R0480).

[0218] Example 6: Selection of CDH17-binding agents with binding capacity and confirmation of binding capacity in a trispecific format The binding of four different CDH17 binding proteins (i.e., CDH17#6, CDH17#4, CDH17#7, and CDH17#1; all produced as described in Example 3; the sequences of the respective binders can be found in Table 1) in either bivalent and monovalent formats was tested. To this end, their binding to a HEK293 cell line recombinantly expressing human CDH17, prepared as described in Example 5, was analyzed by flow cytometry.

[0219] HEK293 cells were stained with increasing concentrations of two-step purified bivalent or monovalent CDH17 binding proteins in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Bound molecules were detected with a FITC-conjugated anti-human secondary antibody (Invitrogen 05-4211). The binding capacity of each CDH17 binding protein was assessed by comparing the binding curves of the respective monovalent format with the bivalent format (Figure 4). Binding proteins showing a significant affinity shift from the monovalent to the bivalent format were selected for subsequent experiments.

[0220] To further test the potential of these CDH17-binding agents to activate T cells, different CDH17-binding agents were assembled into trispecific Trop2 / CDH17 / CD3 binding molecules and T cell activation was assessed in the presence of Hek293 cells expressing CDH17 alone or a combination of CDH17 and Trop2. In this assay, Trop2 / CDH17-positive cell lines were co-cultured with human T cells as effector cells and increasing concentrations of Trop2 / CDH17 / CD3 binding molecules at an effector to target cell ratio of 10:1 for 72 hours. Trop2 / CDH17 / CD3 binding molecules were produced by transient transfection of CHO-E cells with pTT5 vectors carrying the genes encoding the chains as described in Example 4.

[0221] Frozen human peripheral blood mononuclear cells (PBMCs) were obtained from Stemcell Technologies. Cells were washed and resuspended in assay medium containing RPMI-1640 w / o Phenolred (Gibco / Lonza # BE 12-918-F), 5% HiFBS (FBS, HyClone (Thermo Scientific, Cat: SH30071.03), heat inactivated, 56°C, 30 min), Glutamax (Gibco # 35050-061), 27.5 μM beta-mercaptoethanol (Gibco #21985-023). T cells were isolated from washed PBMCs by negative selection using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156). Briefly, peripheral blood mononuclear cells were resuspended in 40 μl of buffer PBS / 0.5% BSA (Gibco ref#041-94553 M) / 2 mM EDTA (Invitrogen ref# 15575-038) per 10 Mio cells and incubated with 10 μl of biotin antibody cocktail per 10 Mio cells for 5 min at 4° C. Then, 30 μl of buffer and 20 μl of anti-biotin MicroBed / 10 million cells were added and incubated for 10 min at 4° C. The mixture was then placed in a pre-rinsed 25LS column (Miltenyi Biotec #130-042-401) in the magnetic field of a suitable MACS separator (Miltenyi Biotec). The flow-through containing the T cells was collected and washed in assay medium.

[0222] Target cells and T cells at a 1:10 ratio were then incubated with the indicated concentrations of Trop2 / CDH17 / CD3 binding proteins for 72 hours (see FIG. 5).

[0223] To determine T cell activation, cells were centrifuged and stained with antibodies against CD4 (Biolegend #31744), CD8 (BD #562428), and CD69 (BD #557745), and fluorescence was measured by flow cytometry. The results are depicted in Figure 5.

[0224] Binding potency was analyzed by comparing the amount of CD8+ T cell activation induced in the presence of HEK293 cells expressing human CDH17 alone (FIG. 5A) or a combination of CDH17 and Trop2 (FIG. 5B). Only binders causing less than 20% T cell activation at 100 nM in the presence of HEK293 cells expressing CDH17 alone were selected for further evaluation.

[0225] Example 7: Selection of Trop2 binders with binding capacity and confirmation of binding capacity in trispecific format The binding of seven different Trop2 binders (Trop2#1 to Trop2#7; the sequences of the respective binders can be found in Table 1) in either bivalent (IgG) or monovalent (knob-in-hole format with dummy binding arms) form was tested. In the monovalent format, one of the binding sites was replaced by a binding site that binds tetranitrophenol (TNP; comprising the VH and VL domains of SEQ ID NOs: 167 and 168), an irrelevant antigen not present in the eukaryotic context. To this end, their binding to a HEK293 cell line recombinantly expressing human Trop2, prepared as described in Example 5, was analyzed by flow cytometry. Trop2 binding proteins were produced as described in Example 4.

[0226] HEK293 cells were stained with increasing concentrations of two-step purified bivalent or monovalent Trop2 binding proteins in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Bound molecules were detected with a FITC-conjugated anti-human secondary antibody (Invitrogen 05-4211). The binding capacity of each Trop2 binding protein was assessed by comparing the binding curves of the respective monovalent format with the bivalent format (Figure 6). Binding proteins showing a significant affinity shift from the monovalent to the bivalent format were selected for further experiments.

[0227] To further test the potential of these Trop2 binding agents to activate T cells, different Trop2 binding agents were assembled into trispecific Trop2 / CDH17 / CD3 binding molecules and T cell activation was assessed in the presence of Hek293 cells expressing either CDH17 alone, Trop2 alone, or a combination of CDH17 and Trop2. In this assay, Trop2 / CDH17 positive HEK293 cell lines were co-cultured with human T cells as effector cells and increasing concentrations of Trop2 / CDH17 / CD3 binding molecules at an effector to target cell ratio of 10:1 for 72 hours. Three different Trop2 binding agents with varying antigen affinities were combined in a trispecific format with the CDH17 binding agent identified in Example 6 (Figure 5A) to induce the lowest T cell activation in the presence of HEK293 cells expressing only CDH17. Trop2 / CDH17 / CD3 binding molecules were produced by transient transfection of CHO-E cells with pTT5 vectors carrying the genes encoding the chains as described in Example 4. Human T cells were isolated as described in Example 6.

[0228] Target cells and T cells at a 1:10 ratio were then incubated with the indicated concentrations of Trop2 / CDH17 / CD3 binding molecules for 72 hours (see FIG. 7).

[0229] T cell activation status was determined as described in Example 6.

[0230] Binding potency was analyzed by comparing the amount of CD69 upregulation on CD8+ T cells induced in the presence of HEK293 cells expressing either human Trop2 alone (Figure 7B), human CDH17 alone (Figure 7C), or a combination of human CDH17 and human Trop2 (Figure 7A). Again, binders causing less than 20% T cell activation at 10 nM in the context of monovalent binding, i.e., with CDH17 (only) or Trop2 (only) expressing cells, were selected for subsequent experiments.

[0231] Binding affinities for Trop2 expressed on HEK293 cells were assessed by the flow cytometry-based Scatchard method, revealing Kds of 2.2 nM for Trop2#1, 0.6 for Trop2#5, and 0.4 for Trop2#7. Binding potency correlated inversely with the affinity determined through this method, indicating that only low affinity binders allow discrimination of single-positive from double-positive cells in a bivalent format.

[0232] Example 8: Analysis of the binding capacity of different trispecific Trop2 / CDH17 / CD3 binder combinations in a cell binding assay To confirm the avidity-based potency increase, target binding of three different Trop2 / CDH17 / CD3 trispecific binding molecules (Trop2#1 / CDH17#1 / CD3#1 Trop2#5 / CDH17#1 / CD3#1, Trop2#7 / CDH17#1 / CD3#1, see FIG. 8) was compared with the binding of bispecific Trop2 / Trop2 / CD3 binding molecules as well as trispecific Trop2 / TNP / CD3 binding molecules. To this end, the binding of these molecules to HEK293 cell lines either recombinantly expressing human Trop2 (FIG. 8A) or co-expressing human Trop2 and human CDH17 (FIG. 8B) was tested by flow cytometry. Trop2 / CDH17 / CD3 Trop2 / TNP / CD3 and Trop2 / Trop2 / CD3 binding proteins were produced as described in Example 4.

[0233] HEK293 cells either recombinantly expressing human Trop2 or co-expressing human Trop2 and human CDH17 were stained with increasing concentrations of two-step purified Trop2 / CDH17 / CD3, Trop2 / Trop2 / CD3, and Trop2 / TNP / CD3 binding molecules in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Binding molecules were detected with a FITC-conjugated anti-human secondary antibody (Invitrogen 05-4211). The binding capacity of each trispecific binding molecule was assessed by comparing monovalent and bivalent binding curves. Binding proteins showing significant binding to Trop2 alone were not selected for the avidity-optimized bispecific binding approach. The results are shown in Figure 8. The sequences of the respective binders can be found in Table 1.

[0234] Example 9: Efficacy in mediating T cell-induced lysis in cells expressing CDH17, Trop2, or co-expressing both To address the selectivity of the Trop2 / CDH17 / CD3 binding protein for human cells co-expressing both Trop2 and CDH17 proteins, we tested the ability to induce lysis by redirecting unstimulated T cells toward cells expressing human CDH17 and / or human Trop2.

[0235] The efficacy of T cell-mediated lysis of Trop2 / CDH17 positive cell lines was determined using lactate dehydrogenase (LDH) release as a readout for cell lysis as described below. In this assay, cell lines expressing either Trop2 or CDH17 or both were co-cultured with human T cells as effector cells and increasing concentrations of Trop2 / CDH17 / CD3 binding molecules for 72 hours at an effector to target cell ratio of 10:1. Trop2 / CDH17 / CD3 binding molecules were produced by transient transfection of CHO-E cells with pTT5 vectors carrying the genes encoding the chains as described in Example 4.

[0236] Human T cells were isolated from frozen PBMCs as described in Example 6.

[0237] Cytotoxic activity was determined using the Cytotoxicity Detection Kit PLUS (Roche) according to the manufacturer's instructions. Briefly, the method is based on the release of LDH from dead or plasma membrane damaged cells. Cell culture supernatants are incubated with the reaction mixture from the kit for 30 min and the formation of formazan, as a result of LDH activity, is measured at 500 nM in a spectrophotometer as a proxy for cell lysis. The percentage of cytotoxicity relative to the maximum lysis control was calculated according to the following formula:

number

[0238] The percentage of cytotoxicity relative to the maximum lysis control was plotted against the corresponding Trop2 / CDH17 / CD3 binding molecule concentration using GraphPad Prism 5 software. Dose-response curves were analyzed using a four-parameter logistic equation model for evaluation of sigmoidal dose-response curves.

[0239] The trispecific Trop2 / CDH17 / CD3 binding molecules mediated lysis of Trop2+ / CDH17+ cells with good efficacy, whereas this was not the case for cell lines expressing only Trop2 or only CDH17, respectively, thus confirming the enhanced avidity biological activity of the trispecific molecules (see FIG. 9A). The specificity of the avidity approach was further supported by the fact that control molecules monovalently binding either Trop2 (Trop2 / TNP / CD3) or CDH17 (TNP / CDH17 / CD3) failed to induce significant target cell lysis. Bivalent binders for Trop2 and CDH17 in the same format were used as controls.

[0240] To validate the avidity approach in a cancer setting (see Figure 9B), the cytotoxicity of redirected T cells was evaluated in the colorectal cancer cell line DLD-1, which co-expresses Trop2 / CDH17, as well as in DLD-1 clones engineered to lack CDH17 and thus express only Trop2. DLD-1 cells were obtained from ATCC to generate CDH17-deficient DLD-1 clones transfected with the reporter protein GFP. After 48 hours, GFP-positive transfectants were isolated by flow cytometry (Sonysorter) and clones were separated by limiting dilution. CDH17 deficiency was confirmed by analyzing clone protein extracts for CDH17 content by Western blotting using a CDH17-specific antibody.

[0241] Cytolysis of Trop2+CDH17- clones was significantly reduced compared to the effect on the Trop2+CDH17+ parental cell line, thereby confirming an avidity-based increase in potency. The activity of Trop2 / CDH17 / CD3 binding molecules on Trop2+CDH17- clones paralleled that of a monovalent Trop2 binder (Trop2 / TNP / CD3), thus confirming that the effect on Trop2+ / CDH17- target cells was Trop2-mediated.

[0242] Figure 9 shows functional data obtained with a selection of molecules representing several exemplary combinations of Trop2 binders (Trop2#1, #8, #9, #10, #11, #12, #13, #14, #15, #16, #17, #18) with CDH17 binders (CDH17#1, #8) and CD3 binders (CD3#1, #2, #3), for which avidity-based potency gains are shown. The sequences of each binder can be found in Table 1.

[0243] Example 10: Confirmation of the contribution of CDH17 binding to avidity-induced increased potency To confirm the contribution of CDH17 binding to the avidity-induced increase in potency, five different Trop2 binders were combined with either the CDH17 binding arm or binders for the irrelevant target tetranitrophenol (TNP) in Trop2 / CDH17 / CD3 or Trop2 / TNP / CD3 binding molecules, respectively. In this assay, the Trop2 / CDH17-positive colorectal cancer cell line SK-CO1 was co-cultured with unstimulated human T cells as effector cells and increasing concentrations of Trop2 / CDH17 / CD3 or Trop2 / TNP / CD3 binding molecules at an effector to target cell ratio of 10:1 for 72 hours. Trop2 / CDH17 / CD3 binding molecules were produced by transient transfection of CHO-E cells with pTT5 vectors carrying the genes encoding the chains as described in Example 4. Cytotoxic activity was determined as described in Example 9. T cells were isolated from healthy donor PBMCs as described in Example 6. The CDH17 binding arm led to a significant increase in potency, with a 40-60 fold increase in EC50 from Trop2 / TNP / CD3 vs. Trop2 / CDH17 / CD3; as shown in FIG. 10 (the sequences of each binder can be found in Table 1).

[0244] Example 11: Comparison of knob-in-hole formats Trispecific molecules were produced in two different knob-in-hole formats (i.e., Trop2 on the hole arm and CDH17 / CD3 on the knob arm, or vice versa) and their effect on T cell-mediated cytotoxicity was evaluated as described in Example 9. While both types of formats performed well, surprisingly, constructs in which both the CD3 and CDH17 binding moieties are located in the hole portion of the molecule were found to be consistently more potent, regardless of the binder used (see FIG. 11 for exemplary results; corresponding binders can be found in Table 1).

[0245] Example 12: Confirmation of tumor growth inhibition in vivo Efficacy studies were performed using a human xenograft mouse model reconstituted with human T cells. Specifically, human HPAF-II pancreatic cancer cells (5×10 7 ) were injected subcutaneously (sc) into the right dorsal flank of NOG mice. In parallel, human T cells were isolated from PBMCs by negative selection using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156) as described above.

[0246] T cells were then expanded for 20 days using the T Cell Activation / Proliferation Kit Human (Miltenyi Biotec Cat#130-091-441, Lot#5170720843). Briefly, anti-biotin MACSiBead™ particles were loaded with CD2-, CD3-, and CD28 biotin and transferred to purified T cells at a ratio of 2 cells per particle and diluted to 0.5-1×10 in the presence of 20 units of recombinant IL-2 (R&D#202-IL-050 / CF). 6 The T cells were incubated at a density of 1000 cells / ml for 20 days. The cells were supplemented with 20 units of fresh IL-2 every 3 days. Three days before injection into the animals, the T cells were restimulated with anti-biotin MACSiBead™ particles loaded with CD2-, CD3-, and CD28 biotin at a ratio of 1 bead per 4 cells for an additional 3 days. Finally, the beads were removed using a MACSiMAG Separator (Miltenyi Biotec) and the T cells were washed in PBS.

[0247] On day 14, animals were randomized into treatment groups based on tumor volume and received 2×10 7Human T cells were injected intraperitoneally (ip). Treatment with the trispecific molecules was initiated on day 17. Trop2 / CDH17 / CD3 binding protein, Trop2 / TNP / CD3 binding protein, TNP / CDH17 / CD3 binding protein, or vehicle buffer (50 mM NaOAc, 100 mM NaCl, pH 5.0) were administered on a weekly dosing regimen by intravenous (iv) bolus injection into the lateral tail vein. Tumor growth was monitored by external caliper measurements and tumor volumes were calculated using the standard semi-ellipsoid formula.

[0248] Human T cell engraftment was confirmed in the spleen by immunohistochemistry (IHC) staining for human CD3 at the end of the study. Only animals that showed human T cell engraftment at the end of the study were included in the statistical analysis. Animals that met the sacrifice criteria were euthanized early during the study for ethical reasons.

[0249] As shown in FIG. 12, treatment of tumor-bearing mice once a week with the trispecific Trop2 / CDH17 / CD3 binding molecule (represented by either the combination of SEQ ID NOs: 436 and 437 or the combination of SEQ ID NOs: 214 and 271) induced significant tumor regression, but not with the control Trop2 / TNP / CD3 binding molecule or the TNP / CDH17 / CD3 binding molecule at the same dose and treatment interval.

Claims

1. (a) at least one antigen-binding site that specifically binds to trophoblast cell surface antigen 2 (TROP2) with a Kd ≥ 1 nM; (b) at least one antigen-binding site that specifically binds to cadherin-17 (CDH17), wherein the at least one antigen-binding site that specifically binds to CDH17 is antigen-binding site (i)-(ii): (i) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 33 (CDR2), and SEQ ID NO: 34 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 35 (CDR1), SEQ ID NO: 36 (CDR2), and SEQ ID NO: 37 (CDR3); and (ii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 32 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 34 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 39 (CDR1), SEQ ID NO: 40 (CDR2), and SEQ ID NO: 37 (CDR3), an antigen-binding site selected from the group consisting of, and (c) at least one antigen-binding site that specifically binds to cluster of differentiation 3 (CD3) A binding molecule comprising.

2. The at least one antigen-binding site that specifically binds to TROP2 is antigen-binding site (i)-(vi): (i) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3); (ii) an antigen-binding site comprising a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2), and SEQ ID NO: 9 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 10 (CDR1), SEQ ID NO: 11 (CDR2), and SEQ ID NO: 12 (CDR3); (iii) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (iv) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO: 15 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 20 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); (v) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 21 (CDR1), SEQ ID NO: 22 (CDR2), and SEQ ID NO: 23 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 24 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 26 (CDR3); and (vi) a heavy chain CDR comprising the amino acid sequences of SEQ ID NO: 27 (CDR1), SEQ ID NO: 28 (CDR2), and SEQ ID NO: 29 (CDR3) and a light chain CDR comprising the amino acid sequences of SEQ ID NO: 30 (CDR1), SEQ ID NO: 25 (CDR2), and SEQ ID NO: 31 (CDR3) The binding molecule according to claim 1, selected from the group consisting of.

3. The at least one antigen-binding site that specifically binds to TROP2 is antigen-binding site (i)-(xii): (i) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 83 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (ii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (iii) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 87 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 88; (iv) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 90; (v) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 91 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 92; (vi) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (vii) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 94; (viii) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96; (ix) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (x) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 97; (xi) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 95 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99; and (xii) An antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 98 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 96 The binding molecule according to claim 1, selected from the group consisting of.

4. The at least one antigen-binding site that specifically binds to CDH17 is antigen-binding sites (i) to (ii): (i) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 100 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 101; and (ii) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 102 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 103 The binding molecule according to claim 1, selected from the group consisting of.

5. The at least one antigen-binding site that specifically binds to CD3 is antigen-binding sites (i) to (xxxi): (i) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3) and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (ii) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3) and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 46 (CDR3); (iii) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 43 (CDR3) and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 47 (CDR3); (iv) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3) and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 50 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (v) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vi) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 49 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 44 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (vii) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 53 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 47 (CDR3); (viii) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 55 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (ix) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (x) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 48 (CDR2), and SEQ ID NO: 54 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xi) a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 58 (CDR2), and SEQ ID NO: 59 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 60 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xii) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xiii) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 62 (CDR2), and SEQ ID NO: 54 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xiv) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 63 (CDR2), and SEQ ID NO: 54 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 56 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xv) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 61 (CDR1), SEQ ID NO: 45 (CDR2), and SEQ ID NO: 51 (CDR3); (xvi) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 41 (CDR1), SEQ ID NO: 42 (CDR2), and SEQ ID NO: 43 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 57 (CDR1), SEQ ID NO: 52 (CDR2), and SEQ ID NO: 51 (CDR3); (xvii) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 64 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); (xviii) An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 70 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 72 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 73 (CDR2), and SEQ ID NO: 70 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 75 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR containing the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 69 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR containing the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 76 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 71 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 78 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); An antigen-binding site comprising a heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 79 (CDR2), and SEQ ID NO: 65 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 80 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3); and A heavy-chain CDR comprising the amino acid sequences of SEQ ID NO: 82 (CDR1), SEQ ID NO: 81 (CDR2), and SEQ ID NO: 70 (CDR3), and a light-chain CDR comprising the amino acid sequences of SEQ ID NO: 66 (CDR1), SEQ ID NO: 67 (CDR2), and SEQ ID NO: 68 (CDR3), an antigen-binding site The binding molecule according to claim 1, selected from the group consisting of

6. The at least one antigen-binding site that specifically binds to CD3 is antigen-binding site (i)-(xvi): (i) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 104 and an immunoglobulin light-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 123, and SEQ ID NO: 129; (ii) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 110 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 111; (iii) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 112 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 113; (iv) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 118 and an immunoglobulin light-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 119 and SEQ ID NO: 122; (v) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 124 and an immunoglobulin light-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; (v) An antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 130 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 127; (vi) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 131 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 132; (vii) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 133 and an immunoglobulin light-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 134 and SEQ ID NO: 135; (viii) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 136 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 137; (ix) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and SEQ ID NO: 156 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 139; (x) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 161 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 141; (xi) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 144 and SEQ ID NO: 146 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 145; (xii) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, and SEQ ID NO: 162 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 148; (xiii) an antigen-binding site comprising an immunoglobulin heavy-chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 149 and SEQ ID NO: 152 and an immunoglobulin light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 150; (xiv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (xv) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 158; and (xvi) an antigen-binding site comprising an immunoglobulin heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and an immunoglobulin light chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 The binding molecule according to claim 1, selected from the group consisting of.

7. The binding molecule, wherein the binding molecule is a modified immunoglobulin (Ig) molecule, and the at least one antigen-binding site that specifically binds to TROP2 and the at least one antigen-binding site that specifically binds to CDH17 are present in the variable region of the Ig molecule, and the at least one antigen-binding site that specifically binds to CD3 is an scFv fused to the TROP2-CDH17 specific Ig molecule.

8. The binding molecule according to claim 7, wherein the scFv is fused to the C-terminus of the heavy chain of the Ig molecule.

9. The binding molecule is: (a) (a-i) to (a-xii): (a-i) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 169 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 170; (a-ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 171 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 172; (a-iii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 173 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 174; (a-iv) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 175 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 176; An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 177 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 178; (a-vi) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 179 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 180; (a-vii) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 181 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 182; (a-viii) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 183 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 184; (a-ix) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 185 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 186; (a-x) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 187 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 188; (a-xi) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 189 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 190; or (a-xii) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 191 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192 A combination of a first immunoglobulin light chain and an immunoglobulin heavy chain, selected from the above and linked together by a peptide linker; and (b) (b-i) to (b-ii): (b-i) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 196 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 197; or (b-ii) An immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 198 and an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 199 A combination of a second immunoglobulin heavy chain and an immunoglobulin light chain, selected from the above and linked together by a peptide linker; and (c) A single-chain variable fragment (scFv) linked to the C-terminus of the immunoglobulin heavy chain of the combination of the second immunoglobulin heavy chain and immunoglobulin light chain, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 222 to 264 The binding molecule according to claim 1, comprising

10. The binding molecule is: (a-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 267; (a-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 267; (a-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 267; (a-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 267; (a-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 267; (a-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 267; (a-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 267; (a-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 267; (a-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 267; (a-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 267; (a-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 267; (a-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 267; (b-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 268; (b-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 268; (b-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 268; (b-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 268; (b-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 268; (b-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 268; (b-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 268; (b-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 268; (b-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 268; (b-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 268; (b-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 268; (b-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 268; (c-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 269; (c-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 269; (c-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 269; (c-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 269; (c-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 269; (c-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 269; (c-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 269; (c-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 269; (c-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 269; (c-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 269; (c-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 269; (c-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 269; (d-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 270; (d-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 270; (d-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 270; (d-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 270; (d-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 270; (d-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 270; (d-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 270; (d-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 270; (d-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 270; (d-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 270; (d-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 270; (d-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 270; (e-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 271; (e-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 271; (e-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 271; (e-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 271; (e-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 271; (e-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 271; (e-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 271; (e-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 271; (e-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 271; (e-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 271; (e-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 271; (e-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 271; (f-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 272; (f-ii) The amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 272; (f-iii) The amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 272; (f-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 272; (f-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 272; (f-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 272; (f-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 272; (f-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 272; (f-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 272; (f-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 272; (f-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 272; (f-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 272; (g-i) The amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 424; (g-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 424; (g-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 424; (g-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 424; (g-v) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 424; (g-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 424; (g-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 424; (g-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 424; (g-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 424; (g-x) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 424; (g-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 424; (g-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 424; (h-i) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 425; (h-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 425; (h-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 425; (h-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 425; (h-v) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 425; (h-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 425; (h-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 425; (h-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 425; (h-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 425; (h-x) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 425; (h-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 425; (h-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 425; (i-i) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 426; (i-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 426; (i-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 426; (i-iv) the amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 426; (i-v) the amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 426; (i-vi) the amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 426; (i-vii) the amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 426; (i-viii) the amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 426; (i-ix) the amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 426; (i-x) the amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 426; (i-xi) the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 426; (i-xii) the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 426; (j-i) the amino acid sequence of SEQ ID NO: 200 and the amino acid sequence of SEQ ID NO: 427; (j-ii) the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 427; (j-iii) the amino acid sequence of SEQ ID NO: 208 and the amino acid sequence of SEQ ID NO: 427; (j-iv) The amino acid sequence of SEQ ID NO: 209 and the amino acid sequence of SEQ ID NO: 427; (j-v) The amino acid sequence of SEQ ID NO: 210 and the amino acid sequence of SEQ ID NO: 427; (j-vi) The amino acid sequence of SEQ ID NO: 211 and the amino acid sequence of SEQ ID NO: 427; (j-vii) The amino acid sequence of SEQ ID NO: 212 and the amino acid sequence of SEQ ID NO: 427; (j-viii) The amino acid sequence of SEQ ID NO: 213 and the amino acid sequence of SEQ ID NO: 427; (j-ix) The amino acid sequence of SEQ ID NO: 214 and the amino acid sequence of SEQ ID NO: 427; (j-x) The amino acid sequence of SEQ ID NO: 215 and the amino acid sequence of SEQ ID NO: 427; (j-xi) The amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 427; (j-xii) The amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 427; or (k) The amino acid sequence of SEQ ID NO: 436 and the amino acid sequence of SEQ ID NO: 437 The binding molecule according to claim 1, comprising or consisting of the same.

11. A nucleic acid molecule encoding the binding molecule according to any one of claims 1 to 10, or a part thereof.

12. An expression vector comprising one or more nucleic acid molecules according to claim 11.

13. A host cell transfected with the expression vector according to claim 12.

14. A method for producing the binding molecule according to any one of claims 1 to 10, said method comprising the steps of: (a) culturing the host cell according to claim 13 under conditions enabling the expression of the binding molecule according to any one of claims 1 to 10; (b) recovering said molecule; and (c) further purifying and / or modifying and / or formulating said binding molecule A method comprising the same. Claim 15 A pharmaceutical composition comprising or consisting of one or more binding molecules according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier. Claim 16 The pharmaceutical composition according to claim 15 for treating, remitting or preventing cancer. Claim 17 The pharmaceutical composition according to claim 16, wherein the cancer is colorectal cancer (CRC), gastric cancer (GC), or pancreatic cancer (PAC). Claim 18 The pharmaceutical composition according to claim 16 for use in combination with an immune checkpoint inhibitor.

Images