Multispecific binding proteins and their use

Abstract

The present invention relates to multispecific binding proteins comprising binding domains with dual-binding specificity for at least one disease-associated antigen, suitably tumor-associated antigen and an immune cell surface antigen. Said binding domains with dual-binding specificity bind to the two targets in a mutually exclusive way, thereby functioning as a molecular switch, such as, e.g., a switch to control activation or deactivation of a therapeutic agent. Said binding proteins are formatted as T cell engagers to optionally comprise a binding domain with co-stimulatory activity, such as CD2. Said binding domains are preferably ankyrin repeat domains, e.g. designed ankyrin repeat domains (DARPins). The invention further relates to nucleic acids, pharmaceutical compositions, recombinant expression vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer.

Description

[0001] MULTISPECIFIC BINDING PROTEINS AND THEIR USE

[0002] FIELD OF THE DISCLOSURE

[0003] The present invention relates to multispecific binding proteins comprising binding domains with dual-binding specificity for at least one disease-associated antigen, suitably tumor-associated antigen and an immune cell surface antigen. Said binding domains with dual-binding specificity bind to the two targets in a mutually exclusive way, thereby functioning as a molecular switch, such as, e.g., a switch to control activation or deactivation of a therapeutic agent. Said binding proteins are formatted as T cell engagers to additionally comprise a binding domain with co-stimulatory activity, such as CD2. Said binding domains are preferably ankyrin repeat domains, e.g., designed ankyrin repeat domains (DARPins). The invention further relates to nucleic acids, pharmaceutical compositions, recombinant expression vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer.

[0004] BACKGROUND

[0005] T cell engagers (TCEs) are a class of protein therapeutics that simultaneously bind to a target antigen, e.g., on a tumor cell, and to the TCR / CD3 complex on a T cell, triggering T cell activation by bypassing the normal TCR-MHC interaction (Ellerman, Methods; 154:102-117 (2019)).

[0006] Currently, T cell engagers typically have the form of bi-specific antibodies or antibody fragments, directed against a constant component of the TCR / CD3 complex, such as CD3, and a tumor-associated antigen (TAA). However, these T cell engaging therapeutic tools present several disadvantages, such as high production costs and the inability to target multiple disease-associated antigens, while also carrying the risk of causing certain severe side effects, such as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer; 6(1):56 (2018); Labrjin etal., Nat Rev Drug Discov; 18(8):585-608 (2019)) and on target / off tumor toxicities (Weiner et al., Cancer Res; 55 (20):4586-4593 (1995); Weiner et al., Cancer Immunol. Immunother.; 4 (3):141— 150 (1996)). For instance, a TCE targeting EGFR was tested in nonhuman primates with safety findings related to engagement with normal tissue expressing lower levels of the antigen (Lutterbuese et al., Proc Natl Acad Sci USA; 107(28):12605-10 (2010)). Additionally, high affinity CD3-binders of HER-targeting TCEs were shown to distribute preferentially to secondary lymphatic tissues, reducing systemic exposure (Mandikian et al., Mol. Cancer Ther.; 17(4): 776 LP-785 (2018)). Antibody-based T cell engagers often show more than 1000-fold higher affinity for CD3, when compared to the natural TCR-MHC interaction (Wu et al., Pharmacol Ther;182:161-75 (2018); WO2014 / 167022; Junntila et al., Cancer Res.; 19:5561-71 (2014); Yang et al, J Immunol Aug.; 15 (137):1097-100 (1986)). This high affinity is correlated to lower efficiency in terms of T cell activation and tumor cell killing (Bortoletto et al., Eur. J. Immunol. 32; 11 : 3102-3107 (2002); Ellerman (supra) Mandikian et al. (supra) Vafa et al., Frontiers in Oncology; 10: 446 (2020)). Thus, finding the optimal balance between drug efficacy and safety is an ongoing challenge in the development of targeted drugs.

[0007] Additionally, the use of T cell engagers in solid tumor indications faces significant challenges, primarily due to the difficulty in identifying antigens not significantly expressed in healthy cells (so called “clean” tumor targets); solid tumors often lack unique antigens that are exclusively expressed on tumor cells. This makes it difficult to target cancer cells without affecting healthy tissues (de Miguel M et al. Clin Can Res (2021) 27(6) 1595-603). Therefore, it is desirable to make tumor targets cleaner in order to avoid on-target off- tumor toxicity of T cell engagers to improve their specificity and efficacy in treating solid tumors.

[0008] Moreover, despite being a potent class of therapeutics, not all patients respond to TCE therapy. For example, recent studies into blinatumomab-treated T cells highlight exhaustion and the lack of costimulation as potential factors in resistance to these drugs. In solid tumors, the immunosuppressive tumor microenvironment is also a major factor while cytokine release is a safety consideration for all T cell engagers with strong affinity.

[0009] Taken together, there remains a need for new drugs which can overcome the above-mentioned drawbacks.

[0010] SUMMARY OF THE DISCLOSURE

[0011] Ankyrin repeat domains with mutually exclusive binding specificity for two targets have been disclosed (WO2023 / 110983). Such dual-specific domains exhibit particularly useful properties in multi-domain immune cell engager DARPin constructs, enabling conditional activation (“switch”) of an immune cell, e.g., a T cell, upon binding to a target antigen, such as a tumor-associated antigen (TAA). Such a TAA- dependent switch mechanism can control CD3 engagement by unmasking or masking the CD3-binding agent, and together with an additional immune cell co-stimulatory component, enables T cell activation and expansion as well as cytokine production over repeated cycles of stimulation, providing targeted, safer, and sustained T cell activation. This precise control of protein activity is particularly beneficial in protein-based therapeutics.

[0012] Accordingly, presently provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second immune cell-surface associated antigen.

[0013] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (A, E). A1 . A multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second immune cell-surface associated antigen.

[0014] A2. The multispecific binding protein according to A1 , wherein said second ankyrin domain reversibly binds said first ankyrin repeat domain.

[0015] A3. The multispecific binding protein according to A1 or A2, wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of the second ankyrin repeat domain to said first tumor associated antigen.

[0016] A4. The multispecific binding protein according to any one of A1 to A3, wherein the first immune cell-surface associated antigen and the second immune cell-surface associated antigen are present on the same immune cell.

[0017] A5. The multispecific binding protein according to A4, wherein said immune cell is a T cell.

[0018] A6. The multispecific binding protein according to any one of A1 to A3, wherein the first immune cell-surface associated antigen is a component of the human T-cell receptor (TOR) complex.

[0019] A7. The multispecific binding protein according to any one of A1 to A6, wherein said first immune cellsurface associated antigen is CD3.

[0020] A8. The multispecific binding protein according to A7, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 3.

[0021] A9. The multispecific binding protein according to A7, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 2.

[0022] A10. The multispecific binding protein according to A7, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 3. A11 . The multispecific binding protein according to any one of A1 to A10, wherein said multispecific binding protein binds CD3 in PBS with a dissociation constant (KD) of or below about 10-7M.

[0023] A12. The multispecific binding protein according to any one of A1 to A11 , wherein the tumor associated antigen is expressed on the surface of a cancer cell.

[0024] A13. The multispecific binding protein according to A12, wherein said cancer cell is a lung cancer cell, a skin cancer cell, a lymphoma cell or an ovarian cancer cell.

[0025] A14. The multispecific binding protein according to A12 or A13, wherein said cancer cell is an ovarian cancer cell.

[0026] A15. The multispecific binding protein according to any one of A1 to A13, wherein said first TAA is EpCAM or MSLN.

[0027] A16. The multispecific binding protein according to A15, wherein said first TAA is EpCAM.

[0028] A17. The multispecific binding protein according to any one of A1 to A16, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 16 to 21.

[0029] A18. The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 16.

[0030] A19. The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 17.

[0031] A20. The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 18.

[0032] A21 . The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 19.

[0033] A22. The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 20. A23. The multispecific binding protein according to any one of A1 to A17, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 21.

[0034] A24. The multispecific binding protein according to A15, wherein said first TAA is MSLN.

[0035] A25. The multispecific binding protein according to any one of A1 to A15 or A24, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 22.

[0036] A26. The multispecific binding protein according to any one of A1 to A25, wherein said second ankyrin repeat domain binds to said first target with a dissociation constant (KD) of or below about 10-5M and / or to said second target with a dissociation constant (KD) of or below about 10-5M.

[0037] A27. The multispecific binding protein according to any one of A1 to A26, wherein said second immune cell-surface associated antigen provides a co-stimulatory signal to a T-cell.

[0038] A28. The multispecific binding protein according to A27, wherein the second immune cell-surface associated antigen is CD2 or CD28.

[0039] A29. The multispecific binding protein according to A28, wherein the second immune cell-surface associated antigen is CD2.

[0040] A30. The multispecific binding protein according to A1 to A29, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 4 to 6.

[0041] A31 . The multispecific binding protein according to A1 to A30, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 4.

[0042] A32. The multispecific binding protein according to A1 to A30, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 5.

[0043] A33. The multispecific binding protein according to A1 to A30, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 6.

[0044] A34. The multispecific binding protein according to A1 to A33, wherein said multispecific binding protein binds CD2 in PBS with a dissociation constant (KD) of or below about 10-7M. A35. The multispecific binding protein according to A1 to A34, comprising a further ankyrin repeat domain with binding specificity for a second tumor associated antigen (TAA).

[0045] A36. A multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second tumor associated antigen, wherein said second ankyrin repeat domain is binding said first ankyrin repeat domain and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to said first tumor associated antigen and (ii) the third ankyrin repeat domain to said second tumor associated antigen.

[0046] A37. The multispecific binding protein according to A36, wherein said second ankyrin domain reversibly binds said first ankyrin repeat domain.

[0047] A38. The multispecific binding protein according to A35 to A37, wherein the first and second tumor associated antigens are present on the same cell.

[0048] A39. The multispecific binding protein according to A36 to A38, comprising a fourth ankyrin repeat domain with binding specificity for a second immune cell-surface associated antigen.

[0049] A40. The multispecific binding protein according to A39, wherein the first immune cell-surface associated antigen and the second immune cell-surface associated antigen are present on the same immune cell.

[0050] A41 . The multispecific binding protein according to A40, wherein said immune cell is a T cell.

[0051] A42. The multispecific binding protein according to any one of A36 to A41 , wherein the first immune cellsurface associated antigen is a component of the human T-cell receptor (TCR) complex.

[0052] A43. The multispecific binding protein according to any one of A36 to A42, wherein said first immune cellsurface associated antigen is CD3.

[0053] A44. The multispecific binding protein according to A43, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 3 or SEQ ID NOs: 106 to 125. A45. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 2.

[0054] A46. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 3.

[0055] A47. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 120.

[0056] A48. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 122.

[0057] A49. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 124.

[0058] A50. The multispecific binding protein according to A44, wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 125.

[0059] A51 . The multispecific binding protein according to any one of A43 to A50, wherein said multispecific binding protein binds CD3 in PBS with a dissociation constant (KD) of or below about 10-7M.

[0060] A52. The multispecific binding protein according to any one of A35 to A51 , wherein the first and second TAA are expressed on the surface of a cancer cell, optionally wherein the first and second TAA are expressed on the surface of the same cancer cell.

[0061] A53. The multispecific binding protein according to A52, wherein said cancer cell is a lung cancer cell, a skin cancer cell, a lymphoma cell or an ovarian cancer cell.

[0062] A54. The multispecific binding protein according to A52 or A53, wherein said cancer cell is an ovarian cancer cell.

[0063] A55. The multispecific binding protein according to any one of A36 to A54, wherein said first TAA is EpCAM or MSLN.

[0064] A56. The multispecific binding protein according to A55, wherein said first TAA is EpCAM. A57. The multispecific binding protein according to any one of A36 to A56, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 16 to 21 or SEQ ID NOs: 133 to 149.

[0065] A58. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 16.

[0066] A59. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 17.

[0067] A60. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 18.

[0068] A61 . The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 19.

[0069] A62. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 20.

[0070] A63. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 21.

[0071] A64. The multispecific binding protein according to any one of A36 to A57, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 133.

[0072] A65. The multispecific binding protein according to any one of A36 to A53, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 134.

[0073] A66. The multispecific binding protein according to any one of A36 to A53, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 135. A67. The multispecific binding protein according to any one of A36 to A53, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 136.

[0074] A68. The multispecific binding protein according to any one of A36 to A53, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 137.

[0075] A69. The multispecific binding protein according to any one of A36 to A53, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 138.

[0076] A70. The multispecific binding protein according to A55, wherein said first TAA is MSLN.

[0077] A71. The multispecific binding protein according to any one of A36 to A51 or A70, wherein the second ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 22.

[0078] A72. The multispecific binding protein according to any one of A36 to A71 , wherein said second ankyrin repeat domain binds to said first target with a dissociation constant (KD) of or below about 10-5M and / or to said second target with a dissociation constant (KD) of or below about 10-5M.

[0079] A73. The multispecific binding protein according to any one of A39 to A72, wherein said second immune cell-surface associated antigen provides a co-stimulatory signal to a T-cell.

[0080] A74. The multispecific binding protein according to A73, wherein the second immune cell-surface associated antigen is CD2 or CD28.

[0081] A75. The multispecific binding protein according to A74, wherein the second immune cell-surface associated antigen is CD2.

[0082] A76. The multispecific binding protein according to A39 to A65, wherein the fourth ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132.

[0083] A77. The multispecific binding protein according to A39 to A75, wherein the fourth ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 4. A78. The multispecific binding protein according to A39 to A75, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 5.

[0084] A79. The multispecific binding protein according to A39 to A75, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 6.

[0085] A80. The multispecific binding protein according to A39 to A75, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 129.

[0086] A81 . The multispecific binding protein according to A39 to A75, wherein the third ankyrin repeat domain comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 130.

[0087] A82. The multispecific binding protein according to A39 to A81 , wherein said multispecific binding protein binds CD2 in PBS with a dissociation constant (KD) of or below about 10-7M.

[0088] A83. The multispecific binding protein according to A35 to A82, wherein said first and second tumor associated antigen are the same.

[0089] A84. The multispecific binding protein according to A35 to A82, wherein said first and second tumor associated antigen are different.

[0090] A85. The multispecific binding protein according to A84, wherein (i) the first TAA is EpCAM and the second TAA is EpCAM or (ii) the first TAA is MSLN and the second TAA is MSLN.

[0091] A86. The multispecific binding protein according to A84, wherein (i) the first TAA is EpCAM and the second TAA is MSLN or (ii) the first TAA is MSLN and the second TAA is EpCAM.

[0092] A87. The multispecific binding protein according to A85 or A86, wherein said second TAA is EpCAM, and wherein said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 10 to 15.

[0093] A88. The multispecific binding protein according to A85 or A86, wherein said second TAA is MSLN, and wherein said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128. A89. The multispecific binding protein according to any one of A1 to A88, wherein said binding agents are covalently linked with a peptide linker.

[0094] A90. The multispecific binding protein according to A89, wherein said peptide linker is a proline-threonine- rich peptide (PT) linker or a poly-Glycine-Serine (G4S) linker.

[0095] A91. The multispecific binding protein according to A89 or A90, wherein the amino acid sequence of said peptide linker has a length from 1 to 50 amino acids.

[0096] A92. The multispecific binding protein according to any one ofA1 to A91 , wherein any of said ankyrin repeat domains additionally comprises a G, a S or a GS at the N-terminus.

[0097] A93. The multispecific binding protein according to any one of A1 to A92, further comprising a half-life extending moiety.

[0098] A94. The multispecific binding protein according to A93, wherein said half-life extending moiety is a binding agent that specifically binds to human serum albumin.

[0099] A95. The multispecific binding protein according to A94, wherein said binding agent that specifically binds to human serum albumin comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0100] A96. The multispecific binding protein according to A95, wherein said ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 39 to 41 .

[0101] A97.The multispecific binding protein according to A94 or A95, wherein said ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 40.

[0102] A98. The multispecific binding protein according to A93, wherein said half-life extending moiety comprises an Fc domain of human lgG1.

[0103] A99. The multispecific binding protein according to A98, wherein the Fc domain comprises a) a first Fc chain comprising the substitution T366W and b) a second Fc chain comprising the substitutions T366S, L368A and T407V (Eu numbering), wherein the first and second chain heterodimerize to form a Fc heterodimer.

[0104] A100. The multispecific binding protein of A99, wherein the first Fc chain further comprises the substitution S354C and the second Fc chain further comprises the substitution Y349C (Eu numbering). A101 . The multispecific binding protein according to A99 or A100, further comprising the substitutions H435R and Y436F in the second Fc chain (Eu numbering).

[0105] A102. The multispecific binding protein according to A98 to A101 , wherein the first and / or second Fc chain comprises the following substitutions: L234A, L235A, and P329G (Eu numbering).

[0106] A103. The multispecific binding protein according to A99 to A102, wherein the first, second, third and optionally fourth ankyrin repeat domains are linearly arranged from N-terminus to C-terminus, wherein the N-terminus of the most N-terminal ankyrin repeat domain is covalently linked to the C-terminus of the first Fc chain, and wherein the second Fc chain is not covalently linked to any ankyrin repeat domain.

[0107] A104. The multispecific binding protein according to A99 to A102, wherein the N-terminus of one or more ankyrin repeat domains is covalently linked to the C-terminus of the first Fc chain, and the N-terminus of one or more ankyrin repeat domains is covalently linked to the C-terminus of the second Fc chain.

[0108] A105. The multispecific binding protein according to A104, wherein said ankyrin repeat domains are arranged, from the N-terminus to C-terminus, in the following order: 1) ankyrin repeat domain with binding specificity for a tumor associated antigen (TAA), optionally wherein said TAA is MSLN, 2) ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, suitably wherein said first immune cell-surface associated antigen is present on T cells, suitably wherein said first immune cell-surface associated antigen is CD3, and 3) ankyrin repeat domain with binding specificity for a second immune cellsurface associated antigen, suitably wherein said second immune cell-surface associated antigen is present on T cells, suitably wherein said second immune cell-surface associated antigen is CD2, and wherein the N-terminus of the ankyrin repeat domain of 1) is covalently linked to the C-terminus of the first Fc chain; and wherein the N-terminus of the ankyrin repeat domain covalently linked to the C-terminus of the second Fc chain is the ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA).

[0109] A106. The multispecific binding protein according to A104 or A105, wherein the one or more ankyrin repeat domains covalently linked to the C-terminus of the first Fc chain are, in this order, a) a MSLN binding domain, optionally comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128, b) a CD3 binding domain, optionally comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NOs: 106 to 125, and c) a CD2 binding domain, optionally comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs: SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132, and wherein the one or more ankyrin repeat domains covalently linked to the C-terminus of the second Fc chain is an ankyrin repeat domain with (i) a first binding specificity for the CD3 binding domain and (ii) a second binding specificity for EpCAM, wherein said ankyrin repeat domain is at least 80% identical to any one of SEQ ID NOs: 16 to 21 or SEQ ID NOs: 133 to 149. A107. The multispecific binding protein according to A99 to A106, wherein the first Fc chain comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 273.

[0110] A108. The multispecific protein according to A99 to A106, wherein the second Fc chain comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 274.

[0111] A109. The multispecific binding protein according to any one of A1 to A108, wherein said ankyrin repeat domains can be arranged in any order.

[0112] A110. The multispecific binding protein according to any one of A1 to 109, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 23 to 31 or SEQ ID NO: 98 to 104, SEQ ID NOs: 147 to 209 or SEQ ID NOs: 285 to 296.

[0113] A111. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0114] A112.The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0115] A113. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0116] A114. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26.

[0117] A115. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.

[0118] A116. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28.

[0119] A117. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0120] A118. The multispecific binding protein according to any one of A1 to A110 wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0121] A119. The multispecific binding protein according to any one of A1 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31 .

[0122] A120. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 98.

[0123] A121. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 99.

[0124] A122. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 100.

[0125] A123. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 101 .

[0126] A124. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 102.

[0127] A125. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 103. A126. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104.

[0128] A127. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 285.

[0129] A128. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 286.

[0130] A129. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 287.

[0131] A130. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 288.

[0132] A131. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 289. A132. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 290.

[0133] A133. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 291 .

[0134] A134. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 292.

[0135] A135. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 293.

[0136] A136. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 294.

[0137] A137. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 295.

[0138] A138. The multispecific binding protein according to any one of A36 to A110, wherein said multispecific binding protein comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 296.

[0139] A139. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 158 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159.

[0140] AMO. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159.

[0141] A141 . The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 161 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159.

[0142] A142. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 162 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.

[0143] A143. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 164 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.

[0144] A144. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163.

[0145] A145. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 166 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167.

[0146] A146. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 168 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167.

[0147] A147. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 169 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167.

[0148] A148. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 170 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 171.

[0149] A149. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 172 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 173.

[0150] A150. The multispecific binding protein according to any of A36 to A109, wherein said multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 174 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 175.

[0151] E1 . A recombinant binding protein comprising an ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said designed repeat domain to said first and second targets is mutually exclusive and wherein the first target is a CD3-specific binding agent and the second target is a tumor associated antigen, suitably an ovarian cancer-specific tumor associated antigen, suitably EpCAM or MSLN.

[0152] E2. The recombinant binding protein according to E1 , wherein said ankyrin repeat domain binds to said first target with a dissociation constant (KD) below 10-5M and / or wherein said ankyrin repeat domain binds to said second target with a dissociation constant (KD) below 10-5M.

[0153] E3. The recombinant binding protein according to any one of E1 to E2, wherein said ankyrin repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module.

[0154] E4. The recombinant binding protein according to any one of E1 to E3, wherein said ankyrin repeat domain comprises between three and six internal repeat modules.

[0155] E5. The recombinant binding protein according to E1 , wherein said second target is EpCAM.

[0156] E6. The recombinant binding protein according to any one of E1 to E5, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with any one of SEQ ID NOs: 16 to 21 or SEQ ID NO: 133 to 149.

[0157] E7. The recombinant binding protein according to any one of E1 to E6, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 16.

[0158] E8. The recombinant binding protein according to any one of E1 to E6, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 17.

[0159] E9. The recombinant binding protein according to any one of E1 to E6, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 18.

[0160] E10. The recombinant binding protein according to any one of E1 to E6, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 19.

[0161] E11 . The recombinant binding protein according to any one of E1 to E6, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 20. E12. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 21 .

[0162] E13. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 133.

[0163] E14. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 134.

[0164] E15. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 135.

[0165] E16. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 136.

[0166] E17. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 137.

[0167] E18. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 138.

[0168] E19. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 139.

[0169] E20. The recombinant binding protein according to any one of E1 to E6, wherein the said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with SEQ ID NO: 138.

[0170] E21 . The recombinant binding protein according to any one of E1 to E4, wherein said second target is MSLN.

[0171] E22. The recombinant binding protein according to E21 , wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with of SEQ ID NO: 22.

[0172] E23. The recombinant binding protein according to any one of E1 to E22, further comprising a binding agent with binding specificity for an immune cell-surface associated antigen.

[0173] E24.The recombinant protein according to E23, wherein said binding agent is an ankyrin repeat domain.

[0174] E25. The recombinant binding protein according to E24, wherein said ankyrin repeat domain has binding specificity for CD3. E26. The recombinant binding protein according to E16 or E25, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with any one of SEQ ID NOs: 2 to 3 or SEQ ID NO: 106 to 125.

[0175] E27. The recombinant binding protein according to any one of E1 to E26, further comprising a binding agent with specificity for a tumor-associated antigen, a cell type-associated antigen and / or an immune cell surface-expressed antigen.

[0176] E28. The recombinant binding protein according to E27, wherein said binding agent has binding specificity for a tumor-associated antigen and / or a cell-type associated antigen.

[0177] E29. The recombinant binding protein according to E28, wherein the tumor-associated antigen and / or the cell type-associated antigen is EpCAM or MSLN.

[0178] E30. The recombinant binding protein according to any one of E25 to E29, wherein the tumor-associated antigen and / or the cell type-associated antigen is EpCAM.

[0179] E31. The recombinant binding protein according to E30, wherein said binding agent with specificity for EpCAM is an ankyrin repeat domain.

[0180] E32. The recombinant binding protein according to E31 , wherein said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 10 to 15.

[0181] E33. The recombinant binding protein according to any one of E27 to E29, wherein the tumor-associated antigen and / or the cell type-associated antigen is MSLN.

[0182] E34. The recombinant binding protein according to E33, wherein said binding agent with specificity for MSLN is an ankyrin repeat domain.

[0183] E35. The recombinant binding protein according to E34, wherein said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128.

[0184] E36. The recombinant binding protein according to any one of E1 to E35, further comprising a binding agent with specificity for a tumor-associated antigen, a cell type-associated antigen and / or an immune cell surface-expressed antigen.

[0185] E37. The recombinant binding protein according to E36, wherein the immune cell surface-expressed antigen is CD2 or CD28. E38. The recombinant binding protein according to any one of E36 or E37, wherein the immune cell surface- expressed antigen is CD2.

[0186] E39. The recombinant binding protein according to any one of E37 or E38, wherein said binding agent with specificity for CD2 is an ankyrin repeat domain.

[0187] E40. The recombinant binding protein according to E39, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132.

[0188] E41 . The recombinant binding protein according to any one of E39 or E40, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 4.

[0189] E42. The recombinant binding protein according to any one of E39 or E40, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 5.

[0190] E43. The recombinant binding protein according to any one of E39 or E40, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 6.

[0191] E44. The recombinant binding protein according to any one of E39 or E40, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 129.

[0192] E45. The recombinant binding protein according to any one of E39 or E40, wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 130.

[0193] E46. An isolated nucleic acid encoding the multispecific binding protein according to any one of A1 to A150 or the recombinant binding protein to any one of E1 to E45.

[0194] E47. A cloning or expression vector comprising the nucleic acid of E46.

[0195] E48. The vector of E47, wherein said vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0196] E49. A host cell comprising the nucleic acid of E46 or the vector according to E47 or E48. E50. A method of producing a multispecific binding protein or recombinant binding protein, the method comprising culturing the cell of E49 and collecting the multispecific binding protein or recombinant binding protein from the cell and / or the culture medium.

[0197] E51 . A pharmaceutical composition comprising the multispecific binding protein according to any one of A1 to A50, recombinant binding protein according to any one of E1 to E45, the isolated nucleic acid of E46, the vector according to any one of E47 or E48 or the host cell of E49 and optionally a pharmaceutically acceptable carrier and / or diluent.

[0198] E52. The multispecific binding protein according to any one of A1 to A150, recombinant binding protein according to any one of E1 to E45, the isolated nucleic acid of E46, the vector according to any one of E47 or E48, the host cell of E49 or the pharmaceutical composition of E51 for use in a method of treating a medical condition.

[0199] E53. The multispecific binding protein according to any one of A1 to A150, recombinant binding protein according to any one of E1 to E45, the isolated nucleic acid of E46, the vector according to any one of E47 or E48, the host cell of E49 or the pharmaceutical composition of E51 , wherein said medical condition is a cancer.

[0200] E54. Use of the multispecific binding protein according to any one of A1 to A150, recombinant binding protein according to any one of E1 to E45, the isolated nucleic acid of E46, the vector according to any one of E47 or E48, the host cell of E49 or the pharmaceutical composition of E51 in the manufacture of a medicament.

[0201] E55. A method of treating a medical condition, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the multispecific binding protein according to any one of A1 to A150, recombinant binding protein according to any one of E1 to E45, the isolated nucleic acid of E46, the vector according to any one of E47 or E48, the host cell of E49 or the pharmaceutical composition of E51 .

[0202] E56. The method of treating a medical condition according to E55, wherein said medical condition is a cancer.

[0203] BRIEF DESCRIPTION OF THE FIGURES

[0204] Figure 1 : Illustration of an exemplary multispecific binding protein comprising a MSLN binding agent (a- MSLN), a 2-in-1 binding domain comprising a first binding region for EpCAM and a second binding region for the CD3 binding agent (“mask”), a CD2 binding agent (a-CD2), and a CD3 binding agent (a-CD3). (A) shows the compound in inactive state (e.g., in the proximity of healthy cells; “off’ state). (B) shows the release of the CD3 specific binding domain due to binding to TAA in the tumor microenvironment, enabling localized T-cell engagement (“on” state). Figure 2: Homogeneous Time Resolved Fluorescence (HTRF) signal x-fold over PBS of DARPin #1 , DARPin #2, DARPin #3, DARPin #4, DARPin #5 and DARPin #6 in absence or presence of 100 nM EpCAM. (A) Binding of DARPin #1 , DARPin #2, DARPin #3, DARPin #4, DARPin #5 and DARPin #6 to human CD3 target in absence of soluble EpCAM. The bio-scCDSsy target is bound by a Terbium-labelled streptavidin molecule, whereas the Switch drug DARPin construct is bound by a d2-labeled anti-His antibody. In absence of EpCAM, the Switch drug DARPin is preferentially in its closed conformation and hence a low signal intensity is expected. (B) Upon addition of soluble EpCAM, the Switch drug DARPin is opened and the binding signal is expected to increase.

[0205] Figure 3: RNA sequencing data analysis in samples from tissue biopsies, cancer cells and T cells. (A) Tissue sample biopsies from TCGA and GTEX which were analyzed for MSLN and EpCAM expression. The upper circulated area represents data for ovarian cancer tissue; lower circulated area represents data from healthy ovarian tissue. (B) Single cancer cell RNA sequencing data, where the fraction of cells coexpressing the genes of interest (EpCAM and MSLN or CD58) was determined. (C) Single cell RNA sequencing data, where the fraction of cells expressing the gene of interest (CD2 or CD28) was determined (n= number of samples; unpaired Wilcoxon test). (D) CD2 and CD28 expression on tumor infiltrated lymphocytes (TILs) isolated from ovarian cancer tumor samples measured by flow cytometry (left: CD4, right: CD8).

[0206] Figure 4: Functional assessment of multispecific binding proteins using a CD28 expressing Jurkat IL2 reporter cell line in the presence of OVCAR-3 WT or OVCAR-3 CD58 KO or without target cells after 24h incubation. (A) MSP2 (multispecific protein with SEQ ID NO: 24, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin); (B) MSP3 (multispecific protein with SEQ ID NO: 25, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin); (C) MSP5 (multispecific protein with SEQ ID NO: 27, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin); (D) MSP7 (multispecific protein with SEQ ID NO: 29, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to EpCAM and a 2-in-1 ankyrin repeat domain with dual binding specificity for MSLN and the anti-CD3 specific DARPin); (E) MSP8 (multispecific protein with SEQ ID NO: 30, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to EpCAM and a 2-in- 1 ankyrin repeat domain with dual binding specificity for MSLN and the anti-CD3 specific DARPin). Luminescence was measured with a Tecan M1000 instrument, and the data were plotted using GraphPad Prism (version 10). RLU: Relative Luminescence Units.

[0207] Figure 5: Functional assessment of multispecific DARPin proteins using a CD28 expressing Jurkat IL2 reporter cell line in the presence of OVCAR-3 WT or OVCAR-3 CD58KO or without target cells after 24h incubation. (A) MSP11 (multispecific protein with SEQ ID NO: 33, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and an ankyrin repeat domain binding to MSLN). (B) MSP12 (multispecific protein with SEQ ID NO: 34, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and an ankyrin repeat domain binding to MSLN). Luminescence was measured with a Tecan M1000 instrument, and the data were plotted using GraphPad Prism (version 10). RLU: Relative Luminescence Units.

[0208] Figure 6: Target specific T cell activation of selected multispecific binding proteins in the presence of OVCAR-3 WT, OVCAR-3 CD58KO or in absence of target cells. (A) corresponds to MSP2 (multispecific protein with SEQ ID NO: 24, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin); (B) corresponds to MSP3 (multispecific protein with SEQ ID NO: 25, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin); (C) corresponds to MSP5 (multispecific protein with SEQ ID NO: 27, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to MSLN and a 2-in-1 ankyrin repeat domain with dual binding specificity for EpCAM and the anti-CD3 specific DARPin). Activation of T cells was assessed by upregulation of CD25, measured by flow cytometry. Data with T cells from 2 different donors shown.

[0209] Figure 7: Target specific T cell activation of selected multispecific binding proteins in the presence of OVCAR-3 WT, OVCAR-3 CD58KO or in absence of target cells. (A) corresponds to MSP7 (multispecific protein with SEQ ID NO: 29, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to EpCAM and a 2-in-1 ankyrin repeat domain with dual binding specificity for MSLN and the anti-CD3 specific DARPin); (B) corresponds to MSP8 (multispecific protein with SEQ ID NO: 30, comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to EpCAM and a 2-in-1 ankyrin repeat domain with dual binding specificity for MSLN and the anti-CD3 specific DARPin); (C) corresponds to MSP9 (multispecific protein with SEQ ID NO: 31 , comprising an ankyrin repeat domain binding to CD2, an ankyrin repeat domain binding to CD3, an ankyrin repeat domain binding to EpCAM and a 2-in-1 ankyrin repeat domain with dual binding specificity for MSLN and the anti-CD3 specific DARPin). Activation of T cells was assessed by upregulation of CD25, measured by flow cytometry.

[0210] Figure 8: Repetitive exposure of T cells to multispecific binding proteins. (A) shows a schematic illustration of the experimental procedure. (B) shows Median fluorescence intensity (MFI) values plotted for CD25 T cell activation marker after four rounds of exposure to multispecific binding proteins MSP1 1 (SEQ ID NO: 33) and MSP12 (SEQ ID NO: 34); (C) shows Median fluorescence intensity (MFI) values plotted for 4-1 BB T cell activation marker after four rounds of exposure to multispecific binding proteins MSP11 and MSP12; (D) shows Median fluorescence intensity (MFI) values plotted percentage of cells double positive for TIM- 3 and PD-1 after four rounds of exposure to multispecific proteins MSP11 and MSP12; (E) shows the cytotoxic effect of MSP3 (SEQ ID NO: 25) and MSP13 (SEQ ID NO: 35) on freshly stimulated T cells and T cells exposed to MSP11 and MSP12 for three stimulation rounds. White and black columns (B-D) correspond to T cells activated with MSP11 and MSP12 coated DARPins, respectively. White and black symbols correspond to cytotoxicity induced by T cells either freshly isolated (inactivated) or previously activated over 3 rounds (as illustrated in (A)), with MSP11 and MSP12, respectively (E).

[0211] Figure 9: Effect of multispecific binding proteins on cytokine release in human whole blood ex vivo. (A) Levels of IFNy, IL-6, TFN-a and IL-10 released in blood. Circles: MSP3 and squares: MSP11 .(B) Levels of IL-2, IL-10, IL-6, IFN-y, TFN-a and Granzyme B released in blood. Squares: MSP11 , circles: MSP3, up- triangles: MSP5, down-triangles: MSP14, small circles: MSP15, empty circles: MSP18 empty squares: MSP16, empty triangles: blood only. Data were analyzed using the MSD discovery software calculating concentrations in pg / mL from interpolation of the standard curves and the output values were plotted as non-linear regression graphs using GraphPad Prism.

[0212] Figure 10: Target specific T cell activation of selected multispecific binding proteins in the presence OVCAR-3 CD58KO or in absence of target cells. (A) MSP3 (SEQ ID NO: 25); (B) MSP11 (SEQ ID NO: 33). CD25 activation marker expressed on CD8+ T cells was measured by flow cytometry.

[0213] Figure 11 : Repetitive activation of T cells previously exposure to multispecific binding proteins. (A) shows a schematic illustration of the experimental procedure. (B) Levels of IFNy after incubation of “exhausted” T cells with MSP3 and MSP13 in the presence of tumor target cells CaoV-3; (C) Levels of IL-2 after incubation of “exhausted” T cells with MSP3 and MSP13 in the presence of tumor target cells CaoV-3; (D) Levels of TNFa after incubation of “exhausted” T cells with MSP3 and MSP13 in the presence of tumor target cells CaoV-3; (E) Levels of activation marker CD25 expressed on CD8+ T cells after incubation of “exhausted” T cells with MSP3 and MSP13 in the presence of tumor target cells CaoV-3; (F) Proliferation of “exhausted” T cells after incubation with MSP3 and MSP13 in the presence of tumor target cells CaoV-3. Square symbol on the graph corresponds to MSP3 and triangle symbol corresponds to MSP13; CR refers to Cytokine Release and MSD to Meso Scale Discovery Electrochemiluminescence.

[0214] Figure 12: In Vivo Efficacy evaluation of multispecific binding proteins in PBMC humanized NXG mice and ovarian tumor model. (A) Effect of the tested proteins MSP3 and MSP11 on the tumor volume as a function of time: circle symbol corresponds to PBS, square symbol corresponds to MSP3, upper triangle corresponds to MSP11 and lower triangle corresponds to a positive control molecule. (B) Cytokine release (IL-6 and TNF-a) after 2 hours of administration and T-cell count of hCD45+ cells in blood by vehicle (circles), MSP3 (squares), MSP15 (triangles) and the control MSLN x CD3 TriTAC (inverted triangles).

[0215] Figure 13: Illustration of an exemplary multispecific binding protein comprising a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen (a-CD3), a second ankyrin repeat domain (2-in-1 domain) with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain (“mask”) and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA1), a third ankyrin repeat domain with binding specificity for a second tumor associated antigen (TAA2), and a fourth ankyrin domain with binding specificity for a second immune cell-surface associated antigen (a-CD2). (A) In absence of a target cell, the mask of the 2-in-1 domain binds to the first ankyrin domain (a-CD3) (closed switch). (B) In the presence of the target cell, the third ankyrin domain binds to TAA2 (arrow). (C) The binding of the third ankyrin domain to TAA2 allows the binding of the second ankyrin domain to TAA1 , releasing the a-CD3 specific binding domain (switch open). (D) Upon release of the first ankyrin domain, this domain binds to CD3, thereby enabling localized T-cell engagement. The fourth ankyrin domain binds to the second immune cell-surface associated antigen (CD2).

[0216] Figure 14: Activity of MSP15 (Switch DARPin) against ovarian cancer cells expressing one or two tumor- associated antigens (TAA). (A) T-cell activation measured as the percentage of CD25 positive CD8 T cells when co-cultured with CHO cells expressing one TAA (EpCAM or MSLN), CHO cells expressing the two TAAs MSLN and EpCAM and Pan T cells alone. (B) T cell mediated cytotoxicity of MSP15 on CHO cells expressing one TAA (EpCAM: black squares or MSLN: grey diamonds) or expressing two TAAs (MSLN and EpCAM: black circles). (C) T-cell mediated cytotoxicity of TriTAC HPN536. (D) T-cell mediated cytotoxicity of Solitomab. Squares: cells expressing EpCAM, diamonds: cells expressing MSLN, circles: cells expressing EpCAM and MSLN. For (A), triangles: Pan T cells.

[0217] Figure 15 Different organization of the ankyrin domains in the multispecific binding protein does not affect T cell mediated cytotoxicity against CHO cells expressing MSLN and EpCAM. (A) Design of eight different confirmations of multispecific proteins. (B) Cytotoxicity of the multispecific binding proteins of (A) measured by Lactate dehydrogenase (LDH) assay on CHO cells expressing both TAAs.

[0218] Figure 16: Effect of the switch activation on the binding of the multispecific binding proteins to ovarian cancer cells expressing one or two tumor associated antigens (TAA), measured as Mean fluorescence intensity (MFI) of anti-DARPin-AF647 antibody. (A) Binding of proteins with or without switch on cells expressing MSLN and EpCAM. (B) Binding of proteins with or without switch on cells expressing MSLN. (C) Binding of proteins with or without switch on cells expressing EpCAM. Up-triangles: MSP11 ; downtriangles: MSP3, circles: MSP15; squares: MSP12; diamonds: MSP13.

[0219] Figure 17: CD3 switch prevents binding to CD3 in absence of TAAs. (A) Competition of switched and nonswitched multispecific proteins having a CD3 domain with the anti-CD3 antibody OKT3. Circles: MSP11 ; triangles: MSP3; squares: MSP15. Black lines represent binding of the OKT3 antibody in presence of increasing concentrations of the DARPin and grey lines represent the binding of the DARPins. (B) Binding of switched (MSP3) and non-switched (MSP11) multispecific binding proteins to T cells expressing CD2 (Jurkat) and non-expressing CD2 (Jurkat CD2 KO).

[0220] Figure 18: In vivo effect of multispecific binding proteins MSP3, MSP15, MSP16 and MSP20 in CAPAN-2 cells. (A) Schematic illustration of the experimental procedure. (B) Effect of the tested proteins on the tumor volume as a function of time (black circles: vehicle PBS, diamonds: MSP15, triangles: MSP20, grey circles: MSP16, squares: MSP3). (C) Cytokine release (IFN-y, TNF-a, IL-6, IL-2, IL-8, Granzyme B, IL12-70p, IL-8 and IP-10 and) after 2 hours of administration. (D) T-cell count of hCD45+ cells in blood. Black circles: PBS (vehicle), diamonds: MSP15, triangles: MSP20, grey circles: MSP16, squares: MSP3. Full symbols: donor A, empty symbols: donor B.

[0221] Figure 19: Illustration of multispecific binding proteins with a half-life extender. A half-life extender (HLE) was added to proteins derived from MSP15 with the domains arranged in different formats. The half-life extender is a Fc fragment knob-in-hole (KiH) wherein all the domains are bound to the knob chain (MSP21 or MSP22, in a linear format) or wherein one or more domains are bound to the knob chain and one or more of the domains are bound to the hole chain (non-linear or asymmetric format, as in MSP23 and MSP26 to MSP27). The chains fused to knob-Fc in the asymmetric format are referenced as “a”, and the chains fused to hole-Fc are referenced as “b”.

[0222] Figure 20: Potency of multispecific binding proteins without (MSP15) and with HLE, either in linear format (MSP21) or in non-linear (MSP23) format. (A) % Cell killing, (B) % T cell proliferation and (C) T cell activation of multispecific proteins MSP15 (circles), MSP21 (squares) and MSP23 (triangles).

[0223] Figure 21 : Cytotoxicity of multispecific binding proteins without (MSP15) and with HLE, either in linear format (MSP21) or in non-linear (MSP23) format on cells expressing EpCAM and MSLN (circles), EpCAM (triangles) or MSLN (squares). (A) % of killing of target cells measured by lactate dehydrogenase (LDH). (B) T cell activation expressed as median fluorescence intensity (MFI) plotted for CD25-positive T cells.

[0224] Figure 22: Functionality of the switch with different ratios of effector (pan-T cells) to tumor cells (OVCAR- 3 cells). (A) Killing of OVCAR-3 cells measured by lactate dehydrogenase assay (LDH). (B) Killing of OVCAR-3 cells measured by Luciferase assay.

[0225] Figure 23: Activity of multispecific binding proteins in the presence of soluble mesothelin (sMSLN). T cell activation by MSP15, MSP21 , MSP23, MSP41 , MSP55, MSP57, HPN-536 (TriTAC-similar) and solitomab. Activation is expressed as median fluorescence intensity (MFI) plotted for CD25 on CD8-positive T cells. Circles: untreated cells; diamonds; 0.1 nM sMSLN; down-triangles: 1 nM sMSLN; up-triangles: 10 nM sMSLN; squares; 100 nM sMSLN.

[0226] Figure 24: Activity of multispecific binding proteins in presence of soluble mesothelin (sMSLN). % Killing of target cells by MSP15, MSP21 , MSP23, MSP41 , MSP55, MSP57, HPN-536 (TriTAC-similar) and Solitomab. Circles: untreated cells; diamonds; 0.1 nM sMSLN; down-triangles: 1 nM sMSLN; up-triangles: 10 nM sMSLN; squares; 100 nM sMSLN.

[0227] Figure 25: Binding of multispecific binding proteins comprising an engineered 2-in-1 domain. (A) Binding of DARPin #21 (SEQ ID NO: 149, squares) compared to its parental sequence, DARPin #1 (circles). (B) Binding of DARPins #14 (SEQ ID NO: 139, triangles) and #16 (SEQ ID NO: 141 , squares) compared to their parental sequence, DARPin #5 (circles). Binding is expressed as median fluorescence intensity (MFI). Figure 26: Multispecific binding proteins comprising engineered versions of the CD2 and MSLN ankyrin repeat domains. Activity of multispecific proteins comprising a MSLN variant (MSP39, triangles) and a CD2 variant (MSP38, squares) measured in a co-culture with OVCAR-3 target cells and effector Jurkat-Lucia NFAT reporter cells and compared to parental protein (MSP15, circles). RLU, relative luminescence.

[0228] Figure 27: T cell activation of multispecific binding proteins comprising engineered versions of the CD2 and MSLN ankyrin repeat domains in the presence of CHO cells expressing a) MSLN and EpCAM, b) MSLN, or c) EpCAM. T-cell activation was measured by staining CD25 on CD8 T-cells and shown as MFI. MSP15 (circles) correspond to the parental protein, MSP38 (squares) comprises a CD2 variant and MSP39 (triangles) comprises a MSLN variant.

[0229] Figure 28: T cell activation of multispecific binding proteins comprising engineered ankyrin repeat domains. Percentage (%) of killing of each engineered protein (MSP21 to MSP71) and the parental (MSP15 and MSP20) was measured in CHO cells expressing MSLN (triangles), EpCAM (squares) or both TAAS (circles).

[0230] Figure 29: Potency of multispecific binding proteins without (MSP15) and with HLE, either in linear format (MSP21) or in non-linear (MSP23) format and also comprising engineered ankyrin repeat domains (MSP28). (A) T cell activation and (B) % Cell killing of multispecific proteins MSP15 (circles), MSP21 (squares), MSP23 (triangles) and MSP28 (diamonds).

[0231] Figure 30: In vivo efficacy of multispecific binding proteins with Fc fusion in OVCAR-3 cells. (A) Mean tumor growth curves after treatment with MSP15, MSP21 and MSP23.Tumor volumes are shown as mean + / - SEM. (B) Cytokine release (IFN-y, TNF-a, IL-6, IL-2, IL-8, Granzyme B, IL12-70p, IL-8 and IP-10 and) after 2 hours of administration. Circles: PBS (vehicle), squares: MSP15, up-triangles: MSP21 administered 3 times per week, diamonds: MSP23, down-triangles: MSP21 administered once per week. Full symbols: donor A, empty symbols: donor B.

[0232] Figure 31 : Pharmacokinetics of multispecific binding proteins MSP21 , MSP22, MSP23, MSP24 and MSP25. Mean of serum concentration of each protein measured at different time points. Dotted line represents the Lower Limit of Quantification (LLOQ).

[0233] DETAILED DESCRIPTION OF THE DISCLOSURE

[0234] “T cell exhaustion” is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, initially in the setting of chronic viral infection, but more recently in response to tumors. Understanding the features of and pathways to T cell exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell stimulation therapies. In certain cancer, e.g., ovarian cancer, currently available immunotherapy approaches demonstrated limited effectiveness, since T cells are present but often in an exhausted and inactive state. Additionally, a highly immunosuppressive tumor microenvironment is observed and about 90% of ovarian cancer cells show CD58 genetic aberrations, or lowered CD58 expression levels (Zhao et al. Seminars in Cancer Biology 2024; 104-105:1-15).

[0235] In response to the above-mentioned challenges, Applicant has designed multispecific proteins formatted as conditionally-activatable T cell engagers additionally comprising a co-stimulatory binding domain, suitably CD2. By co-engaging CD3 with the co-stimulatory effect of CD2, tumor infiltrating lymphocytes (TILs) are rescued or prevented from exhaustion / senescence, a mechanism which will also allow activity on CD58 negative / low target cells, while decreasing or preventing off-tumor toxicity, and allowing higher dosing and increased potency. Activation of the T cell, suitably via stimulation of CD3, is conditional on the presence of at least one tumor associated antigen (TAA). This is accomplished by designing a binding domain having binding specificity for two targets, i.e. for an immune cell-surface associated antigen, e.g. CD3 and at least one TAA, suitably EpCAM or MSLN, wherein binding of such repeat domain to its two targets is mutually exclusive. Such a conformation exhibits reduced cytokine release syndrome compared to conventional T cell engagers, as CD3 is only activated in the presence of the TAA. Moreover, said multispecific proteins may comprise a binding domain with binding specificity for a second TAA, suitably EpCAM or MSLN. Said binding domains are suitably ankyrin repeat domains.

[0236] Ankyrin repeat proteins (DARPins) are a class of binding molecules with the potential to overcome the limitations of monoclonal antibodies, offering novel therapeutic approaches. These proteins can consist of a single ankyrin repeat domain or multiple domains — two, three, four, five, or more — targeting the same or different molecules (Stumpp et al. Drug Discov. Today 2008; 13: 695-701 ; U.S. Patent No. 9,458,211). Single-domain ankyrin repeat proteins are small (14 kDa) but exhibit high affinity and specificity for their target proteins. The ability to combine multiple domains in one protein enhances their versatility, making them ideal candidates for agonistic, antagonistic, or inhibitory drugs. Additionally, ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extending agents, enabling the development of entirely new drug formats. Ankyrin repeat domains, as structural units, can be selected from libraries (W02002 / 020565; Binz et al., Nat. Biotechnol. 2004; 22: 575-582; Stumpp et al. 2008 (supra)) for their ability to bind specific targets with high affinity, making them valuable components in recombinant proteins for disease treatment.

[0237] The binding specificity of an ankyrin repeat domain is dictated by the nature of the repeat modules comprised in each repeat domain. Both internal repeat modules and terminal capping modules (C-cap or N-cap modules) can contribute to said specificity.

[0238] The ankyrin repeat domains with dual binding specificity described herein can be genetically fused to further components, such as, e.g., a therapeutic agent, and such fusions are also referred to as “multispecific binding protein” or “constructs” interchangeably. In such constructs, the ankyrin repeat domain with dual binding specificity described herein may act as on- or off-switch for a fused therapeutic agent. Such a therapeutic agent is a binding agent. Such binding agents encompass any molecule capable of specifically binding a target, and include, for example, antibodies, antibody fragments, aptamers, peptides, alternative scaffolds, antibody mimics, repeat proteins, e.g., ankyrin repeat proteins and receptor proteins, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues.

[0239] The present disclosure provides novel multispecific binding proteins comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second immune cell-surface associated antigen. Additionally, this multispecific protein can comprise (4) a fourth binding domain with binding specificity for a second disease-associated antigen, suitably a tumor associated antigen (TAA).

[0240] The disclosure also provides ankyrin repeat domains and recombinant proteins comprising said ankyrin repeat domains. At least one of the ankyrin repeat domains has binding specificity for two targets, wherein binding of such repeat domain to its two targets is mutually exclusive, thereby functioning as a molecular switch, such as, e.g., a switch to control activation or deactivation of a therapeutic agent. The disclosure further relates to nucleic acids, pharmaceutical compositions, vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer.

[0241] The present disclosure additionally addresses the critical issue of "dirty targets" in cancer therapy through an approach designed to avoid on-target off-tumor toxicity of immune cell engagers to improve their specificity and efficacy in treating tumors, e.g. solid tumors. As shown herein, T-cell activation occurs preferentially on target cells expressing two tumor-associated antigens, thereby providing spatial control over cytotoxicity. This enables targeting of previously unsuitable proteins, expanding the repertoire of addressable cancer targets beyond those with strictly tumor-specific expression patterns, while maintaining a favorable therapeutic index through its contextual activation mechanism.

[0242] AND-gates in T cell engagers represent a sophisticated safety mechanism that requires dual antigen recognition before T cell activation occurs. This design addresses one of the most critical challenges in T cell immunotherapy: achieving tumor-specific activation while minimizing off-target toxicity.

[0243] Traditional bispecific tumor cell engagers bind to a single tumor-associated antigen (TAA) and CD3 on T cells. However, most TAAs are also expressed at low levels on healthy tissues, leading to potential on- target, off-tumor toxicity. This has been a major dose-limiting factor in clinical applications.

[0244] AND-gate T cell engagers are engineered to require binding to two different tumor antigens simultaneously before productive T-cell activation can occur. By targeting two antigens, the probability of finding both on healthy cells decreases exponentially, improving the therapeutic window. Additionally, healthy tissues expressing only one of the target antigens remain largely unaffected, allowing for higher dosing and improved efficacy. Finally, the requirement for dual engagement creates multiple fail-safes against off-target activation.

[0245] This approach has shown particular promise in solid tumors where single antigen targeting has been challenging due to antigen expression on healthy tissues. The AND-gate design allows targeting of antigen combinations that would be too toxic to address with conventional bispecific TCEs. The development of AND-gate systems represents a significant advancement in precision immunotherapy, offering a path toward safer and more effective T-cell engagers for treating cancer.

[0246] Based on this rationale, the present disclosure also provides a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second tumor associated antigen, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to said first tumor associated antigen and (ii) the third ankyrin repeat domain to said second tumor associated antigen.

[0247] If the target cell expresses both the first and the second TAA, binding of the third binding domain to the second TAA allows the second binding domain to bind to its TAA, thereby increasing the binding avidity of the full molecule to the target cell. When the second binding domain binds to its TAA, the first binding domain is released from the second binding domain due to steric hindrance. In the absence of the first TAA, the binding of the second binding domain to its TAA is strongly reduced by steric hindrance caused by the first binding domain binding to the second binding domain. Hence, the increased binding avidity of the second binding domain upon binding of the third binding domain allows stronger binding of the second binding domain to its TAA, displacing the first binding domain from the second binding domain due to steric hindrance, thereby releasing the first binding domain, allowing the binding of the first binding domain to the first immune cell-surface associated antigen, providing an AND-gate (Figure 13).

[0248] In one embodiment, said second ankyrin domain reversibly binds said first ankyrin repeat domain.

[0249] In one embodiment, the first and second tumor-associated antigens (TAA) are present on the same cell.

[0250] In one embodiment, the first immune cell-surface associated antigen and the second immune cell-surface associated antigen are present on the same immune cell. Suitably, said immune cell is a T cell. Suitably, said first immune cell-surface associated antigen is a component of the human T-cell receptor (TCR) complex. In a preferred embodiment, said first immune cell-surface associated antigen is CD3. In one embodiment, said second ankyrin domain comprised reversibly binds said first ankyrin repeat domain. In one embodiment, said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of the second ankyrin repeat domain to said tumor associated antigen. In one embodiment, said second repeat domain has a first binding specificity for a first target, wherein said first target is a CD3-specific binding agent. In one embodiment, said CD3-specific binding agent is a CD3- specific ankyrin repeat domain. In one embodiment, said second target is a tumor associated antigen (TAA), e.g. EpCAM or MSLN, wherein binding of said ankyrin repeat domain comprising first and second binding regions to the first and second targets is mutually exclusive.

[0251] Suitably, said second immune cell-surface associated antigen provides a co-stimulatory signal to a T-cell, e.g. CD2 or CD28. In a preferred embodiment, said second immune cell-surface associated antigen is CD2. Suitably, said first and second tumor associated antigen are the same. In an alternative embodiment, said first and second tumor associated antigen are different. In one embodiment (i) the first TAA is EpCAM and the second TAA is EpCAM or (ii) the first TAA is MSLN and the second TAA is MSLN. In one embodiment (i) the first TAA is EpCAM and the second TAA is MSLN or (ii) the first TAA is MSLN and the second TAA is EpCAM.

[0252] Immune recognition of therapeutic proteins represents a critical challenge in biotechnology and pharmaceutical development that significantly impacts the safety, efficacy, and commercial viability of protein-based therapeutics. This phenomenon occurs when the human immune system identifies administered therapeutic proteins as foreign substances, triggering unwanted immune responses that can compromise treatment outcomes.

[0253] Methods for / n silico prediction of immunogenicity are known, e.g., using MHC Binding prediction tools, e.g., provided by the Immune Epitope Database (IEDB) (www.iedb.org), NetMHCHpan (Nielsen M et al. 2008. PLoS Comput Biol. Jul 4;4(7):e1000107) or EpiBase (Lonza). A suitable version of the NetMHCHpan algorithm is version 4.1 (Kaabinejadian et al., 2002. Frontiers in Immunology, 13:835454) or version 4.3 (Nilsson et al. (2023). Sci Adv 9(47); eadj6367).

[0254] In certain embodiments, the multispecific binding proteins disclosed herein, or the designed ankyrin repeat domains comprised in the multispecific binding protein disclosed herein, or the designed ankyrin repeat domains disclosed herein have reduced immunogenicity potential, e.g., as determined by in silico analysis, e.g. as described herein. Accordingly, in one embodiment, said designed ankyrin repeat domain with binding specificity for CD3, or the 2-in- 1 domain, or designed ankyrin repeat domain with binding specificity for CD2, or designed ankyrin repeat domain with binding specificity for MSLN has an immunogenicity score of less than 2.0, less than 1 .0, less than 1 .8, less than 1 .7, less than 1 .6, less than 1 .5, less than 1 .4, less than 1 .3, less than 1 .2, less than 1 .1 , less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 or substantially 0.

[0255] EpCAM specific binding agents Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Additionally, it has oncogenic potential via its capacity to upregulate c-myc, e-fabp, and cyclins A & E.

[0256] In one aspect the disclosure relates to an ankyrin repeat domain with binding specificity for EpCAM.

[0257] In one embodiment said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 10 to 15.

[0258] Thus, in one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 10 to 15. In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 10 to 15. In another embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 10 to 15. In a further embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 10 to 15. In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 10 to 15. In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 10 to 15.

[0259] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 10. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0260] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 1 1. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 11 . In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 12. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0261] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 13. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0262] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 14. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0263] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 15. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0264] In one embodiment, said ankyrin repeat domain with binding specificity for EpCAM binds to human EpCAM with a dissociation constant (KD) of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M. Thus, in one embodiment, said recombinant binding protein binds to EpCAM with a KD of or below about 10-6M. In another embodiment, said recombinant binding protein binds to EpCAM with a KD of or below about 10-7M. In another embodiment, said recombinant binding protein binds to EpCAM with a KD of or below about 10-8M. In another embodiment, said recombinant binding protein binds to EpCAM with a KD of or below about 10-9M. Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR). Accordingly, in one embodiment, said ankyrin repeat domain binds human EpCAM in PBS with a KD of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 10 to 15.

[0265] CD3-specific binding agents

[0266] The term “CD3" or "Cluster of Differentiation 3" refers to a multimeric protein complex composed of four distinct chains. In mammals, the complex contains a CD3y (gamma) chain, a CD36 (delta) chain, and two CD3s (epsilon) chains. These chains associate with the T cell receptor (TCR) and the CD3 (zeta) chain to generate an activation signal in T cells. CD3 is critical for T cell activation and the initiation of T cell effector functions, such as cytokine production, cytotoxicity, and the release of other immune mediators. It enables T cells to recognize and respond to specific antigens, thereby orchestrating adaptive immune responses. The amino acid sequences of human CD3 gamma, delta, epsilon and zeta chains are shown in NCBI Ref. Seq. NP_000064.1 , NP_000723.1 , NP_000724.1 and NP_932170.1 , respectively. The term CD3-specific binding agent encompasses any molecule capable of specifically binding CD3, and include, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al. J Biol Chem 1991 ; 266:5182- 5190), alternative scaffolds, antibody mimics, repeat proteins, e.g., ankyrin repeat proteins, receptor proteins and any other naturally occurring interaction partners of CD3, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues. Ankyrin repeat domains specifically binding CD3 are disclosed in WO2022 / 129428 (incorporated by reference).

[0267] In a particular embodiment, said CD3-specific binding agent is an ankyrin repeat domain. In some embodiments said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 3. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2 to 3.

[0268] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0269] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0270] In one embodiment said ankyrin repeat domain with binding specificity for CD3 has reduced immunogenicity risk. Suitably, said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 106 to 125. Optionally, said designed ankyrin repeat domain with binding specificity for CD3 has an immunogenicity score of less than 2.0, less than 1 .0, less than 1 .8, less than 1 .7, less than 1 .6, less than 1 .5, less than 1 .4, less than 1 .3, less than 1 .2, less than 1 .1 , less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 or substantially 0.

[0271] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 106. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 106.

[0272] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 107. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 107.

[0273] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 108.

[0274] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 109. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 109.

[0275] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 110. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 10.

[0276] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 111. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 11. In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 112. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 12.

[0277] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 113. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 13.

[0278] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 114. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 14.

[0279] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 115. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 15.

[0280] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 116. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 16.

[0281] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 117. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 17.

[0282] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 118. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 18.

[0283] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 119. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 1 19.

[0284] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 120. In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 121. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 121.

[0285] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 122. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 122.

[0286] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 123. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 123.

[0287] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 124. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 124.

[0288] In some embodiment, said CD3-specific ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 125. In one embodiment said ankyrin repeat domain with binding specificity for CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 125.

[0289] In one embodiment said CD3-specific ankyrin repeat domain binds CD3 with a dissociation constant (KD) or of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M, or of or below about 1 O-10M. Thus, in one embodiment, said recombinant binding protein binds to CD3 with a KD of or below about 10-6M. In another embodiment, said recombinant binding protein binds to

[0290] CD3 with a KD of or below about 10-7M. In another embodiment, said recombinant binding protein binds to

[0291] CD3 with a KD of or below about 10-8M. In another embodiment, said recombinant binding protein binds to

[0292] CD3 with a KD of or below about 10-9M. In another embodiment, said recombinant binding protein binds to

[0293] CD3 with a KD of or below about 10'10M. Preferably, said binding is measured in PBS, suitably using surface plasmon resonance (SPR). Accordingly, in one embodiment, said ankyrin repeat domain binds human CD3 in PBS with a KD of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M, or of or below about 10-10M, wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NOs: 106 to 125.

[0294] MSLN-specific binding agents

[0295] Mesothelin, also known as MSLN, is a 40 kDa protein that is expressed in mesothelial cells. It constitutes an attractive therapeutic target for the treatment of certain cancers, particularly in certain cancers that overexpress mesothelin, e.g., ovarian cancer, mesothelioma or pancreatic ductal adenocarcinoma. Ankyrin repeat domains specifically binding mesothelin are disclosed in WO2025 / 163144 (incorporated by reference).

[0296] In one embodiment said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 7 to 9.

[0297] Thus, in one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 7 to 9. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 7 to 9. In another embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 7 to 9. In a further embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 7 to 9. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 7 to 9. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 7 to 9.

[0298] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 7. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0299] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 8. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0300] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 9. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0301] In one embodiment said ankyrin repeat domain with binding specificity for MSLN has reduced immunogenicity risk. Suitably, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 126 to 128. Optionally, said designed ankyrin repeat domain with binding specificity for MSLN has an immunogenicity score of less than 2.0, less than 1 .0, less than 1 .8, less than 1 .7, less than 1 .6, less than 1 .5, less than 1 .4, less than 1 .3, less than 1 .2, less than 1 .1 , less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 or substantially 0.

[0302] Thus, in one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 126 to 128. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 126 to 128. In another embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 126 to 128. In a further embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 126 to 128. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 126 to 128. In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 126 to 128.

[0303] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 126. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 126.

[0304] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 127. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 127.

[0305] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with the amino acid sequence of SEQ ID NO: 128. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 128.

[0306] In one embodiment, said ankyrin repeat domain with binding specificity for MSLN binds to human MSLN with a dissociation constant (KD) of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M. Thus, in one embodiment, said recombinant binding protein binds to MSLN with a KD of or below about 10-6M. In another embodiment, said recombinant binding protein binds to MSLN with a KD of or below about 10-7M. In another embodiment, said recombinant binding protein binds to MSLN with a KD of or below about 10-8M. In another embodiment, said recombinant binding protein binds to MSLN with a KD of or below about 10-9M. Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR).

[0307] Accordingly, in one embodiment, said ankyrin repeat domain binds human MSLN in PBS with a KD of or below about 10-6M, or of or below about 10-7M, or of or below about 10-8M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128.

[0308] 2-in-1 binding domains

[0309] Ankyrin repeat domains with mutually exclusive binding specificity for two targets have been described (see WO2023 / 110983). Such dual-specific repeat domains (also referred to as “2-in-1 domains”) can be created by combining repeat modules of two parental repeat domains. The binding specificity to the respective targets of the parental repeat domains therefore confers the binding specificity for each of the two targets. In one embodiment, said repeat domain has a first binding specificity for a first target, wherein said first target is a CD3-specific binding agent. In one embodiment, said CD3-specific binding agent is a CD3- specific ankyrin repeat domain as described above in “CD3 specific binding agents”.

[0310] In one embodiment, said first target is an ankyrin repeat domain with binding specificity for CD3, e.g., as described in “CD3 specific binding agents”, and said second target is a tumor associated antigen (TAA), e.g. EpCAM, as described in “EpCAM-specific binding agents" or MSLN, as described in “MSLN-specific binding agents", wherein binding of said ankyrin repeat domain comprising first and second binding regions to the first and second targets is mutually exclusive. Said ankyrin repeat domain binds the first target, i.e., the CD3-binding ankyrin repeat domain (“masking target”) and blocks its activity in the absence of TAA (“sensor target”). Upon binding to TAA, steric hindrance induces the release of the CD3-binding ankyrin repeat domain, enabling binding of this ankyrin repeat domain to its target, stimulating T cell activation. In some embodiments, the affinity of said ankyrin repeat domain for the sensor target is higher than the affinity of said ankyrin repeat domain for the masking target. In any of the embodiments and aspects herein, the first binding region with binding specificity for a CD3-binding ankyrin repeat domain may be located N- terminally of the second binding region with binding specificity for TAA, or the first binding region with binding specificity for a CD3-binding ankyrin repeat domain may be located C-terminally of the second binding region with binding specificity for TAA within said ankyrin repeat domain. An example of such ankyrin repeat domains with (i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive are described e.g., in Example 1 .

[0311] In one embodiment, said ankyrin repeat domain with a first binding specificity for a first target, wherein said first target is a CD3-specific ankyrin repeat domain, comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1 , or any one of SEQ ID NO: 297 to 299.

[0312] In one embodiment, said ankyrin repeat domain with a first binding specificity for a first target, wherein said first target is a CD3-specific ankyrin repeat domain, comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1 .

[0313] In one embodiment, said ankyrin repeat domain with a first binding specificity for a first target, wherein said first target is a CD3-specific ankyrin repeat domain, comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 297.

[0314] In one embodiment, said ankyrin repeat domain with a first binding specificity for a first target, wherein said first target is a CD3-specific ankyrin repeat domain, comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 298.

[0315] In one embodiment, said ankyrin repeat domain with a first binding specificity for a first target, wherein said first target is a CD3-specific ankyrin repeat domain, comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 299.

[0316] In one embodiment, said first repeat domain having binding specificity for a CD3-specific binding agent binds to said agent with a dissociation constant (KD) of or below about 10-7M, of or below about 10-8M or of or below about 10-9M. Thus, in one embodiment, said first repeat domain binds to said agent with a KD of or below about 10-7M. In another embodiment, said first repeat domain binds to said agent with a KD of or below about 10-8M. In another embodiment, said first repeat domain binds to said agent with a KD of or below about 10-9M. Preferably, said binding is measured in PBS.

[0317] In one embodiment, said second ankyrin repeat domain binds to each of said first and second targets with KD1 and KD2 being independently about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10_8M or less, about 10-9M or less, about 1 O-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 1 O-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 1 O-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about I O-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In exemplary embodiments, KD1 and KD2 are independently equal to or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, KD1 and KD2 are independently less than or equal to about 1 nM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 100 pM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 10 pM. In yet another exemplary embodiment, said first ankyrin repeat domain binds to each of the first and second target with KD1 and KD2 being independently less than or equal to about 1 pM.

[0318] In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 16 to 21 or SEQ ID NOs: 133 to 149. In one embodiment said second ankyrin repeat domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 16 to 21 or SEQ ID NOs: 133 to 149.

[0319] In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 16.

[0320] In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0321] In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 17. In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0322] In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 18. In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0323] In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 19. In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 20. In one embodiment, said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 21.

[0324] In one embodiment said ankyrin repeat domain with mutually exclusive binding specificity for two targets has reduced immunogenicity risk. Suitably, said ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 133 to 149. Optionally, said designed ankyrin repeat domain with binding specificity for ankyrin repeat domain with mutually exclusive binding specificity for two targets has an immunogenicity score of less than 2.0, less than 1.0, less than 1.8, less than 1.7, less than 1.6, less than 1 .5, less than 1 .4, less than 1 .3, less than 1 .2, less than 1.1 , less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 or substantially 0.

[0325] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 133. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 133.

[0326] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 134. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 134. In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 135. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 135.

[0327] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 136. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 136.

[0328] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 137. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 137.

[0329] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 138. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 138.

[0330] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 139. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 139.

[0331] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 140. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 140.

[0332] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 141. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 141.

[0333] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 142. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 142.

[0334] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 143. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 143. In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 144. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 144.

[0335] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 145. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 145.

[0336] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 146. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 146.

[0337] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 147. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 147.

[0338] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 148. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 148.

[0339] In one embodiment, the ankyrin repeat domain with mutually exclusive binding specificity for two targets comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 149. In one embodiment, said ankyrin repeat domain comprises or consists of an amino acid sequence of SEQ ID NO: 149.

[0340] In one embodiment, said second ankyrin repeat domain binds to EpCAM with a dissociation constant (KD) below 10-5M. In some embodiments, said second ankyrin repeat domain binds to EpCAM with a KD of about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10'1° M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10_14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10_14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 10-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10'13M. In further embodiments, said second ankyrin repeat domain binds to EpCAM with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, said second ankyrin repeat domain binds to EpCAM with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said second ankyrin repeat domain binds to EpCAM with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said second ankyrin repeat domain binds to EpCAM with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said second ankyrin repeat domain binds to EpCAM with a KD value of less than or equal to about 1 pM.

[0341] In some embodiments, said second ankyrin repeat domain binds to CD3 with a dissociation constant (KD) below 10-5M. In certain embodiments, said second ankyrin repeat domain binds to CD3 with a KD of about 10'5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10'1° M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 1 O-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10_14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10_14M, from about 10-9M to about 10-14M, from about 1 O-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 1 O-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In further embodiments, said second ankyrin repeat domain binds to CD3 with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said second ankyrin repeat domain binds to CD3-specific with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said second ankyrin repeat domain binds to the CD3 with a KD value of less than or equal to about 1 pM.

[0342] In some embodiments, the binding affinity of said second ankyrin repeat domain to each of CD3 (first target) and EpCAM (second target) is described in terms of two dissociation constant (KD) values, wherein KD1 represents the dissociation constant for CD3 and KD2 represents the dissociation constant for EpCAM. In exemplary embodiments, said second ankyrin repeat domain binds to each of said first and second targets with KD1 and KD2 being independently about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 10-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In exemplary embodiments, KD1 and KD2 are independently equal to or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, KD1 and KD2 are independently less than or equal to about 1 nM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 100 pM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 10 pM. In yet another exemplary embodiment, said first ankyrin repeat domain binds to each of the first and second target with KD1 and KD2 being independently less than or equal to about 1 pM.

[0343] In one embodiment, said second ankyrin repeat domain binds to CD3 with a first binding affinity and to EpCAM with a second binding affinity, wherein the ratio of said first binding affinity to said second binding affinity is between 103:1 and 1 :106. In further embodiments, said ratio is equal to about 103:1 , 102:1 , 10:1 , 1 :1 , 1 :10, 1 :102, 1 :103, 1 :104, 1 :105or 1 :106.

[0344] In one embodiment said second ankyrin repeat domain comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22. In one embodiment said second ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0345] In one embodiment, said second ankyrin repeat domain binds to MSLN with a dissociation constant (KD) below 10-5M. In some embodiments, said second ankyrin repeat domain binds to MSLN with a KD of about 10'5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10'1° M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10_14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10_14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 10-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10'13M. In further embodiments, said second ankyrin repeat domain binds to EpCAM with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, said second ankyrin repeat domain binds to MSLN with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said second ankyrin repeat domain binds to MSLN with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said second ankyrin repeat domain binds to MSLN with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said second ankyrin repeat domain binds to MSLN with a KD value of less than or equal to about 1 pM. In some embodiments, said second ankyrin repeat domain binds to CD3 with a dissociation constant (KD) below 10-5M. In certain embodiments, said second ankyrin repeat domain binds to CD3 with a KD of about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 1 O'10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 1 O-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10_14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10_14M, from about 10-9M to about 10-14M, from about 1 O-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 1 O-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In further embodiments, said second ankyrin repeat domain binds to CD3 with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said second ankyrin repeat domain binds to CD3 with a KD value of less than or equal to about 1 pM.

[0346] In some embodiments, the binding affinity of said second ankyrin repeat domain to each of CD3 (first target) and MSLN (second target) is described in terms of two dissociation constant (KD) values, wherein KD1 represents the dissociation constant for CD3 and KD2 represents the dissociation constant for MSLN. In exemplary embodiments, said second ankyrin repeat domain binds to each of said first and second targets with KD1 and KD2 being independently about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 10-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In exemplary embodiments, KD1 and KD2 are independently equal to or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one exemplary embodiment, KD1 and KD2 are independently less than or equal to about 1 nM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 100 pM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 10 pM. In yet another exemplary embodiment, said first ankyrin repeat domain binds to each of the first and second target with KD1 and KD2 being independently less than or equal to about 1 pM.

[0347] In one embodiment, said second ankyrin repeat domain binds to CD3 with a first binding affinity and to MSLN with a second binding affinity, wherein the ratio of said first binding affinity to said second binding affinity is between 103:1 and 1 :106. In further embodiments, said ratio is equal to about 103:1 , 102:1 , 10:1 , 1 :1 , 1 :10, 1 :102, 1 :103, 1 :104, 1 :105or 1 :106.

[0348] CD2-specific binding agents

[0349] CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells as well as by the majority of T cell lymphomas and leukemias. CD2 functions as a cell adhesion and co-stimulatory molecule. It is known to bind lymphocyte function-associated antigen-3 (LFA-3 / CD58), a surface molecule expressed by antigen presenting cells and epithelial cells. The CD2 / CD58 interaction promotes the initial stages of cell contact and facilitates T-cell receptor (TOR) triggering. CD2 is used as a specific marker for T cells and NK cells, and to distinguish B cell neoplasms from T cell lymphomas and leukemias. Ankyrin repeat domains specifically binding CD2 are disclosed in WO2024 / 251742 (incorporated by reference).

[0350] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4 to 6.

[0351] Thus, in one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 4 to 6. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 4 to 6. In another embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 4 to 6. In a further embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 4 to 6. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 4 to 6. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 4 to 6.

[0352] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 4. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0353] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 5. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0354] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 6. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0355] In one embodiment said ankyrin repeat domain with binding specificity for CD2 has reduced immunogenicity risk. Suitably, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 129 to 132. Optionally, said designed ankyrin repeat domain with binding specificity for CD2 has an immunogenicity score of less than 2.0, less than 1 .0, less than 1 .8, less than 1 .7, less than 1 .6, less than 1 .5, less than 1 .4, less than 1 .3, less than 1 .2, less than 1 .1 , less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 or substantially 0. Thus, in one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 129 to 132. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 129 to 132. In another embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 129 to 132. In a further embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 129 to 132. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 129 to 132. In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 129 to 132.

[0356] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 129. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 129.

[0357] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 130. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 130.

[0358] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 131. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 131 .

[0359] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical with SEQ ID NO: 132. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 132.

[0360] In one embodiment, said ankyrin repeat domain with binding specificity for CD2 binds to human CD2 with a dissociation constant (KD) of or below about 10-6M, or of or below about 10-7M, or of or below about 1 O'8M, or of or below about 10-9M. Thus, in one embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10'6M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 107M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10'8M. In another embodiment, said recombinant binding protein binds to CD2 with a KD of or below about 10'9M. Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR).

[0361] Accordingly, in one embodiment, said ankyrin repeat domain binds human CD2 in PBS with a KD of or below about 10'6M, or of or below about 10'7M, or of or below about 10-8M, and wherein said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132.

[0362] Multispecific binding proteins

[0363] Accordingly, in one aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for EpCAM, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and EpCAM is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for CD2. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for MSLN. In a preferred embodiment, the multispecific binding protein comprises (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for EpCAM, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and EpCAM is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD2 and (4) a fourth ankyrin repeat domain with binding specificity for MSLN. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0364] In another aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for MSLN, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and MSLN is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for CD2. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for EpCAM.

[0365] In a preferred embodiment, the multispecific binding protein comprises (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for MSLN, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and MSLN is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD2 and (4) a fourth ankyrin repeat domain with binding specificity for EpCAM. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0366] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NO: 106 to 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 16 to 22 or SEQ ID NOs: 133 to 149, (3) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 4 to 6 or SEQ ID NOs: 129 to 132, and optionally (4) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128.

[0367] Suitably, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin (HSA) comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 39 to 41.

[0368] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NO: 106 to 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for MSLN, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 22, (3) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 4 to 6 or SEQ ID NOs: 129 to 132, and optionally (4) an ankyrin repeat domain with binding specificity for EpCAM comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 10 to 15. Suitably, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin (HSA) comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 39 to 41 .

[0369] In another aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for EpCAM, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and EpCAM is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for MSLN, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to EpCAM and (ii) the third ankyrin repeat domain to MSLN. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for CD2. In a preferred embodiment, the multispecific binding protein comprises (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for EpCAM, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and EpCAM is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for MSLN and (4) a fourth ankyrin repeat domain with binding specificity for CD2, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to EpCAM and (ii) the third ankyrin repeat domain to MSLN. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0370] In another aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for MSLN, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and MSLN is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for EpCAM, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to MSLN and (ii) the third ankyrin repeat domain to EpCAM. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for CD2. In a preferred embodiment, the multispecific binding protein comprises (1) a first ankyrin repeat domain with binding specificity for CD3, (2) a second ankyrin repeat domain with (i) a first binding specificity for the CD3-binding ankyrin repeat domain and (ii) a second binding specificity for MSLN, and wherein binding of said second ankyrin repeat domain to said CD3-binding ankyrin repeat domain and MSLN is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for EpCAM and (4) a fourth ankyrin repeat domain with binding specificity for CD2, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to MSLN and (ii) the third ankyrin repeat domain to EpCAM. Optionally, said multispecific binding protein further comprises an ankyrin repeat domains with binding specificity for human serum albumin.

[0371] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NO: 106 to 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 16 to 22 or SEQ ID NOs: 133 to 149, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 7 to 9 or SEQ ID NOs: 126 to 128, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132, wherein said second ankyrin repeat domain binds said first ankyrin repeat domain, and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to EpCAM and (ii) the third ankyrin repeat domain to MSLN.

[0372] Suitably, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin (HSA) comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 39 to 41.

[0373] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 2 to 3 or SEQ ID NO: 106 to 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for MSLN, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 22, (3) an ankyrin repeat domain with binding specificity for EpCAM comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10 to 15, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132. Suitably, said multispecific binding protein further comprises an ankyrin repeat domains with binding specificity for human serum albumin (HSA) comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 39 to 41. In one embodiment, the multispecific binding protein comprises said first, second, and third ankyrin repeat domains, and wherein said multispecific protein is capable of binding the respective targets of said first, second, and third ankyrin repeat domains simultaneously.

[0374] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 120, 122, 123, 124 or 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NO: 133 to 138, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126 or 127, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NOs: 129 or 130.

[0375] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 124, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 133, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0376] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 120, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 133, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0377] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 122, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 133, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0378] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 124, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 135, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0379] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 120, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 135, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0380] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 122, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 135, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0381] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 124, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 137, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0382] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 120, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 137, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0383] In one embodiment, provided is a multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 122, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 137, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 126, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 129.

[0384] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 134, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 127, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 130.

[0385] In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 136, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 127, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 130. In one embodiment, the multispecific binding protein comprises (1) an ankyrin repeat domain with binding specificity for CD3 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 125, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD3 and a second binding region with binding specificity for EpCAM, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 138, (3) an ankyrin repeat domain with binding specificity for MSLN comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 127, and optionally (4) an ankyrin repeat domain with binding specificity for CD2 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 130.

[0386] In one embodiment, the multispecific binding protein comprises said first, second, third and fourth ankyrin repeat domains, and wherein said multispecific protein is capable of binding the respective targets of said first, second, third and fourth ankyrin repeat domains simultaneously.

[0387] In one embodiment, the multispecific binding protein comprises said first, second, third, fourth and fifth ankyrin repeat domains, and wherein said multispecific protein is capable of binding the respective targets of said first, second, third, fourth and fifth ankyrin repeat domains simultaneously.

[0388] Suitably, said ankyrin repeat domain binding human serum albumin is located C-terminally within said multispecific binding protein. Suitably, said ankyrin repeat domain binding human serum albumin is located N-terminally within said multispecific binding protein. In some embodiments, the multispecific binding protein provided herein comprises two or more serum albumin binding ankyrin repeat domains. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the N-terminus of the multispecific binding protein provided herein. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the C-terminus of the multispecific binding protein provided herein. In some embodiments, a first serum albumin binding ankyrin repeat domain is located at the N-terminus of the multispecific binding protein provided herein, and a second serum albumin binding ankyrin repeat domains is located at the C-terminus of the multispecific binding protein provided herein.

[0389] In one embodiment, said multispecific binding protein comprises a first, a second, and a third ankyrin repeat domain, wherein said first, second and third ankyrin repeat domain can be arranged in any order from the N-terminus to the C-terminus. For example, the first ankyrin repeat domain can be at the N-terminal position, or the first ankyrin repeat domain can be at the C-terminal position, or the first ankyrin repeat domain can be positioned between the second and the third ankyrin repeat domains. In another example, the second ankyrin repeat domain can be at the N-terminal position or the second ankyrin repeat domain can be at the C-terminal position, or the second ankyrin repeat domain can be positioned between the first and the third ankyrin repeat domain. In another example, the third ankyrin repeat domain can be at the N- terminal position or the third ankyrin repeat domain can be at the C-terminal position, or the third ankyrin repeat domain can be positioned between the first and the second binding agent.

[0390] In one embodiment, said multispecific binding protein comprises first, second, third and fourth ankyrin repeat domains, wherein said first, second, third and fourth ankyrin repeat domains can be arranged in any order from the N-terminus to the C-terminus. For example, the first ankyrin repeat domain can be at the N- terminal position or the first ankyrin repeat domain can be at the C-terminal position, or the first ankyrin repeat domain can be positioned between any of the other ankyrin repeat domains. In another example, the second ankyrin repeat domain can be at the N-terminal position or the second ankyrin repeat domain can be at the C-terminal position, or the second ankyrin repeat domain can be positioned between any of the other ankyrin repeat domains. In another example, the third ankyrin repeat domain can be at the N- terminal position or the third ankyrin repeat domain can be at the C-terminal position, or the third ankyrin repeat domain can be positioned between any of the other ankyrin repeat domains. In another example, the fourth ankyrin repeat domain can be at the N-terminal position or the fourth ankyrin repeat domain can be at the C-terminal position, or the fourth ankyrin repeat domain can be positioned between any of the other ankyrin repeat domains.

[0391] In one embodiment, said ankyrin repeat domains are covalently linked with a peptide linker. Said peptide linker may be a proline-threonine-rich peptide linker (PT linker) or a Glycine-Serine linker (GS linker). Suitably, said peptide linker comprises between 1 and 50 amino acids. Suitably, said peptide linker comprises between 1 and 30 amino acids. In one embodiment, the PT linker comprises or consists of SEQ ID NO: 97 or SEQ ID NO: 281 . In another embodiment, the GS linker comprises or consists of SEQ ID NO: 275 or SEQ ID NO: 276.

[0392] In one embodiment, any of said ankyrin repeat domains additionally comprises a G, a S or a GS at the N- terminus.

[0393] Accordingly, in one aspect said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 23 to 35, SEQ ID NOs: 98 to 104 or SEQ ID NOs: 147 to 209. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 23 to 35, SEQ ID NOs: 98 to 104 or SEQ ID NO: 147 to 209.

[0394] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23. In one embodiment said multispecific t binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 23.

[0395] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0396] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 25.

[0397] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0398] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 27.

[0399] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 28. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0400] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0401] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0402] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 31 .

[0403] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0404] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 33. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 33.

[0405] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 34.

[0406] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0407] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 98. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 98.

[0408] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 99. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 99.

[0409] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 100. In one embodiment said multispecific t binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 100.

[0410] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 101. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 101.

[0411] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 102. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 102.

[0412] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 103. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 103.

[0413] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104. In one embodiment said multispecific t binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 104.

[0414] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 285. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 285.

[0415] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 286. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 286.

[0416] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 287. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 287.

[0417] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 288. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 288.

[0418] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 289. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 289.

[0419] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 290. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 290.

[0420] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 291. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 291.

[0421] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 292. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 292.

[0422] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 293. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 293.

[0423] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 294. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 294.

[0424] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 295. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 295.

[0425] In one embodiment said multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 296. In one embodiment said multispecific binding protein comprises or consists of the amino acid sequence of SEQ ID NO: 296.

[0426] Half-life extending moieties

[0427] When developing protein-based therapeutics, one of the most significant challenges is their inherently short persistence in the bloodstream. Most therapeutic proteins, peptides, and small biologies are rapidly cleared from circulation through several mechanisms.

[0428] A “half-life extending moiety” extends the serum half-life in vivo of the binding proteins described herein, compared to the same protein without the half-life extending moiety. Examples of half-life extending moieties include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin domain, maltose binding protein (MBP), a human serum albumin (HSA) binding domain, or polyethylene glycol (PEG).

[0429] In one embodiment, the binding proteins provided herein further comprise one or more half-life extending moieties. Preferably, said half-life extending moiety binds to human serum albumin. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin.

[0430] In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin, said ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 39 to 41.

[0431] In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 39 to 41 . In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 39.

[0432] In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 40.

[0433] In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 41 .

[0434] In some embodiments, the binding protein provided herein comprises two or more serum albumin binding ankyrin repeat domains. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the N-terminus of the binding proteins provided herein. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the C-terminus of the binding proteins provided herein. In some embodiments, a first serum albumin binding ankyrin repeat domain is located at the N-terminus of the binding proteins provided herein, and a second serum albumin binding ankyrin repeat domain is located at the C-terminus of the binding proteins provided herein.

[0435] In some embodiments, the half-life extending moiety comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an Fc domain. In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes: IgG (y), IgM (p), IgD (6), IgE (s), or IgA (a). In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes or subtypes: lgG1 (y1), lgG2 (y2), lgG3 (y3), lgG4 (y4), lgA1 (a1), lgA2 (a2). In some embodiments, the Fc domain is the Fc domain of human lgG1. In some embodiments, the Fc domain comprises an uninterrupted native sequence (i.e., wild type sequence) of an Fc domain. In some embodiments, the immunoglobulin Fc domain comprises a variant Fc domain resulting in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain so as to reduce or eliminate the effector activity (e.g., International Patent Publication No. W02005 / 063815), and / or to increase the homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain is the Fc domain of human lgG1 and comprises one or more of the following effector-null substitutions: L234A, L235A, and P329G (Eu numbering). In some embodiments, the Fc domain does not comprise the lysine located at the C-terminal position of human IgG 1 (i.e., K447 by Eu numbering). The absence of the lysine may increase homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain comprises the lysine located at the C-terminal position (K447, Eu numbering).

[0436] In one embodiment, the half-life extending moiety comprises an Fc variant domain. Suitable variant Fc domains are disclosed in WO2012130831 , WO2021234402, WO9428027, WO2012130831 , EP3428194, W02005018572, W02010151792A1 or US8216805, incorporated herein by reference.

[0437] In one embodiment, the half-life extending moiety comprises an Fc domain of human lgG1.

[0438] “Fc” refers to a polypeptide corresponding to the final two constant domains (CH2-CH3) of the human immunoglobulin heavy chain. The reference sequence used for human Fc is derived from Uniprot ID P01857-1 , residues 104-330, corresponding to residues 221-447 according to the conventional Eu numbering system.

[0439] The CH3 domains in the Fc-region of the heavy chains of an antibody can be altered by "knob-into-holes" technology, which is described in detail with several examples in e.g., WO 96 / 027011 , Ridgway JB, et al. Protein Eng. 9 (1996) 617-621 ; and Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681 . In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole".

[0440] For the knob-in-hole design, complementary mutations are introduced into the two different heavy chains of the Fc. On one chain, designated as the "knob" chain (or first Fc chain), a relatively small amino acid at the CH3 interface is mutated to a much larger amino acid. The most common mutation is threonine 366 to tyrosine (T366Y), which introduces a bulky aromatic side chain that protrudes into the interface. This creates a structural protrusion, the "knob," (or second Fc chain) that extends toward where the partner chain would bind (Ridgway JB (1996), supra). On the complementary chain, designated as the "hole" chain, mutations are introduced to create a cavity that can accommodate the knob. The typical mutations involve replacing larger amino acids with smaller ones. Common hole mutations include threonine 366 to serine (T366S), leucine 368 to alanine (L368A), and tyrosine 407 to valine (Y407V). These substitutions create a depression or cavity in the surface of this chain. When a knob chain and a hole chain are co-expressed, the knob naturally fits into the hole, creating a stable and favorable interaction. In an embodiment, the Fc domain comprises a) a first Fc chain comprising the substitution T366W and b) a second Fc chain comprising the substitutions T366S, L368A and T407V (Eu numbering), wherein the first and second chain heterodimerize to form a Fc heterodimer. In a preferred embodiment, the first Fc chain further comprises the substitution S354C and the second Fc chain further comprises the substitution Y349C (Eu numbering). In an embodiment, the second Fc chain further comprises the substitutions H435R and Y436F (Eu numbering). In an embodiment, the first and / or second Fc chain comprises the following substitutions: L234A, L235A, and P329G (Eu numbering).

[0441] The ankyrin repeat domains comprised in the multispecific binding proteins disclosed herein may be fused to any of the knob or hole Fc chains, in any possible formatting: e.g., four domains fused, in any order, to the knob-Fc and no domains fused to the hole-Fc; or three domains fused, in any order, to the knob-Fc and one domain fused to the hole-Fc; or two domains fused, in any order, to the knob-Fc and two domains fused, in any order, to the hole-Fc; or one domain fused to the knob-Fc and three domains fused, in any order, to the hole-Fc; or no domains fused to the knob-Fc and four domains fused, in any order, to the hole- Fc.

[0442] When all domains of the multispecific binding proteins are bound to the knob-Fc or the hole-Fc chain, the format is called “linear”, as all the binding domains are arranged consecutively. When one or more domains of the multispecific binding protein are covalently bound to the knob-Fc and one or more domains of the multispecific binding protein are bound to the hole-Fc chain, the format is called “asymmetric”, “non-linear”, “branched”, “split” or any equivalent term.

[0443] In an embodiment, the present disclosure refers to the multispecific binding protein according to any of the previous embodiments, wherein the first, second, third and optionally fourth ankyrin repeat domains are linearly arranged from N-terminus to C-terminus, wherein the N-terminus of the most N-terminal ankyrin repeat domain is covalently linked to the C-terminus of the first Fc chain, and wherein the second Fc chain is not covalently linked to any ankyrin repeat domain.

[0444] In another embodiment, the present disclosure refers to the multispecific binding protein according to any of the previous embodiments, wherein the N-terminus of one or more ankyrin repeat domains is covalently linked to the C-terminus of the first Fc chain, and the N-terminus of one or more ankyrin repeat domain is covalently linked to the C-terminus of the second Fc chain.

[0445] In one embodiment, the N-terminus of a first binding domain is fused covalently to the C-terminus of the knob- or hole- Fc via a linker. An example of linker is a “(G4S)3” linker, as defined in SEQ ID NO: 275.

[0446] In one embodiment, the half-life extending moiety comprises a knob-Fc comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the SEQ ID NO: 273 and a hole-Fc comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the SEQ ID NO: 274.

[0447] In one embodiment, the half-life extending moiety comprises a knob-Fc comprising an amino acid sequence of SEQ ID NO: 273 and a hole-Fc comprising an amino acid sequence of SEQ ID NO: 274.

[0448] Accordingly, in one embodiment the multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 150 or 151 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 274. In one embodiment said multispecific binding protein comprises a first polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 150 or 151 , and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 274.

[0449] In another embodiment, the multispecific binding protein comprises a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least ab...

Claims

1. CLAIMS1. A multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for a first immune cell-surface associated antigen, (2) a second ankyrin repeat domain with(i) a first binding specificity for a first target, wherein said first target is the first ankyrin repeat domain and (ii) a second binding specificity for a second target, wherein said second target is a first tumor associated antigen (TAA), and wherein binding of said second ankyrin repeat domain to said first and second targets is mutually exclusive, and (3) a third ankyrin repeat domain with binding specificity for a second tumor associated antigen, wherein said second ankyrin repeat domain is binding said first ankyrin repeat domain and wherein said first ankyrin repeat domain is released from binding to the second ankyrin repeat domain upon binding of (i) the second ankyrin repeat domain to said first tumor associated antigen and (ii) the third ankyrin repeat domain to said second tumor associated antigen.

2. The multispecific binding protein according to claim 1 , wherein said second ankyrin domain reversibly binds said first ankyrin repeat domain.

3. The multispecific binding protein according to any one of the preceding claims, wherein said multispecific binding protein comprises a further ankyrin repeat domain with binding specificity for a second immune cell-surface associated antigen, optionally wherein the first immune cell-surface associated antigen and the second immune cell-surface associated antigen are present on the same immune cell and wherein said immune cell is a T cell.

4. The multispecific binding protein according to any one of the preceding claims, wherein said first immune cell-surface associated antigen is CD3, and wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 3 or SEQ ID NOs: 106 to 125.

5. The multispecific binding protein according to any one of the preceding claims, wherein (i) the first TAA is EpCAM and the second TAA is EpCAM or (ii) the first TAA is MSLN and the second TAA is MSLN, or (i) the first TAA is EpCAM and the second TAA is MSLN or (ii) the first TAA is MSLN and the second TAA is EpCAM, optionally wherein the first and second tumor associated antigens are present on the same cell, and wherein said cell is a cancer cell.

6. The multispecific binding protein according to any one of the preceding claims, wherein said second ankyrin repeat domain with (i) a first binding specificity for the first ankyrin repeat domain and(ii) a second binding specificity for EpCAM comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 16 to 21 or SEQ ID NOs: 133 to 149.

7. The multispecific binding protein according to any one of the preceding claims, wherein said third ankyrin repeat domain with binding specificity to MSLN comprises an amino acid sequence at least about 80% identical to the amino sequence of any one of SEQ ID NOs: 7 to 9 or SEQ ID NOs: 126 to 128.

8. The multispecific binding protein according to any one of claims 3 to 7, wherein said second immune cell-surface associated antigen is CD2, and wherein said ankyrin repeat domain with binding specificity for CD2 comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 4 to 6 or SEQ ID NOs: 129 to 132.

9. The multispecific binding protein according to any one of the preceding claims, further comprising a half-life extending moiety, wherein i) said half-life extending moiety is a binding agent that specifically binds to human serum albumin, optionally an ankyrin repeat domain with binding specificity for human serum albumin, or ii) said half-life extending moiety is an immunoglobulin Fc domain.

10. The multispecific binding protein according to claim 9, wherein the half-life extending moiety is an immunoglobulin Fc domain comprising a knob-Fc comprising the amino acid sequence of SEQ ID NO: 273 and a hole-Fc comprising the amino acid sequence of SEQ ID NO: 274.11 . The multispecific binding protein of claim 10, wherein the knob-Fc or hole-Fc is covalently linked to the N-terminal end of the first ankyrin repeat domain.

12. The multispecific binding protein of claims 10 or 11 , wherein the multispecific binding protein comprises an amino acid sequence at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 150 or 151 and an amino acid sequence comprising SEQ ID NO: 274.

13. The multispecific binding protein according to claims 10 to 12, wherein one or more of the ankyrin repeat domains are covalently linked to the knob-Fc chain and one or more of the ankyrin repeat domains are covalently linked to the hole-Fc chain.

14. The multispecific binding protein of claim 13, wherein multispecific binding protein comprises:(i) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 158 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159; or(ii) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159; or(iii) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 161 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 159; or(iv) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 162 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163; or(v) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 164 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163; or(vi) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 165 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 163; or(vii) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 166 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167; or(viii) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 168 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167; or(ix) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 169 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 167; or(x) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 170 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 171 ; or(xi) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 172 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 173; or(xii) a first polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 174 and a second polypeptide at least about 80% or at least about 81 % or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% at least about 88% or at least about 89% or at least about 90% or at least about 91 % or at least about 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO: 175.

15. An isolated nucleic acid encoding the multispecific binding protein according to any one of claims 1 to 14.

16. A cloning or expression vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 12 or the vector of claim 16.

18. A method for treating a medical condition comprising administering to a subject in need thereof a therapeutically effective amount of the multispecific binding protein according to any one of claims 1 to 14, the isolated nucleic acid of claim 15, the vector according to claim 16 or the host cell according to claim 17.

19. The method of claim 18, wherein the medical condition is cancer.

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