Recombinant mesothelin binding proteins and their use

Abstract

The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain with binding specificity for soluble mesothelin or membrane-bound mesothelin. In addition, the invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases, such as cancer in a mammal, including a human.

Description

[0001] RECOMBINANT MESOTHELIN BINDING PROTEINS AND THEIR USE

[0002] TECHNICAL FIELD

[0003] The present invention relates to recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for mesothelin (MSLN). In addition, the invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such recombinant binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods of treating diseases, such as cancer, in a subject in need thereof.

[0004] BACKGROUND

[0005] Mesothelin (MSLN) is predominantly expressed in specific types of epithelial cells such as the mesothelial lining of the pleura, pericardium, and peritoneum, but it is significantly upregulated in several solid tumors, including mesothelioma, ovarian carcinoma, pancreatic adenocarcinoma, and some gastric and lung cancers. Aberrant MSLN expression on tumor cells plays a role in tumor progression, metastasis, drug resistance, and immune evasion by interacting with MUC16 / CA125, which is a mucin-like glycoprotein found on the surface of tumor cells and identified as an ovarian cancer antigen (Rump A et al, J Biol Chem. 2004;279(10):9190-8; Chen SH et al, Sci Rep. 2013;3:1870). Elevated MSLN expression has been correlated with increased tumor burden and poorer overall survival in clinical observations (Kachala SS et al, Clin Cancer Res. 2014;20(4): 1020-8; Tozbikian G et al, PLoS One. 2014;9(12):e114900.).

[0006] The MSLN gene coding for mesothelin encodes a 622-residue (69 kDa) precursor protein, known as pre- pro mesothelin. Upon insertion into the cell membrane, the N-terminal signal peptide is excised, followed by truncation of several C-terminal residues, allowing for the addition of a GPI anchor. A proteolytic processing site is recognized by furin protease and other proteases, and facilitates cleavage of the precursor protein. This proteolytic cleavage yields a GPI-anchored 303 amino acid residue (40 kDa) fragment (“membrane-bound mesothelin”) and a 31 kDa secreted fragment, also referred to as “megakaryocyte-potentiating factor” (MPF). Similar to other GPI-anchored proteins, mesothelin undergoes shedding in the extracellular environment, which is rich in proteases. Alternative splicing and protease cleavage by different enzymes at several cleavage sites close to the membrane anchor on mesothelin- expressing cells results in the generation of soluble mesothelin (sMSLN) as well as soluble mesothelin- related peptides (SMRP). Soluble forms of mesothelin, also referred to as “shed mesothelin”, have gained particular attention as a biomarker for cancer detection and monitoring. Elevated levels of soluble MSLN in the blood or other bodily fluids have been associated with the presence of cancer, making it a valuable tool for early diagnosis and disease management. The non-invasive nature of soluble MSLN measurement offers significant advantages over traditional cancer diagnostics, providing a potential avenue for improving patient outcomes through early intervention. Beyond its utility as a diagnostic biomarker, the expression of mesothelin has also been linked to the prognosis of cancer patients. In many cases, higher levels of mesothelin expression correlate with more aggressive disease and poorer clinical outcomes. The unique attributes of mesothelin, particularly its restricted expression in cancer cells and limited presence in normal tissues, make it an appealing target for cancer therapy. Various approaches have been explored to harness the potential of mesothelin as a therapeutic target, including antibody-drug conjugates (ADCs), immunotherapies including chimeric antigen receptor (CAR) T-cell therapy, and targeted drug delivery systems. However, soluble mesothelin can act as a significant antigen sink. Targeting membranebound mesothelin in particular, on the other hand, is not amenable to all therapeutic approaches. Therefore, non-antibody-based approaches binding mesothelin by targeting epitopes close to the membrane may be beneficial. Taken together, in view of the role of mesothelin in diagnostic and therapeutic approaches, there is a need for novel mesothelin-specific binding proteins, including binding proteins with specificity for membrane-bound mesothelin but not for soluble mesothelin as well as binding proteins with specificity for soluble mesothelin.

[0007] SUMMARY

[0008] Designed Ankyrin Repeat Proteins (DARPins) are uniquely suitable as cancer therapeutics as their small size, specificity and high affinity can lead to high tumor uptake and tumor residence. Current approaches that rely on activation of the immune-system to target tumors can be circumvented using radioligand therapies. In addition, the systemic clearance of DARPins can be fine-tuned. DARPins are also easily amenable to linkage and surface engineering, making them ideal molecules for radiotherapeutic and radiodiagnostic purposes. Such binding proteins may be useful in therapeutic and diagnostic approaches for the treatment and characterization of diseases, including cancer. The present disclosure relates to recombinant binding proteins comprising an ankyrin repeat domain with binding specificity for mesothelin (MSLN), e.g., expressed by tumor cells. More specifically, the present disclosure provides recombinant binding proteins comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin (mbMSLN). The present disclosure also relates to recombinant binding proteins comprising an ankyrin repeat domain with binding specificity for soluble mesothelin (sMSLN). Such binding proteins can serve as a tool or as a building block for the generation of new therapeutic or diagnostic agents with enhanced tumor-targeting capabilities and / or reduced off-target effects to improve therapeutic efficacy. Also disclosed herein are recombinant binding proteins, in which the MSLN-specific ankyrin repeat domains are combined with one or more other functional moieties in one molecule. Such other functional moieties include a half-life extending moiety and / or a binding moiety, for example ankyrin repeat domains, with binding specificity for a target present on immune cells.

[0009] 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 (E).

[0010] Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination:

[0011] E1. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for mesothelin (MSLN). E2. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin (sMSLN).

[0012] E3. The recombinant binding protein of E1 or E2, wherein said ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids.

[0013] E4. The recombinant binding protein of any one of E1 to E3, wherein said ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module.

[0014] E5. The recombinant binding protein of E4, wherein said first and said second ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids.

[0015] E6. The recombinant binding protein of any one of E1 to E5, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 46 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 47 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 48, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 48 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 49, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 49 are substituted with other amino acids;

[0016] Hi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 50, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 50 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 51 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 51 are substituted with other amino acids; iv. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 58 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 59, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 59 are substituted with other amino acids; or v. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 60, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 60 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 61 are substituted with other amino acids.

[0017] E7. The recombinant binding protein of any one of E1 to E3, wherein said ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module.

[0018] E8. The recombinant binding protein of E7, wherein said first, second and third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids.

[0019] E9. The recombinant binding protein of any one of E1 to E3 or E7 to E8, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 43, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 43 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 44, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 44 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 45, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 45 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 52, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 52 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 53, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 53 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 54 are substituted with other amino acids; or

[0020] Hi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 55, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 55 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 56, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 56 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 57, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 57 are substituted with other amino acids.

[0021] E10. The recombinant binding protein of any one of E3 to E9, further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79 to 86, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79 to 86 are substituted by other amino acids and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 to 97, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 to 97 are substituted by other amino acids.

[0022] E11. The recombinant binding protein of any one of E1 to E10, 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: 1 to 8.

[0023] E12. The recombinant binding protein of any one of E1 to E11 , wherein said mesothelin is human mesothelin.

[0024] E13. The recombinant binding protein of any one of E1 to E12, wherein said ankyrin repeat domain binds soluble human mesothelin in PBS with a dissociation constant (KD) 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, or of or below about 10-11M.

[0025] E14. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin (mbMSLN).

[0026] E15. The recombinant binding protein of E1 and E14, wherein said ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids.

[0027] E16. The recombinant binding protein of any one of E1 , E14 to E15, wherein said ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module.

[0028] E17. The recombinant binding protein of any one of E1 , E14 to E16, wherein said first and said second ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids, or wherein said first ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 101 to 105, and wherein said second ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 106 to 109.

[0029] E18. The recombinant binding protein of any one of E1 , E14 to E17, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids;

[0030] Hi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 69 are substituted with other amino acids, and a second ankyrin repeat module, comprising an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 70 are substituted with other amino acids; or iv. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 71 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids; or v. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids, and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids.

[0031] E19. The recombinant binding protein of any one of E1 , E14 to E15, wherein said ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module. E20. The recombinant binding protein of E19, wherein said first, second and third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids, or wherein said first ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 101 to 105, said second ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 106 to 109, and said third ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 110 to 112.

[0032] E21 . The recombinant binding protein of any one of E1 , E14 to E15 or E19 to E20, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 62 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 63, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 63 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 64 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 73 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 75 are substituted with other amino acids; or

[0033] Hi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 76 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 77 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids.

[0034] E22. The recombinant binding protein of any one of E15 to E21 , further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79, 87 to 90, or 201 , or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79, 87 to 90, or 201 are substituted by other amino acids; and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 , 98 to 100, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 , 98 to 100 are substituted by other amino acids.

[0035] E23. The recombinant binding protein of any one of E1 , E14 to E22, 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: 9 to 15, 195, 198, or 199.

[0036] E24. The recombinant binding protein of any one of E14 to E23, wherein said mesothelin is human mesothelin.

[0037] E25. The recombinant binding protein of any one of E1 , E14 to E24, wherein said recombinant binding protein binds membrane-bound human mesothelin in PBS with a dissociation constant (KD) 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, or of or below about 10-11M and / or wherein said recombinant binding protein binds human mesothelin with an EC50 of about 0.1 to about 10 nM.

[0038] E26. The recombinant binding protein of any one of E1 , E14 to E24, wherein said ankyrin repeat domain comprises an engineered ankyrin repeat module.

[0039] E27. The recombinant binding protein of E26, wherein said engineered ankyrin repeat module comprises fewer basic amino acids and / or a reduced isoelectric point (pl) compared to the non-engineered ankyrin repeat module.

[0040] E28. The recombinant binding protein of E26 or E27, wherein said ankyrin repeat domain comprises an N- terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 121 to 131 , or 202, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 121 to 131 , or 202 are substituted by other amino acids; at least one, at least two, or at least three internal repeat modules, each module independently comprising an amino acid sequence of any one of SEQ ID NOs: 75, 132 to 151 or 203 to 205, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 75, 132 to 151 or 203 to 205 are substituted with other amino acids and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 , 152 to 160, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 , 152 to 160 are substituted by other amino acids.

[0041] E29. The recombinant binding protein of any one of E26 to E28, 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: 16 to 35, 196, 197, or 200.

[0042] E30. The recombinant binding protein of any one of E1 to E29, wherein said recombinant binding protein comprises at least one ankyrin repeat domain with binding specificity for soluble mesothelin as defined in any one of E2 to E13 and at least one ankyrin repeat domain with binding specificity for membrane-bound mesothelin as defined in any one of E14 to E29.

[0043] E31. The recombinant binding protein of any one of E1 to E30, further comprising at least one binding moiety with binding specificity for a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell).

[0044] E32. The recombinant binding protein of E31 , wherein said binding moiety comprises an ankyrin repeat domain with binding specificity for CD3.

[0045] E33. The recombinant binding protein of E32, said ankyrin repeat domain with binding specificity for CD3 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: 36 to 40.

[0046] E34. The recombinant binding protein of any one of E1 to E33, further comprising at least one half-life extending moiety.

[0047] E35. The recombinant binding protein of E34, wherein said half-life extending moiety comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0048] E36. The recombinant binding protein of E35, wherein said ankyrin repeat domain with binding specificity for human serum albumin 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: 41 to 42.

[0049] E37. The recombinant binding protein of any one of E1 to E36, further comprising a drug moiety.

[0050] E38. The recombinant binding protein of E37, wherein said drug moiety is a radionuclide or a cytotoxin.

[0051] E39. A nucleic acid encoding the recombinant binding protein of any one of E1 to E38.

[0052] E40. A vector comprising the nucleic acid of E39.

[0053] E41 . The vector of E40, wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector. E42. A cell comprising the nucleic acid of E39 or the vector of any one of E40 to E41 .

[0054] E43. A pharmaceutical composition comprising the recombinant binding protein of any one of E1 to E38, the nucleic acid of E39, the vector of any one of E40 to E41 , or the cell of E42, and a pharmaceutically acceptable carrier and / or diluent.

[0055] E44. A method of producing a recombinant binding protein, the method comprising culturing the cell of E42 and collecting the recombinant binding protein from the cell and / or the culture medium.

[0056] E45. The recombinant binding protein of any one of E1 to E38, the nucleic acid of E39, the vector of any one of E40 to E41 , the cell of E42, or the pharmaceutical composition of E43, for use as a medicament.

[0057] E46. Use of the recombinant binding protein of any one of E1 to E38, the nucleic acid of E39, the vector of any one of E40 to E41 , the cell of E42, or the pharmaceutical composition of E43, in the manufacture of a medicament.

[0058] E47. The recombinant binding protein of E45, the nucleic acid of E45, the vector of E45, the cell of E45, or the pharmaceutical composition of E45 or the use of E46, wherein said medicament is a medicament for the treatment of cancer.

[0059] E48. A method of treatment in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant binding protein of any one E1 to E38, the nucleic acid of E39, the vector of any one of E40 to E41 , the cell of E42, or the pharmaceutical composition of E43.

[0060] E49. The method of E48, wherein said treatment is the treatment of cancer.

[0061] E50. A method of detecting the presence of mesothelin-expressing cells, suitably cancer cells, in a subject, the method comprising:

[0062] (a) contacting a sample comprising one or more cells obtained from said subject with the recombinant binding protein of any of E1 to E38, the nucleic acid of E39, the vector of any one of E40 to E41 , the cell of E42, or the pharmaceutical composition of E43, and

[0063] (b) detecting binding of said recombinant binding protein to said one or more cells.

[0064] E51. The method of E50, wherein the detection of binding of said recombinant binding protein to said one or more cells is indicative of the presence of cancer cells in said sample.

[0065] BRIEF DESCRIPTION OF THE FIGURES

[0066] Figure 1 : Binding profile of recombinant binding proteins comprising ankyrin repeat domains with binding specificity for membrane-bound mesothelin on OVCAR-3 cancer cells. Serial dilutions of His-tagged ankyrin repeat monodomains with binding specificity for membrane-bound mesothelin were incubated with target cells, and binding of the constructs to the target cells was determined by flow cytometry through detection of the His-tag. Shown are median fluorescence intensity (MFI) values for the ankyrin repeat monodomains mbMSLN DARPin #1 to mbMSLN DARPin #7 binding to membrane-bound mesothelin (mbMSLN). Shown are resulting median fluorescent intensity (MFI) values with a standard non-linear regression three parameter logistic model fit.

[0067] Figure 2: Binding of mbMSLN DARPin #6 and reference antibody to biotinylated mesothelin targets. Plates were coated with biotinylated human, cynomolgus and mouse mesothelin, and serial dilutions of ankyrin repeat domain specifically binding to membrane-bound mesothelin, mbMSLN DARPin #6, as well as reference antibody 15B06 were added. (A) Ankyrin repeat domain was detected with anti-DARPin 1.1.1 antibody. MbMSLN DARPin #6 is cross-reactive with human, cynomolgus and mouse mesothelin. (B) Reference antibody 15B06 was detected with goat anti-mouse IgG HRP. 15B06 is cross-reactive with human and cynomolgus mesothelin, but not with mouse mesothelin.

[0068] Figure 3: Alignment of the amino acid sequences for human (residues 296-596), cynomolgus (residues 296-596) and mouse (residues 298-600) membrane-bound mesothelin. The C-terminal peptide epitope is marked in the box.

[0069] Figure 4: Kidney uptake of indium-labeled DARPins. Radiolabeled DARPins were injected into wild-type mice. Data are shown as mean % injected activity / gram of tissue mass (% lA / g). Measures were taken 4 hours after injection. Error bars show standard deviations.

[0070] Figure 5: Binding to membrane-proximal MSLN peptide fragments by ELISA. Binding of mbMSLN DARPins #2 (A), #3 (B), #4 (C), #5 (D) and #6 (E), sMSLN DARPin #1 (F) and comparator antibody 15B6 (G) to peptide fragments of membrane-proximal MSLN (residues 580-595) (SEQ ID NO: 190), residues 580-592 (SEQ ID NO: 191), residues 580-589 (SEQ ID NO: 192), residues 580-586 (SEQ ID NO: 193), residues 580-583 (SEQ ID NO: 194), residues 296-580 of SEQ ID NO: 166 (“soly ECD”), and residues 296- 597 of SEQ ID NO: 167 (“mb ECD”) was assessed by ELISA. Also shown are binding to carrier (Fc-KiH) and control samples not comprising any target or binder, respectively.

[0071] Figure 6: Binding competition of selected DARPins in the presence of soluble MSLN mimics. Binding of mbMSLN DARPins # 6 and #2.1 as well as sMSLN DARPin #7 to immobilised full length MSLN was determined in the presence of sMSLN mimics, which result from different protease cleavage sites within the MSLN C-terminal peptide region (short (amino acid residues 296-580 of SEQ ID NO: 166, medium (amino acid residues 296-586 of SEQ ID NO: 167), long (amino acid residues 296-591 of SEQ ID NO: 167) or full-length (residues 296-597 of SEQ ID NO: 167).

[0072] Figure 7: Cell binding competition assay. Selected DARPins and control antibody anetumab were preincubated with recombinant MSLN versions (full-length (residues 296-597 of SEQ ID NO: 167), long (residues 296-591 of SEQ ID NO: 167), medium (residues 296-586 of SEQ ID NO: 167) and short (residues 296-580 of SEQ ID NO: 166), and subsequently incubated with OVCAR-8 cells. Cell binding was assessed by Flow Cytometry. Graphs show the Mean Fluorescence Intensity of the live cell population.

[0073] Figure 8: Cell binding of selected mbMSLN DARPins to hMSLN on OVCAR-3 cells. DARPins were detected with anti-DARPin antibody 1.4.8. MFI: median fluorescent intensity. DETAILED DESCRIPTION OF THE INVENTION

[0074] Mesothelin is 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. The recombinant binding proteins described herein comprise an ankyrin repeat domain comprising at least one ankyrin repeat module that specifically bind to mesothelin. More specifically, in some embodiments the recombinant binding proteins described herein comprise an ankyrin repeat domain comprising at least two or at least three ankyrin repeat modules that specifically bind to the soluble form of mesothelin. In other embodiments the recombinant binding proteins described herein comprise an ankyrin repeat domain comprising at least two ankyrin or at least three repeat modules that specifically bind to membrane-bound mesothelin. Optionally, said ankyrin repeat domain comprising at least two or at least three designed ankyrin repeat modules that specifically bind to membrane-bound mesothelin do not bind soluble mesothelin.

[0075] Ankyrin repeat domains

[0076] Designed ankyrin repeat protein libraries (W02002 / 020565; Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701 , 2008) can be used for the selection of target-specific designed ankyrin repeat domains that bind to their target with high affinity. Such target-specific designed ankyrin repeat domains in turn can be used as valuable components of recombinant binding proteins for the treatment of diseases. Ankyrin repeat proteins are a class of binding molecules which have the potential to overcome limitations of monoclonal antibodies, hence allowing novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain or may comprise a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13, 695-701 , 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins comprising only a single designed ankyrin repeat domain are small proteins (14 kDa) which can be selected to bind a given target protein with high affinity and specificity. These characteristics, and the possibility of combining two or more ankyrin repeat domains in one protein, make ankyrin repeat proteins ideal agonistic, antagonistic and / or inhibitory drug candidates. Furthermore, such ankyrin repeat proteins can be engineered with various effector functions, e.g., cytotoxic agents, radionuclides, half-life extending agents and / or immune-cell engagers, enabling completely new drug formats. By simultaneously targeting antigens on tumor cells and activating receptors on immune cells, such immune cell engager molecules strengthen the interaction between the two cell types and increase immune cell effector functions. Tumor-localized activation of the immune cells can therefore be achieved.

[0077] Ankyrin repeat domains with binding specificity for soluble mesothelin

[0078] In one aspect, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin. Specifically, said ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 8 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 7 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 or an amino acid sequence wherein up to 6 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 5 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 4 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 3 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 2 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 1 amino acid in any one of SEQ ID NOs: 43 to 61 is substituted with other amino acids. In one embodiment, all of said 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions occur in framework positions of said ankyrin repeat module(s). In one embodiment, said ankyrin repeat module comprises an amino acid sequence of anyone of SEQ ID NOs: 43 to 61 .

[0079] In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 43 or a sequence in which one or two amino acids in SEQ ID NO: 43 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 44 or a sequence in which one or two amino acids in SEQ ID NO: 44 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 45 or a sequence in which one or two amino acids in SEQ ID NO: 45 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 46 or a sequence in which one or two amino acids in SEQ ID NO: 46 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 47 or a sequence in which one or two amino acids in SEQ ID NO: 47 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 48 or a sequence in which one or two amino acids in SEQ ID NO: 48 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 49 or a sequence in which one or two amino acids in SEQ ID NO: 49 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 50 or a sequence in which one or two amino acids in SEQ ID NO: 50 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 51 or a sequence in which one or two amino acids in SEQ ID NO: 51 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 52 or a sequence in which one or two amino acids in SEQ ID NO: 52 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 53 or a sequence in which one or two amino acids in SEQ ID NO: 53 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 54 or a sequence in which one or two amino acids in SEQ ID NO: 54 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 55 or a sequence in which one or two amino acids in SEQ ID NO: 55 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 56 or a sequence in which one or two amino acids in SEQ ID NO: 56 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 57 or a sequence in which one or two amino acids in SEQ ID NO: 57 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 58 or a sequence in which one or two amino acids in SEQ ID NO: 58 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 59 or a sequence in which one or two amino acids in SEQ ID NO: 59 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 60 or a sequence in which one or two amino acids in SEQ ID NO: 60 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 61 or a sequence in which one or two amino acids in SEQ ID NO: 61 are substituted with other amino acids.

[0080] In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 43. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 44. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 45. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 46. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 47. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 48. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 49. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 50. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 51. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 52. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 53. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 54. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 55. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 56. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 57. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 58. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 59. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 60. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 61 .

[0081] In one embodiment, said ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module and a second ankyrin repeat module. In one embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain. In one embodiment, said first and said second ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids and further comprises a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids.

[0082] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 46 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 47 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 46 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 47 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 46 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 47, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0083] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 48, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 48 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 49, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 49 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 48, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 48 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 49, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 49 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 48 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 49, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0084] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 50, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 50 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 51 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 51 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 50, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 50 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 51 , or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 51 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 50 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 51 , wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0085] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 58 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 59, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 59 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 58 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 59, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 59 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 58 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 59, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 60, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 60 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 61 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 60, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 60 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 61 , or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 61 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 60 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 61 , wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0086] In one embodiment, said ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module, a second ankyrin repeat module and a third ankyrin repeat module. In one preferred embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain, and said second ankyrin repeat module is located N-terminally of said third ankyrin repeat module within said ankyrin repeat domain.

[0087] In one embodiment, said first, second and third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids and a third ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted with other amino acids.

[0088] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 43, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 43 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 44, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 44 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 45, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 45 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 43, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 43 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 44, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 44 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 45, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 45 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 43, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 44 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 45, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0089] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 52, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 52 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 53, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 53 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 54 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 52, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 52 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 53, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 53 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 54 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 52, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 53 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 54, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0090] In one particular embodiment, the ankyrin repeat domain specifically binding soluble mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 55, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 55 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 56, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 56 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 57, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 57 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 55, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 55 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 56, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 56 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 57, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 57 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 55, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 56 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 57, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0091] In one embodiment, all of said amino acid substitutions of said ankyrin repeat module(s) as described and referred to herein occur in framework positions of said ankyrin repeat module(s), wherein typically the overall structure of the module(s) is not affected by the substitutions.

[0092] The ankyrin repeat domains specifically binding soluble mesothelin as disclosed herein preferably comprise a N-terminal and / or a C-terminal capping module (thereafter also referred to as “capping repeats”). Capping modules are located at the N-and / or C-terminal end of an ankyrin repeat domain, typically forming tight tertiary interactions (i.e. tertiary structure interactions) with the ankyrin repeat module(s) in between, thereby providing a cap that shields the hydrophobic core of the ankyrin repeat domain at the side from exposure to the solvent. Examples of capping sequences are described in International Patent Publication Nos. WO 2002 / 020565 and WO 2012 / 069655, in U.S. Patent Publication No. US20130296221 , and by Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54. Examples of amino acid sequences of N-terminal capping modules (i.e. N-terminal capping repeats) are provided in SEQ ID NOs: 79 to 86 and examples of amino acid sequences of C-terminal capping modules (i.e. C-terminal capping repeats) are provided in SEQ ID NOs: 91 to 97. Accordingly, in some embodiments, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin as described herein, wherein said ankyrin repeat domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79 to 86, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79 to 86 are substituted by other amino acids. Alternatively or in addition, said ankyrin repeat domain comprises a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 to 97, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 to 97 are substituted by other amino acids.

[0093] In one preferred embodiment, said ankyrin repeat domain specifically binding soluble mesothelin comprises from N-terminus to C-terminus: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat module(s) as described more specifically herein; and a C-terminal capping module.

[0094] In one embodiment, said ankyrin repeat domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79 to 86, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79 to 86 are substituted by other amino acids; at least one, at least two, or at least three ankyrin repeat module independently comprising an amino acid sequence of any one of SEQ ID NOs: 43 to 61 , or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 43 to 61 are substituted by other amino acids; a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 to 97, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 to 97 are substituted by other amino acids.

[0095] In another aspect, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin, 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: 1 to 8. Optionally, A at the second last position of SEQ ID NOs: 1 to 8 is substituted with L, and / or optionally A at the last position of SEQ ID NOs: 1 to 8 is substituted with N.

[0096] Thus, in one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 1 to 8. In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 1 to 8. In another embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 1 to 8. In a further embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 1 to 8. In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 1 to 8. In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 8.

[0097] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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: 1 . In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0098] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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: 2. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0099] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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: 3. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0100] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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.

[0101] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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.

[0102] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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.

[0103] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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: 7. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0104] In one embodiment, said ankyrin repeat domain with binding specificity for soluble mesothelin 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: 8. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0105] Also provided is a recombinant binding protein with binding specificity for soluble mesothelin that competes with a reference binding protein comprising an ankyrin repeat domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8. In one embodiment thereof, said competing recombinant binding protein with binding specificity for soluble mesothelin comprises an ankyrin repeat domain. Also provided is a recombinant binding protein with binding specificity for soluble mesothelin that binds to the same epitope as a reference binding protein comprising an ankyrin repeat domain comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 8. In one embodiment thereof, said competing recombinant binding protein with binding specificity for soluble mesothelin comprises an ankyrin repeat domain.

[0106] In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin binds to human soluble mesothelin with a dissociation constant (KD) 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 10'1°M, or of or below about 10-11M. Thus, in one embodiment, said recombinant binding protein binds to soluble mesothelin with a KD of or below about 10-7M. In another embodiment, said recombinant binding protein binds to soluble mesothelin with a KD of or below about 10-8M. In another embodiment, said recombinant binding protein binds to soluble mesothelin with a KD of or below about 10-9M. In another embodiment, said recombinant binding protein binds to soluble mesothelin with a KD of or below about 10'10M. In another embodiment, said recombinant binding protein binds to soluble mesothelin with a KD of or below about 10_11M. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble human mesothelin binding also binds to soluble cynomolgus mesothelin. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain with binding specificity for mesothelin binds to soluble cynomolgus mesothelin with a dissociation constant (KD) 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 or of or below about Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR), e.g. as described in Example 1 and 2.

[0107] Accordingly, in one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said binding protein binds human soluble mesothelin in PBS with a KD 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 or of or below about 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: 1 to 8.

[0108] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 1 . In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 1 .

[0109] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 2. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 2. In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 3. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 3.

[0110] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 4. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 4.

[0111] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 5. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 5.

[0112] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 6. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 6.

[0113] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 7. In one embodiment, said recombinant binding protein comprising an ankyrin repeat binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 7.

[0114] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain, wherein said recombinant binding protein binds soluble human mesothelin in PBS with a KD 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 SEQ ID NO: 8. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds soluble human mesothelin in PBS with a KD of or below about 10-8M, wherein said ankyrin repeat domain comprises or consists of the amino acid sequence with SEQ ID NO: 8.

[0115] In one embodiment, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for soluble mesothelin, wherein said ankyrin repeat domain has a melting temperature (Tm) higher than about 65°C, higher than about 70°C, higher than about 72°C, higher than about 75°C, higher than about 78°C, higher than about 80°C, of about 75°C, of about 80°C, of about 82°C, of about 85°C, of between about 65°C and about 95°C, of between about 70°C and about 90°C, or of between about 72°C and about 88°C. Thus, in one embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 65°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 70°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 75°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 80°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) of between about 65°C and about 95°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) of between about 72°C and about 88°C.

[0116] Ankyrin repeat domains with binding specificity for membrane-bound mesothelin

[0117] In one aspect, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds membrane-bound mesothelin, wherein membrane-bound mesothelin refers to amino acid sequence 296-597 with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 580-598 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 580-598 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 596-598 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 596-598 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 593-598 of membranebound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 593-598 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 580-595 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 580-595 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 593-595 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 593-595 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In a preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to amino acid residues 590-592 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In another preferred embodiment, said recombinant binding protein comprising an ankyrin repeat domain binds to an epitope comprised within amino acid residues 590-592 of membrane-bound mesothelin with reference to UniProt ID Q13421-3 (SEQ ID NO: 167). In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin does not bind to soluble mesothelin (sMSLN). The recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin is not inhibited by the presence of sMSLN. Specifically, said ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 8 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 7 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 6 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 5 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 4 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 3 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 2 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 1 amino acid in any one of SEQ ID NOs: 62 to 78 is substituted with other amino acids. In one embodiment, all of said 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions occur in framework positions of said ankyrin repeat module(s). In one embodiment, said ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78.

[0118] In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 62 or a sequence in which one or two amino acids in SEQ ID NO: 62 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 63 or a sequence in which one or two amino acids in SEQ ID NO: 63 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 64 or a sequence in which one or two amino acids in SEQ ID NO: 64 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 65 or a sequence in which one or two amino acids in SEQ ID NO: 65 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 66 or a sequence in which one or two amino acids in SEQ ID NO: 66 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 67 or a sequence in which one or two amino acids in SEQ ID NO: 67 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 68 or a sequence in which one or two amino acids in SEQ ID NO: 68 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 69 or a sequence in which one or two amino acids in SEQ ID NO: 69 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 70 or a sequence in which one or two amino acids in SEQ ID NO: 70 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 71 or a sequence in which one or two amino acids in SEQ ID NO: 71 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 72 or a sequence in which one or two amino acids in SEQ ID NO: 72 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 73 or a sequence in which one or two amino acids in SEQ ID NO: 73 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 74 or a sequence in which one or two amino acids in SEQ ID NO: 74 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 75 or a sequence in which one or two amino acids in SEQ ID NO: 75 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 76 or a sequence in which one or two amino acids in SEQ ID NO: 76 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 77 or a sequence in which one or two amino acids in SEQ ID NO: 77 are substituted by other amino acids. In one embodiment, said ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 78 or a sequence in which one or two amino acids in SEQ ID NO: 78 are substituted by other amino acids.

[0119] In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 62. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 63. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 64. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 65. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 66. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 67. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 68. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 69. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 70. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 71. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 72. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 73. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 74. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 75. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 76. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 77. In one embodiment, said ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 78.

[0120] In one embodiment, said ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module and a second ankyrin repeat module. In one embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module within said ankyrin repeat domain. In one embodiment, said first and said second ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids and further comprises a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids.

[0121] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 65 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 66, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0122] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 68 or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 67 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 68, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0123] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 69 or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 69 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 70 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 69 or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 69 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 70 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 69 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 70, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0124] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 71 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 71 , or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module. In one embodiment, said first ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 71 and said second ankyrin repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 72, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0125] In another embodiment, said ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 101 to 105, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 101 to 105 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 106 to 109, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 106 to 109 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 106 to 109, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of any one of SEQ ID NOs: 106 to 109 are substituted with other amino acids and said second ankyrin repeat module comprises an amino acid sequence of any one of SEQ ID NOs: 106 to 109, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of any one of SEQ ID NOs: 106 to 109 are substituted with other amino acids, wherein said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module.

[0126] In one embodiment, said ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module. In one preferred embodiment, said first ankyrin repeat module is located N-terminally of said second ankyrin repeat module, and said second ankyrin repeat module is located N-terminally of said third ankyrin repeat module within said ankyrin repeat domain.

[0127] In one embodiment, said first, second and third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids. Thus, in one embodiment, said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids.

[0128] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 62 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 63, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 63 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 64 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 62 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 63 or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 63 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 64 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 62, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 63, and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 64, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0129] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 73 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 75 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 73 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 74 or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 75 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 73, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 74 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 75, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0130] In one particular embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 76 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 77 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids. In one embodiment, said first ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 76 are substituted with other amino acids, said second ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 77 are substituted with other amino acids, and said third ankyrin repeat module comprises an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain. In one embodiment, said ankyrin repeat domain comprises (i) a first ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 76, (ii) a second ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 77 and (iii) a third ankyrin repeat module comprising or consisting of the amino acid sequence of SEQ ID NO: 78, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0131] In another embodiment, said ankyrin repeat domain specifically binding membrane-bound mesothelin comprises a first ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 101 to 105, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 101 to 105 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 106 to 109, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 106 to 109 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 110 to 112, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 110 to 112 are substituted with other amino acids, wherein said first, second and third ankyrin repeat modules are arranged as first module-second module-third module in N-terminal to C-terminal direction within said ankyrin repeat domain.

[0132] In another embodiment, the ankyrin repeat domain specifically binding membrane-bound mesothelin comprising said first, optionally said second and optionally said third ankyrin repeat module independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, or an amino acid sequence wherein up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or 1 framework residue in any one of SEQ ID NOs: 62 to 78 or 101 to 112 are substituted with other amino acids.

[0133] The ankyrin repeat domains specifically binding membrane-bound mesothelin preferably comprise a N- terminal and / or a C-terminal capping module (thereafter also referred to as “capping repeats”). Examples of amino acid sequences of N-terminal capping modules (i.e. N-terminal capping repeats) are provided in SEQ ID NOs: 79, 87 to 90, 113 to 115, and 201 and examples of amino acid sequences of C-terminal capping modules (i.e. C-terminal capping repeats) are provided in SEQ ID NOs: 91 , 98 to 100, and 116 to 120.

[0134] Accordingly, in some embodiments, provided is a recombinant binding protein comprising an ankyrin repeat domain as described herein with binding specificity for membrane-bound mesothelin, wherein said ankyrin repeat domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79, 87 to 90, 113 to 115, or 201 , or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79, 87 to 90, 113 to 115, or 201 are substituted by other amino acids. Alternatively or in addition, said ankyrin repeat domain comprises a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 , 98 to 100 or 116 to 120, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to

[0135] 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 , 98 to 100 or 116 to 120 are substituted by other amino acids.

[0136] In one preferred embodiment, said ankyrin repeat domain specifically binding membrane-bound mesothelin comprises from N-terminus to C-terminus: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat module(s) specifically binding membrane-bound mesothelin as described herein; and a C-terminal capping module.

[0137] Accordingly, in some embodiments, provided is an ankyrin repeat domain, wherein said ankyrin repeat domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79, 87 to 90, 113 to 115, or 201 , or an amino acid sequence wherein to 9, up to 8, up to 7, up to

[0138] 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79, 87 to 90, 113 to 115, or 201 are substituted by other amino acids; at least one, at least two, or at least three internal ankyrin repeat modules independently comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, and 101 to 112, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 or 101 to 112 are substituted by other amino acids; a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 , 98 to 100 or 116 to 120, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 , 98 to 100 or 116 to 120 are substituted by other amino acids.

[0139] In one embodiment, all of said amino acid substitutions of said ankyrin repeat module(s) as described and referred to herein occur in framework positions, e.g., in positions other than the randomized positions 3, 4, 6, 11 , 14 and 15 of said ankyrin repeat module(s) of SEQ ID NOs: 62 to 78, and 101 to 112, e.g., in positions otherthan positions 4, 8, 11 and 12 of SEQ ID NOs: 79, 87 to 90, 113 to 115, and 201 , and e.g., in positions other than positions 3, 4, 6, 14 and 15 of SEQ ID NOs: 91 , 98 to 100, and 116 to 120, wherein typically the overall structure of the module(s) is not affected by the substitutions. Such an embodiment of substitution in framework positions shall apply to all embodiments irrespective of whether such substitution is explicitly described. In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Tyr or Trp in position 4, (ii) His in position 6, (iii) Leu or Vai in position 11 , and / or (iv) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0140] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Tyr or Trp in position 4, (ii) His in position 6, (iii) Leu in position 11 , and / or (iv) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0141] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Trp in position 4, (ii) His in position 6, (iii) Leu in position 11 , and / or (iv) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0142] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4, (ii) His in position 6, (iii) Vai in position 11 , and / or (iv) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0143] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4, (ii) His in position 6, (iii) Leu in position 11 , and / or (iv) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0144] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) His in position 6, and / or (ii) Trp or Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0145] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) His in position 6, and (ii) Tyr in position 14, numbered relative to SEQ ID NO: 101.

[0146] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) His in position 6, and (ii) Trp in position 14, numbered relative to SEQ ID NO: 101.

[0147] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe, Tyr or Trp in position 4, (ii) He in position 6, (iii) Vai in position 11 , (iv) Tyr in position 14, and / or (v) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0148] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe or Tyr in position 4, (ii) He in position 6, (iii) Vai in position 11 , (iv) Tyr in position 14, and / or (v) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0149] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe in position 4, (ii) lie in position 6, (iii) Vai in position 11 , (iv) Tyr in position 14, and / or (v) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0150] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4, (ii) lie in position 6, (iii) Vai in position 11 , (iv) Tyr in position 14, and / or (v) Arg in position 15 numbered relative to SEQ ID NO: 106. In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Trp in position 4, (ii) He in position 6, (iii) Vai in position 11 , (iv) Tyr in position 14, and / or (v) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0151] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe or Tyr in position 4, (ii) Tyr in position 14, and / or (iii) Arg or Lys in position 15 numbered relative to SEQ ID NO: 106.

[0152] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4, (ii) Tyr in position 14, and / or (iii) Lys in position 15 numbered relative to SEQ ID NO: 106.

[0153] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe in position 4, (ii) Tyr in position 14, and / or (iii) Lys in position 15 numbered relative to SEQ ID NO: 106.

[0154] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4, (ii) Tyr in position 14, and / or (iii) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0155] In some embodiments, the internal ankyrin repeat module, suitably the second internal ankyrin repeat module, comprises amino acid residues (i) Phe in position 4, (ii) Tyr in position 14, and / or (iii) Arg in position 15 numbered relative to SEQ ID NO: 106.

[0156] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises amino acid residues (i) Vai or Arg in position 3, (ii) Glu or Tyr in position 4, (iii) Trp in position 6, (iv) He in position 11 , (v) Gin or Glu in position 14 and / or (vi) Ser or His in position 15 numbered relative to SEQ ID NO: 110.

[0157] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises amino acid residues (i) Vai or Arg in position 3, (ii) Glu in position 4, (iii) Trp in position 6, (iv) lie in position 11 , (v) Gin in position 14 and / or (vi) Ser or His in position 15 numbered relative to SEQ ID NO: 110.

[0158] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises amino acid residues (i) Vai in position 3, (ii) Glu or Tyr in position 4, (iii) Trp in position 6, (iv) lie in position 11 , (v) Gin or Glu in position 14 and / or (vi) His in position 15 numbered relative to SEQ ID NO: 110.

[0159] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises amino acid residues (i) Tyr in position 4 and (ii) His in position 15 numbered relative to SEQ ID NO: 110.

[0160] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises amino acid residues (i) Trp in position 6, (ii) Gin or Glu in position 14 and / or (iii) His in position 15 numbered relative to SEQ ID NO: 110. In some embodiments, the N-terminal capping module comprises amino acid residues (i) Ala in position 11 and / or (ii) Arg, Gin or Lys in position 12 numbered relative to SEQ ID NO: 115.

[0161] In some embodiments, the N-terminal capping module comprises amino acid residues Ala in position 11 numbered relative to SEQ ID NO: 115.

[0162] In some embodiments, the C-terminal capping module comprises amino acid residues (i) Glu in position 3, (ii) Ala in position 4, (iii) Leu in position 6, (iv) Arg or Ala in position 14, (v) Leu in position 15 and / or (vi) Glu or Asp in position 18 numbered relative to SEQ ID NO: 119.

[0163] In some embodiments, the C-terminal capping module comprises amino acid residues (i) Ala in position 4, (ii) Leu in position 6, and / or (iii) Leu in position 15 numbered relative to SEQ ID NO: 119.

[0164] In some embodiments, the C-terminal capping module comprises amino acid residues (i) Lys in position 3, (ii) Ser in position 4, (iii) Lys in position 6, (iv) Arg in position 14, (v) Ala in position 15 and / or (vi) Gin in position 18 numbered relative to SEQ ID NO: 119.

[0165] In another aspect, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for human membrane-bound mesothelin, 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: 9 to 15, 195, 198, or 199. Thus, in one embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In one embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In another embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In a further embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In one embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In one embodiment, said ankyrin repeat domain comprises an amino acid sequence with any one of SEQ ID NOs: 9 to 15, 195, 198, or 199.

[0166] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin, 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 SEQ ID NO: 9. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 10. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0167] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 11 . In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 11 .

[0168] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 12. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0169] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 13. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0170] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 14. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0171] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 15. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0172] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 195. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 195.

[0173] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 198. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 198.

[0174] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 199. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 199.

[0175] Also provided is a recombinant binding protein with binding specificity for membrane-bound mesothelin that competes with a reference binding protein comprising an ankyrin repeat domain comprising the amino acid sequence of any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In one embodiment thereof, said recombinant binding protein with binding specificity for membrane-bound mesothelin comprises an ankyrin repeat domain. Also provided is a recombinant binding protein with binding specificity for membrane-bound mesothelin that binds to the same epitope as a reference binding protein comprising an ankyrin repeat domain comprising the amino acid sequence of any one of SEQ ID NOs: 9 to 15, 195, 198, or 199. In one embodiment thereof, said recombinant binding protein with binding specificity for membrane-bound mesothelin comprises an ankyrin repeat domain.

[0176] In some embodiments, the ankyrin repeat modules described herein are engineered further to reduce the number of basic amino acids (positive charge) and / or reduce the isoelectric point (pl) compared to the nonengineered ankyrin repeat modules. This approach is further described in PCT / EP2023 / 071188. Recombinant binding proteins comprising such engineered ankyrin repeat modules have been found to reduce renal accumulation of a linked drug moiety (such as a radionuclide) upon administration thereof.

[0177] The charge characteristics of said engineered ankyrin repeat domain can alternatively or in addition be defined by a ratio between the sum of Arg (R) and Lys (K) residues over the sum of Asp (D) and Glu (E) residues comprised in said engineered ankyrin repeat domain (also referred to as KR / DE ratio). The KR / DE ratio can be computed based on the amino acid sequences of the ankyrin repeat domains described herein.

[0178] Accordingly, in some embodiments, said engineered ankyrin repeat domain has a KR / DE ratio equal to or lower than 0.44, equal to or lower than 0.42, equal to or lower than 0.40, equal to or lower than 0.38, equal to or lower than 0.36, equal to or lower than 0.34, equal to or lower than 0.32, equal to or lower than 0.30, equal to or lower than 0.28, equal to or lower than 0.26, equal to or lower than 0.24, equal to or lower than 0.22, equal to or lower than 0.20, equal to or lower than 0.18, equal to or lower than 0.16, equal to or lower than 0.14, equal to or lower than 0.12, equal to or lower than 0.10, or equal to or lower than 0.08. Suitably, said KR / DE ratio is equal to or lower than 0.3. Unless specified otherwise, the KR / DE ratio is determined by considering all residues comprised in the engineered ankyrin repeat domain. In other embodiments, the KR / DE ratio is computed among the framework residues of the ankyrin repeat domain only. The positions of framework residues are described herein. Accordingly, in some embodiments, said engineered ankyrin repeat domain has a KR / DE ratio among the framework residues equal to or lower than 0.36, equal to or lower than 0.34, equal to or lower than 0.32, equal to or lower than 0.30, equal to or lower than 0.28, equal to or lower than 0.26, equal to or lower than 0.24, equal to or lower than 0.22, equal to or lower than 0.20, equal to or lower than 0.18, equal to or lower than 0.16, equal to or lower than 0.14, equal to or lower than 0.12, equal to or lower than 0.10, or equal to or lower than 0.08. Suitably, said KR / DE ratio among the framework residues is equal to or lower than 0.25.

[0179] In some embodiments, said engineered ankyrin repeat domain has an isoelectric point (pl) at least 0.1 pH points lower than the parental (non-engineered) ankyrin repeat domain. In some embodiments, said pl is at least 0.1 , at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1 .0 or at least 1 .1 pH points lower than the parental (non-engineered) ankyrin repeat domain.

[0180] In some embodiments, the engineered ankyrin repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0%, equal to or lower than 7.5%, equal to or lower than 7.0%, equal to or lower than 6.5%, or equal to or lower than 6.0%, equal to or lower than 5.5%, equal to or lower than 5.0%, equal to or lower than 4.5%, equal to or lower than 4.0%, or equal to or lower than 3.5%.

[0181] In some embodiments, the engineered ankyrin repeat domain has a percentage of Arg and Lys residues among the framework residues equal to or lower than 7.5%, equal to or lower than 7.0%, equal to or lower than 6.5%, or equal to or lower than 6.0%, equal to or lower than 5.5%, equal to or lower than 5.0%, equal to or lower than 4.5%, equal to or lower than 4.0%, or equal to or lower than 3.5%.

[0182] In some embodiments, the engineered ankyrin repeat domain comprises one or more internal repeat modules each comprising one or more amino acid residues of (i) Ser or Glu in position 1 , (ii) Leu in position 25, (iii) Glu in position 26 and / or (iv) Ala in position 27. In some embodiments, the engineered ankyrin repeat domain comprises one or more internal repeat modules each comprising one or more amino acid residues of (i) Ser or Glu in position 1 , and / or (ii) Glu in position 26.

[0183] In some embodiments, the engineered ankyrin repeat domain comprises an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and / or (v) Gin in position 23.

[0184] In some embodiments, the engineered ankyrin repeat domain comprises an N-terminal capping module comprising one or more amino acid residues of (i) Gin in position 5, (ii) Thr in position 17, and / or (iii) Gin in position 23.

[0185] In some embodiments, the engineered ankyrin repeat domain comprises a C-terminal capping module comprising one or more amino acid residues of (i) Thr in position 3, (ii) Ser in position 6, (iii) Asp in position 14, (iv) Asn in position 17, (v) Glu in position 18, and / or (vi) Gin in position 26.

[0186] In some embodiments, the engineered ankyrin repeat domain comprises a C-terminal capping module comprising one or more amino acid residues of (i) Asn in position 17, (ii) Glu in position 18, and (iii) Gin in position 26.

[0187] In some embodiments, the engineered ankyrin repeat domain comprises

[0188] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23, and / or

[0189] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , (ii) Leu in position 25, (iii) Glu in position 26 and (iv) Ala in position 27, and / or

[0190] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0191] 3, (ii) Ser in position 6, (iii) Asp in position 14, (iv) Asn in position 17, (v) Glu in position 18, and (vi) Gin in position 26.

[0192] In some embodiments, the engineered ankyrin repeat domain comprises

[0193] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0194] 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23, and / or

[0195] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , (ii) Leu in position 25, (iii) Glu in position 26 and (iv) Ala in position 27, and / or

[0196] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Asn in position 17, (ii) Glu in position 18, and (iii) Gin in position 26.

[0197] In some embodiments, the engineered ankyrin repeat domain comprises

[0198] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23; and / or (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Gin in position 5, (ii) Thr in position 17, and / or (iii) Gin in position 23; and / or

[0199] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0200] 3, (ii) Ser in position 6, (iii) Asp in position 14, (iv) Asn in position 17, (v) Glu in position 18, and (vi) Gin in position 26.

[0201] In some embodiments, the engineered ankyrin repeat domain comprises

[0202] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0203] 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23; and / or

[0204] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Gin in position 5, (ii) Thr in position 17, and / or (iii) Gin in position 23; and / or

[0205] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Asn in position 17, (ii) Glu in position 18, and (iii) Gin in position 26.

[0206] In some embodiments, the engineered ankyrin repeat domain comprises

[0207] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23, and / or

[0208] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , and / or (ii) Glu in position 26, and / or

[0209] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0210] 3, (ii) Ser in position 6, (iii) Asp in position 14, (iv) Asn in position 17, (v) Glu in position 18, and (vi) Gin in position 26.

[0211] In some embodiments, the engineered ankyrin repeat domain comprises

[0212] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0213] 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23, and / or

[0214] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , and / or (ii) Glu in position 26, and / or

[0215] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Asn in position 17, (ii) Glu in position 18, and (iii) Gin in position 26.

[0216] In some embodiments, the engineered ankyrin repeat domain comprises

[0217] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23; and / or

[0218] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , and / or (ii) Glu in position 26; and / or (3) a C-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0219] 3, (ii) Ser in position 6, (iii) Asp in position 14, (iv) Asn in position 17, (v) Glu in position 18, and (vi) Gin in position 26.

[0220] In some embodiments, the engineered ankyrin repeat domain comprises

[0221] (1) an N-terminal capping module comprising one or more amino acid residues of (i) Thr in position

[0222] 4, (ii) Gin in position 5, (iii) Glu in position 8, (iv) Thr in position 17, and (v) Gin in position 23; and / or

[0223] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more amino acid residues of (i) Ser or Glu in position 1 , and / or (ii) Glu in position 26; and / or

[0224] (3) a C-terminal capping module comprising one or more amino acid residues of (i) Asn in position 17, (ii) Glu in position 18, and (iii) Gin in position 26.

[0225] In some embodiments, the engineered ankyrin repeat domain comprises one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 , position

[0226] 25, position 26 and / or position 27, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27.

[0227] In some embodiments, the engineered ankyrin repeat domain comprises one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 and / or position 26, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , and / or (ii) Glu at position

[0228] 26.

[0229] In some embodiments, the engineered ankyrin repeat domain comprises an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N- terminal capping module further comprising one or more amino acid substitution at position 4, position 5, position 8, position 17 and / or position 23, wherein said amino acid is substituted for (i) Thr at position 4, (ii) Gin at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gin at position 23.

[0230] In some embodiments, the engineered ankyrin repeat domain comprises an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N- terminal capping module further comprising one or more amino acid substitution at position 5, position 17 and / or position 23, wherein said amino acid is substituted for (i) Gin at position 5, (ii) Thr at position 17, and / or (iii) Gin at position 23.

[0231] In some embodiments, the engineered ankyrin repeat domain comprises a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 3, position 6, position 14, position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Thr at position 3, (ii) Ser at position 6, (iii) Asp at position 14, (iv) Asn at position 17, (v) Glu at position 18, and / or (vi) Gin at position 26. In some embodiments, the engineered ankyrin repeat domain comprises a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gin at position 26.

[0232] In some embodiments, the engineered ankyrin repeat domain comprises

[0233] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 4, position 5, position 8, position 17 and / or position 23, wherein said amino acid is substituted for (i) Thr at position 4, (ii) Gin at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gin at position 23; and / or

[0234] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 , position 25, position 26 and / or position 27, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27; and / or

[0235] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 3, position 6, position 14, position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Thr at position 3, (ii) Ser at position 6, (iii) Asp at position 14, (iv) Asn at position 17, (v) Glu at position 18, and / or (vi) Gin at position 26.

[0236] In some embodiments, the engineered ankyrin repeat domain comprises

[0237] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 4, position 5, position 8, position 17 and / or position 23, wherein said amino acid is substituted for (i) Thr at position 4, (ii) Gin at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gin at position 23; and / or

[0238] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 , position 25, position 26 and / or position 27, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27; and / or

[0239] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gin at position 26.

[0240] In some embodiments, the engineered ankyrin repeat domain comprises

[0241] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 5, position 17 and / or position 23, wherein said amino acid is substituted for (i) Gin at position 5, (ii) Thr at position 17, and / or (iii) Gin at position 23; and / or

[0242] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 , position 25, position 26 and / or position 27, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27; and / or

[0243] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO:

[0244] 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 3, position 6, position 14, position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Thr at position 3, (ii) Ser at position 6, (iii) Asp at position 14, (iv) Asn at position 17, (v) Glu at position 18, and / or (vi) Gin at position 26.

[0245] In some embodiments, the engineered ankyrin repeat domain comprises

[0246] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 5, position 17 and / or position 23, wherein said amino acid is substituted for (i) Gin at position 5, (ii) Thr at position 17, and / or (iii) Gin at position 23; and / or

[0247] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 , position 25, position 26 and / or position 27, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27; and / or

[0248] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO:

[0249] 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gin at position 26.

[0250] In some embodiments, the engineered ankyrin repeat domain comprises

[0251] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 4, position 5, position 8, position 17 and / or position 23, wherein said amino acid is substituted for (i) Thr at position 4, (ii) Gin at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gin at position 23; and / or

[0252] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 and / or position 26, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , and / or (ii) Glu at position 26; and / or

[0253] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO:

[0254] 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 3, position 6, position 14, position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Thr at position 3, (ii) Ser at position 6, (iii) Asp at position 14, (iv) Asn at position 17, (v) Glu at position 18, and / or (vi) Gin at position 26.

[0255] In some embodiments, the engineered ankyrin repeat domain comprises

[0256] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 4, position 5, position 8, position 17 and / or position 23, wherein said amino acid is substituted for (i) Thr at position 4, (ii) Gin at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gin at position 23; and / or

[0257] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 and / or position 26, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , and / or (ii) Glu at position 26; and / or

[0258] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gin at position 26.

[0259] In some embodiments, the engineered ankyrin repeat domain comprises

[0260] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 5, position 17 and / or position 23, wherein said amino acid is substituted for (i) Gin at position 5, (ii) Thr at position 17, and / or (iii) Gin at position 23; and / or

[0261] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 and / or position 26, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , and / or (ii) Glu at position 26; and / or

[0262] (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 3, position 6, position 14, position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Thr at position 3, (ii) Ser at position 6, (iii) Asp at position 14, (iv) Asn at position 17, (v) Glu at position 18, and / or (vi) Gin at position 26.

[0263] In some embodiments, the engineered ankyrin repeat domain comprises

[0264] (1) an N-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 , said N-terminal capping module further comprising one or more amino acid substitution at position 5, position 17 and / or position 23, wherein said amino acid is substituted for (i) Gin at position 5, (ii) Thr at position 17, and / or (iii) Gin at position 23; and / or

[0265] (2) one or more internal repeat modules comprising the amino acid sequence of any one of SEQ ID NOs: 62 to 78 or 101 to 112, said one or more internal repeat module further comprising one or more amino acid substitution at position 1 and / or position 26, wherein said amino acid is substituted for (i) Ser or Glu at position 1 , and / or (ii) Glu at position 26; and / or (3) a C-terminal capping module comprising the amino acid sequence of any one of SEQ ID NO: 91 , 98 to 100 or 116 to 120, said C-terminal capping module further comprising one or more amino acid substitution at position 17, position 18 and / or position 26, wherein said amino acid is substituted for (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gin at position 26.

[0266] In some embodiments, the engineered ankyrin repeat domain comprises one or more internal repeat modules, wherein each internal repeat module independently comprises an amino acid sequence of any one of SEQ ID NOs: 132 to 151 or 203 to 205, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 132 to 151 or 203 to 205 are substituted by other amino acids.

[0267] Alternatively or in addition, in some embodiments, the engineered ankyrin repeat domain comprises an N- terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 121 to 131 , or 202, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 121 to 131 , or 202 are substituted by amino acids.

[0268] Alternatively or in addition, in some embodiments, the engineered ankyrin repeat domain comprises a C- terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 152 to 160, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 152 to 160 are substituted by amino acids.

[0269] Accordingly, in some embodiments, the engineered ankyrin repeat domain comprises

[0270] (1) N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 121 to 131 , or 202, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 121 to 131 , or 202 are substituted by amino acids; and / or

[0271] (2) one or more internal repeat modules, wherein each internal repeat module independently comprises an amino acid sequence of any one of SEQ ID NOs: 132 to 151 or 203 to 205, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 132 to 151 or 203 to 205 are substituted by other amino acids; and / or

[0272] (3) a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 152 to 160, or an amino acid sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 152 to 160 are substituted by amino acids.

[0273] In another aspect, provided is a recombinant binding protein comprising an engineered ankyrin repeat domain with binding specificity for human membrane-bound mesothelin, 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: 16 to 35, 196, 197, or 200. Thus, in one embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 16 to 35, 196, 197, or 200. In one embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence at least about 90% identical with any one of SEQ ID NOs: 16 to 35, 196, 197, or 200. In another embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence at least about 93% identical with any one of SEQ ID NOs: 16 to 35,

[0274] 196, 197, or 200. In a further embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence at least about 95% identical with any one of SEQ ID NOs: 16 to 35, 196, 197, or 200. In one embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence at least about 98% identical with any one of SEQ ID NOs: 16 to 35, 196, 197, or 200. In one embodiment, said engineered ankyrin repeat domain comprises an amino acid sequence with any one of SEQ ID NOs: 16 to 35, 196,

[0275] 197, or 200. In another embodiment, said engineered ankyrin repeat domain comprises or consists of an amino acid sequence with SEQ ID NO: 161 or 162.

[0276] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 16. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0277] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 17. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0278] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 18. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0279] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 19. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 19.

[0280] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 20. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0281] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 21 . In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 21 .

[0282] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 22. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0283] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 23. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 23.

[0284] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 24. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0285] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 25. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 25.

[0286] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 26. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0287] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 27. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 27.

[0288] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 28. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 28. In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 29. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0289] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 30. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0290] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 31 . In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 31 .

[0291] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 32. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0292] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 33. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 33.

[0293] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 34. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 34.

[0294] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 35. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0295] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 196. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 196.

[0296] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 197. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 197.

[0297] In one embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin 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 SEQ ID NO: 200. In one embodiment, said ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 200.

[0298] In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds to human membrane-bound mesothelin with a dissociation constant (KD) 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, or of or below about 10-11M. Thus, in one embodiment, said recombinant binding protein binds to membrane-bound mesothelin with a KD of or below about 10-7M. In another embodiment, said recombinant binding protein binds to membrane-bound mesothelin with a KD of or below about 10-8M. In another embodiment, said recombinant binding protein binds to membrane-bound mesothelin with a KD of or below about 10-9M. In another embodiment, said recombinant binding protein binds to membrane-bound mesothelin with a KD of or below about In another embodiment, said recombinant binding protein binds to membrane-bound mesothelin with a KD of or below about In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound human mesothelin also bind to cynomolgus mesothelin. In one embodiment, said recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds to cynomolgus membrane-bound mesothelin with a dissociation constant (KD) 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 or of or below about Suitably, said dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR), e.g. as described in Examples 1 to 4.

[0299] Accordingly, in one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds membrane-bound human mesothelin in PBS with a KD 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 or of or below about 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: 9 to 35 or 195 to 200.

[0300] In one embodiment, said recombinant binding protein binds membrane-bound human mesothelin in PBS with a KD 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 SEQ ID NO: 9.

[0301] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 10.

[0302] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 11 .

[0303] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 12.

[0304] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 13.

[0305] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 14.

[0306] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 15.

[0307] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 16. In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 17.

[0308] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 18.

[0309] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 19.

[0310] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 20.

[0311] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 21 .

[0312] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 22.

[0313] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 23.

[0314] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 24.

[0315] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 25.

[0316] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 26.

[0317] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 27.

[0318] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 28.

[0319] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 29.

[0320] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 30.

[0321] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 31 .

[0322] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 32. In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 33.

[0323] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 34.

[0324] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 35.

[0325] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 195.

[0326] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 196.

[0327] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 197.

[0328] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 198.

[0329] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 199.

[0330] In one embodiment, said recombinant binding protein binds human membrane-bound mesothelin in PBS with a KD 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 SEQ ID NO: 200.

[0331] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 of less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM.

[0332] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM. In another embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds human MSLN with an EC50 from about 0.1 nM to about 1 nM. In a further embodiment, said ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds human MSLN with an EC50 from about 0.1 nM to about 0.5 nM. Suitably, binding of the ankyrin repeat domain with binding specificity for membrane-bound human mesothelin is performed on OVCAR-3 cells, e.g., as described in Example 4.

[0333] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 of less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, less than about 0.2 nM, 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: 9 to 35 or 195 to 200.

[0334] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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: 9 to 35 or 195 to 200.

[0335] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 9.

[0336] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 10. In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 11 .

[0337] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 12.

[0338] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 13.

[0339] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 14.

[0340] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 15.

[0341] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 16.

[0342] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 17.

[0343] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 18.

[0344] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 19.

[0345] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 20.

[0346] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 21 .

[0347] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 22.

[0348] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 23.

[0349] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 24.

[0350] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 25.

[0351] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 26.

[0352] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 27.

[0353] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 28.

[0354] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 29. In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 30.

[0355] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 31 .

[0356] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 32.

[0357] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 33.

[0358] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 34.

[0359] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 35.

[0360] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 195.

[0361] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 196.

[0362] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 197.

[0363] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 198.

[0364] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 199.

[0365] In one embodiment, said recombinant binding protein comprises an ankyrin repeat domain with binding specificity for membrane-bound mesothelin, wherein said recombinant binding protein binds human mesothelin with an EC50 from about 0.1 nM to about 10 nM, 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 SEQ ID NO: 200.

[0366] In another embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds to membrane-bound mesothelin as described herein, and does not substantially bind to soluble mesothelin. Accordingly, in a preferred embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin has a higher binding affinity for membrane-bound mesothelin than to soluble mesothelin. Thus, in one embodiment the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds to membrane-bound mesothelin with a Kothat is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 102times, at least 103times, at least 104times or at least 105times lower than the binding affinity of said recombinant binding protein for soluble mesothelin.

[0367] In one embodiment, provided is a recombinant binding protein comprising an ankyrin repeat domain comprising an amino acid sequence at least about 80% identical to any one of SEQ ID NO: 9 to 35 or 195 to 200 with binding specificity for membrane-bound mesothelin, wherein said ankyrin repeat domain has a melting temperature higher than about 65°C, higher than about 70°C, higher than about 72°C, higher than about 75°C, higher than about 78°C, higher than about 80°C, of about 75°C, of about 80°C, of about 82°C, of about 85°C, of between about 65°C and about 95°C, of between about 70°C and about 90°C, or of between about 72°C and about 88°C. Thus, in one embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 65°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 70°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 75°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) higher than about 80°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) of between about 65°C and about 95°C. In another embodiment, said ankyrin repeat domain has a melting temperature (Tm) of between about 72°C and about 88°C.

[0368] In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin binds to human, mouse and cynomolgus mesothelin. In such a case, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin is considered to be “cross-reactive”.

[0369] Multivalent and multiparatopic ankyrin repeat domains with binding specificity for human mesothelin

[0370] Recombinant binding proteins which are multiparatopic, i.e., containing recombinant binding domains recognizing two, three, four or more epitopes on the same target protein, suitably human mesothelin are also provided. Accordingly, in a further aspect, provided is a recombinant biparatopic binding protein with binding specificity for mesothelin, wherein said recombinant binding protein comprises at least one ankyrin repeat domain with binding specificity for membrane-bound mesothelin as described herein and at least one ankyrin repeat domain with binding specificity for soluble mesothelin as described herein. In another aspect, provided is a recombinant bivalent binding protein with binding specificity for mesothelin, wherein said recombinant binding protein comprises at least two ankyrin repeat domains with binding specificity for membrane-bound mesothelin as described herein. In another aspect, provided is a recombinant bivalent binding protein with binding specificity for mesothelin, wherein said recombinant binding protein comprises at least two ankyrin repeat domains with binding specificity for soluble mesothelin as described herein.

[0371] Immune Cell Engaging Moieties

[0372] Binding proteins specific for mesothelin may be useful as a component of a bi- or multi-specific binding molecule that is directed against disease-associated cells. Binding proteins specific for mesothelin may be used in a bi- or multi-specific binding protein further comprising at least one binding moiety specific for immune cells, e.g. CD3. Such a bi- or multi-specific binding protein may recruit immune cells, such as T cells, to the cancer cells.

[0373] In a further aspect, provided is a recombinant binding protein with binding specificity for mesothelin as described herein, further comprising at least one binding moiety with binding specificity for a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell). Suitably, said protein expressed on the surface of an immune cell, e.g., a T cell, is an activating receptor of the immune cell. Examples of such activating receptors of immune cells include CD3. Thus, in a particular embodiment, provided is a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for mesothelin as described herein, further comprising a binding moiety with binding specificity for CD3. Suitably, said recombinant binding protein comprises a CD3-binding moiety and an ankyrin repeat domain with binding specificity for mesothelin, wherein said CD3-binding moiety is located C-terminally of said ankyrin repeat domain with binding specificity for mesothelin. In one embodiment, said recombinant binding protein comprises a CD3-binding moiety and an ankyrin repeat domain with binding specificity for mesothelin, wherein said CD3-binding moiety is located N-terminally of said ankyrin repeat domain with binding specificity for mesothelin.

[0374] In one embodiment, said binding agent with binding specificity for CD3 is an ankyrin repeat domain. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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: 36 to 40. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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 SEQ ID NO: 36. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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 SEQ ID NO: 37. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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 SEQ ID NO: 38. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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 SEQ ID NO: 39. In one embodiment, said ankyrin repeat domain with binding specificity for CD3 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 SEQ ID NO: 40.

[0375] Half-Life Extending Moieties A “half-life extending moiety” extends the serum half-life in vivo of the recombinant binding proteins described herein, compared to the same protein without the half-life extending moiety. Examples of halflife 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).

[0376] In one embodiment, the recombinant binding proteins provided herein further comprise one or more halflife extending moieties. Thus, in a particular embodiment, the recombinant binding protein comprises an ankyrin repeat domain with binding specificity for mesothelin as described herein and further comprises one or more half-life extending moieties. Preferably, said half-life extending moiety binds to human serum albumin. In one embodiment, said half-life extending moiety binding to human serum albumin is an ankyrin repeat domain.

[0377] 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: 41 to 42. 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: 41 to 42. 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 . 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: 42.

[0378] Suitably, said ankyrin repeat domain binding human serum albumin is located C-terminally of said ankyrin repeat domain with binding specificity for mesothelin. Suitably, said ankyrin repeat domain binding human serum albumin is located N-terminally of said ankyrin repeat domain with binding specificity for mesothelin. In some embodiments, the recombinant 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 recombinant binding protein provided herein. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the C-terminus of the recombinant binding protein provided herein. In some embodiments, a first serum albumin binding ankyrin repeat domain is located at the N-terminus of the recombinant binding protein provided herein, and a second serum albumin binding ankyrin repeat domain is located at the C-terminus of the recombinant binding protein provided herein.

[0379] 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 (5), IgE (E), or IgA (a). In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes or subtypes: IgGi (y1), lgG2 (y2), lgG3 (y3), lgG4 (y4), IgAi (a1), lgA2 (a2). In some embodiments, the Fc domain is the Fc domain of human IgGi.

[0380] 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., WO 2005 / 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 IgGi and comprises one or more of the following effector-null substitutions: L234A, L235A, and G237A (Eu numbering). In some embodiments, the Fc domain does not comprise the lysine located at the C-terminal position of human lgG1 (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).

[0381] Conjugates

[0382] In a further aspect, also provided are recombinant binding proteins with binding specificity for soluble or membrane-bound mesothelin fused or chemically conjugated (including both covalent and non-covalent conjugations) to a drug moiety. The drug moiety may be used for diagnostic purposes or for therapeutic purposes. Such a drug moiety can be a heterologous protein, polypeptide or peptide possessing a desired biological activity to generate fusion proteins. Such proteins may include, for example, toxins; antibiotics; proteins; or a biological response modifier such as, for example, a lymphokine. Methods for fusing or conjugating proteins, polypeptides, or peptides are known in the art.

[0383] Moreover, the recombinant binding protein can be conjugated to radionuclides, e.g., a radioactive metal ion, e.g., alpha-emitters or beta-emitters. In a more particular embodiment, the radionuclide is conjugated to the recombinant binding protein by a chelator. Appropriate chelators may be selected depending on the purpose of the application, e.g., diagnostic purpose or therapeutic purpose.

[0384] In one embodiment, recombinant binding proteins described herein are conjugated to a drug moiety that serves as a diagnostic or detectable moiety. Such conjugates can be useful for monitoring or prognosing the onset, development, progression and / or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy and / or for the diagnosis of a certain disease. Such diagnosis and detection can accomplished by coupling the recombinant binding protein described herein to detectable substances including, but not limited to, various enzymes; prosthetic groups; fluorescent materials; luminescent materials; radioactive materials and radionuclides; and positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. In a more particular embodiment, said diagnostic moiety is a fluorophore, a chromophore, an imaging agent or a radionuclide.

[0385] Nucleic acids and vectors

[0386] Also provided herein are nucleic acids encoding a recombinant binding protein or ankyrin repeat domain described herein. Such nucleic acids can encode polypeptides comprising segments or domains of the recombinant binding protein or ankyrin repeat domain described above. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each of the ankyrin repeat domain sequences described herein. The nucleic acid can comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 175 to 189, or a sequence substantially identical thereto (e.g., a sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, orwhich differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences in any one of SEQ ID NOs: 175 to 189).

[0387] In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 35 or 195 to 200. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 . In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 . In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of the ankyrin repeat binding domains or any one of the ankyrin repeat binding modules disclosed herein.

[0388] Also provided are nucleic acid molecules that derive from any one of SEQ ID NOs: 175 to 189, having been optimized for protein expression in a suitable host cell, such as a prokaryotic host cell, e.g., E.coli. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector. Accordingly, also provided is a cloning or expression vector comprising one or more nucleic acid sequences comprising any one of SEQ ID NOs: 175 to 189, wherein the vector is suitable for the recombinant production of the recombinant binding protein as described herein.

[0389] For expression of the recombinant binding protein described herein, standard techniques can be applied to transfect a host cell with the expression vector, suitably comprising the nucleic acid sequence of any one of SEQ ID NOs: 175 to 189 encoding a recombinant protein as described herein. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calciumphosphate precipitation, DEAE-dextran transfection and the like.

[0390] In one embodiment, a cloning or expression vector as described herein comprises the nucleic acid sequence of any one of SEQ ID NOs: 175 to 189, operatively linked to suitable promoter sequences.

[0391] Also provided is a vector comprising the nucleic acid encoding any of the recombinant binding proteins described herein, wherein said vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0392] When recombinant expression vectors encoding the recombinant binding proteins disclosed herein are introduced into suitable host cells, e.g., prokaryotic host cells, the recombinant binding proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the recombinant binding protein in the host cells or secretion of the recombinant binding proteins into the culture medium in which the host cells are grown. Recombinant binding proteins can be recovered from the host cells and / or the culture medium using standard protein purification methods. In one embodiment, the method comprises culturing a suitable host cell, e.g., a prokaryotic host cell as described herein and collecting the recombinant binding protein from the host cells and / or the culture medium.

[0393] Compositions, Uses and Methods of Treatment

[0394] In one aspect, also provided is a pharmaceutical composition comprising a recombinant binding protein as described herein as described herein, and / or a nucleic acid encoding a recombinant binding protein as described herein, and a pharmaceutically acceptable carrier, stabilizer and / or diluent. Pharmaceutically acceptable carriers, excipients, and stabilizers, are, for example, described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed., 1980.

[0395] Accordingly, in one aspect, provided is a pharmaceutical composition comprising a recombinant binding protein comprising an ankyrin repeat domain with binding specificity for mesothelin, wherein said ankyrin repeat domain comprises an amino acid sequence at least 80% identical with any one of SEQ ID NOs: 1 to 35 or 195 to 200, and a pharmaceutically acceptable carrier, stabilizer and / or diluent. Pharmaceutically acceptable carriers and / or diluents are known to the person skilled in the art and are explained in more detail below.

[0396] Suitable carriers, diluents, excipients or stabilizers include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. A pharmaceutically acceptable excipient typically has no significant adverse effect on the patient receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. A pharmaceutical composition may also be a combination formulation, comprising an additional active agent, such as an anticancer agent or an anti-angiogenic agent, or an additional bioactive compound. The compositions to be used for in vivo administration must be aseptic or sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0397] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein and a detergent, such as non-ionic detergent, e.g. Tween-20, a buffer, such as phosphate buffer, and a sugar, such as sucrose. In one aspect, such a pharmaceutical composition comprises a recombinant binding protein as described herein and PBS.

[0398] The recombinant binding proteins with binding specificity for mesothelin described herein have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of disorders with mesothelin-dependent pathophysiology. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders with mesothelin-dependent pathophysiology. Accordingly, in one aspect provided herein are methods of treating a medical condition, e.g., a disease, e.g., cancer, in a subject in need thereof, comprising administering a therapeutically effective amount of the recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell.

[0399] In another aspect, a method of treating a subject, e.g., reducing or ameliorating, a hyperproliferative condition or disorder (e.g., a cancer), e.g., solid tumor, a hematological cancer, soft tissue tumor, or a metastatic lesion, is provided. Accordingly, in one embodiment, provided is a method of inhibiting the growth of tumor cells, comprising administering to a subject in need thereof a therapeutically effective amount of the recombinant binding proteins with binding specificity for mesothelin as described herein alone or in combination with other agents, e.g., therapeutic agents, or therapeutic modalities. In some embodiments, such methods can further comprise a step of assaying expression levels of mesothelin, suitably soluble mesothelin, in a sample obtained from a subject, e.g., tissue biopsy of the subject.

[0400] Also provided is a recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell, for use in the treatment of a medical condition, e.g., a disease, e.g., cancer.

[0401] In one aspect, provided is the use of recombinant binding protein described herein, a nucleic acid encoding such a recombinant binding protein, a vector comprising a nucleic acid encoding such a recombinant binding protein, a cell comprising such a nucleic acid or vector, or a pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell, in the manufacture of a medicament, e.g., for treatment of a cancer. In one embodiment, the recombinant binding protein with binding specificity for mesothelin as described herein further comprises an immune-cell activating moiety, suitably a CD3-binding ankyrin repeat domain as described herein, a nucleic acid encoding said recombinant binding protein or a pharmaceutical composition comprising said recombinant binding protein. In one embodiment, said activation is tumor- localized. In one embodiment, said immune cells are adaptive immune cells. In one embodiment, said adaptive immune cells are T cells.

[0402] Administration may include topical administration, oral administration, or parenteral administration. Atypical route of administration is parenteral administration. In parental administration, the pharmaceutical composition is formulated in a unit dosage injectable form such as a solution, suspension, or emulsion, in association with suitable pharmaceutically acceptable excipients, e.g., as described above. The dosage and mode of administration depends on the individual to be treated and the disease.

[0403] The recombinant binding protein with binding specificity for mesothelin described herein, the nucleic acid encoding such a recombinant binding protein, or the vector or cell comprising a nucleic acid encoding such a recombinant binding protein, or the pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector, or cell, can be administered to the subject parenterally, e.g., through intravenous, intratumoral or subcutaneous route. In some embodiments, such a recombinant binding protein, nucleic acid, vector, cell, or pharmaceutical composition, is administered intravenously.

[0404] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0405] A “medical condition” may be one that is characterized by inappropriate cell proliferation. A medical condition may be a hyperproliferative condition. In one embodiment said medical condition is a neoplastic disease. The term "neoplastic disease", as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly proliferating cell growth or neoplasm. In one embodiment said medical condition is a malignant neoplastic disease. Suitably, said medical condition or disease is a cancer. In a preferred embodiment, the cancer is a cancer which expresses mesothelin. In one embodiment, said cancer is ovarian cancer, fallopian tube cancer, peritoneal cancer, mesothelioma or pancreatic ductal adenocarcinoma (PDAC). In one embodiment, said cancer is ovarian cancer. In one embodiment, said cancer is mesothelioma. In one embodiment, said cancer is pancreatic ductal adenocarcinoma (PDAC). The recombinant binding protein described herein, nucleic acid encoding such a recombinant binding protein, vector comprising a nucleic acid encoding such a recombinant binding protein, cell comprising such a nucleic acid or vector, or pharmaceutical composition comprising such a recombinant binding protein, nucleic acid, vector or cell may also be used in combination with one or more other therapies known in the art. The term “use in combination with”, as used herein, refers to a co-administration, which is carried out under a given regimen. This includes synchronous administration of the different compounds as well as time-shifted administration of the different compounds (e.g., compound A is given once and compound B is given several times thereafter, or vice versa, or both compounds are given synchronously and one of the two is also given at later stages).

[0406] In one aspect, also provided is a kit comprising the recombinant binding protein comprising an ankyrin repeat domain with binding specificity for mesothelin described herein. In one aspect, provided is a kit comprising a nucleic acid encoding the recombinant binding protein disclosed herein. In one aspect, provided is a kit comprising the pharmaceutical composition described herein. In one aspect, provided is a kit comprising the recombinant protein described herein, and / or the nucleic acid described herein, and / or the pharmaceutical composition described herein. In one aspect, provided is a kit comprising the recombinant protein comprising an ankyrin repeat domain with binding specificity for mesothelin, for example a recombinant binding protein comprising an ankyrin repeat domain comprising the amino acid of any one of SEQ ID NOs: 1 to 35 or 195 to 200, and / or a nucleic acid encoding the recombinant protein comprising an ankyrin repeat domain with binding specificity for mesothelin, for example a recombinant binding protein comprising an ankyrin repeat domain comprising the amino acid of any one of SEQ ID NOs: 1 to 35 or 195 to 200, and / or a pharmaceutical composition comprising the recombinant protein comprising an ankyrin repeat domain with binding specificity for mesothelin, for example a recombinant binding protein comprising an ankyrin repeat domain comprising the amino acid of any one of SEQ ID NOs: 1 to 35 or 195 to 200. In one aspect, provided is a kit comprising a recombinant protein as described herein, for example a recombinant binding protein comprising an ankyrin repeat domain comprising the amino acid sequences of any one of SEQ ID NOs: 1 to 35 or 195 to 200, and / or a nucleic acid encoding said recombinant protein, and / or a pharmaceutical composition comprising said recombinant protein.

[0407] DEFINITIONS

[0408] Selected terms are defined below and throughout the specification. It is understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art in the technical field of the invention.

[0409] All publications, patents, and accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0410] The use of any and all examples, or exemplary language (e g., "such as") provided herein, is intended merely to better illustrate the disclosure and does not pose a limitation on the scope unless otherwise claimed. As used herein, the articles "a" and "an” can mean “one”, but their use is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0411] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or", unless context clearly indicates otherwise and is to be interpreted as an inclusive “or” meaning any one or any combination.

[0412] Throughout this specification and the claims which follow, and unless the context requires otherwise, the terms “comprising”, “having” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments of the invention.

[0413] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 10% of a given value or range of values. For example, where a dosage is mentioned as “about” a particular value, it is intended to include a range around the specified value of plus or minus 10%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”.

[0414] It is also to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

[0415] As used herein, the term “polypeptide” refers to a molecule comprising a chain of multiple amino acids linked via peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked via peptide bonds. Furthermore, the term also encompasses peptides modified by, e.g., glycosylation, and proteins comprising two or more polypeptide chains, cross-linked by, e.g., disulfide bonds.

[0416] As used herein, the term "protein" refers to a molecule comprising a polypeptide, wherein at least part of the polypeptide has, or is able to acquire, a defined three-dimensional arrangement by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be linked non-covalently or covalently, e.g., by a disulfide bond between two polypeptides. A part of a protein, which individually has, or is able to acquire, a defined three-dimensional arrangement by forming secondary and / or tertiary structure, is termed "protein domain”.

[0417] The term “recombinant” as used in “recombinant protein”, “recombinant polypeptide” and the like, means that said protein or polypeptide is produced by the use of recombinant DNA technologies known to the practitioner skilled in the art. For example, a “recombinant DNA molecule” (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or a DNA enabling in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., Escherichia coli), these bacteria can produce the polypeptide(s) encoded by this recombinant DNA. The correspondingly produced polypeptide or protein is called a “recombinant polypeptide” or “recombinant protein”.

[0418] As used herein, the term "binding protein" refers to a protein comprising at least one binding domain. A binding protein may also comprise two, three, four, five or more binding domains. Preferably, said binding protein is a recombinant binding protein. Recombinant binding proteins of the present disclosure comprise an ankyrin repeat domain with binding specificity for human mesothelin, e.g. membrane-bound mesothelin or soluble mesothelin. Furthermore, any such binding protein may comprise additional polypeptides (such as e.g., polypeptide tags, peptide linkers, fusion to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (such as coupling to polyethylene-glycol, toxins (e.g., DM1), small molecules, antibiotics and alike) known to the person skilled in the art.

[0419] The term “binding domain” refers to a protein domain with binding specificity for a target. Preferably, said binding domain is a recombinant binding domain.

[0420] As used herein, the term "target" refers to an individual molecule such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, orto complexes of two or more of such molecules, orto a whole cell or a tissue sample, or to any non-natural compound. Preferably, a target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing chemical modifications, for example, naturally occurring or non-natural phosphorylation, acetylation, or methylation. In the context of the present disclosure, mesothelin protein expressed on tumor cells is a target of mesothelin-specific binding proteins. Preferably, the target is human mesothelin.

[0421] Mesothelin may be derived from a number of different isoforms of the mesothelin precursor proteins. The recombinant binding proteins described herein may bind to mesothelin derived from any precursor isoform. The MSLN gene encodes a 622-residue (69 kDa) precursor protein recognized as pre-pro mesothelin (e.g. the major isoform referenced in UniProt ID Q13421-3; SEQ ID NO: 167). Upon insertion into the cell membrane, the N-terminal signal peptide, comprising residues 1-33 of SEQ ID NO: 167, undergoes excision. Truncation of C-terminal residues 599-622 prompts the addition of a GPI anchor at S598. Cleavage of the precursor protein generates a GPI-anchored 303 amino acid residue (40 kDa) fragment termed “membrane-bound mesothelin”, alongside a 31 kDa secreted fragment, also referred to as megakaryocyte-potentiating factor (MPF). Mesothelin can undergo membrane shedding through proteolytic cleavage at one of several cleavage sites at the membrane-proximal region of mesothelin, resulting in truncated mesothelin protein that remains on the cell surface as well as soluble mesothelin. As used herein, the term “soluble mesothelin” refers to amino acid sequences 296-580 of human mesothelin, while “membrane-bound mesothelin” refers to amino acid sequences 296-597, both with reference to UniProt ID Q13421-3.

[0422] 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 CD35 (delta) chain, and two CD3E (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. Ankyrin repeat domains specifically binding CD3 are disclosed in WO 2022 / 129428 (incorporated by reference).

[0423] Patent application WO 2002 / 020565 and Forrer et al., (FEBS Letters 539, 2-6, 2003), contain a general description of repeat protein features and repeat domain features, techniques and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, said repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers. The repeat domains can be binding domains.

[0424] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein said repeat modules have structural and sequence homology. Preferably, a repeat domain further comprises an N-terminal and / or a C-terminal capping module. For clarity, a capping module can be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are known to the practitioner in the art from examples of ankyrin repeat domains (WO 2002 / 020565), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, E.R., et al., Structure 11 (5), 497-508, 2003), and armadillo repeat domains (WO 2009 / 040338). It is further known to the practitioner in the art that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).

[0425] The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units. Ankyrin repeat domains may be modularly assembled into larger ankyrin repeat proteins, optionally with half-life extension domains, using standard recombinant DNA technologies (see, e.g., Forrer, P„ et al., FEBS letters 539, 2-6, 2003); WO 2002 / 020565, WO 2016 / 156596; WO 2018 / 054971).

[0426] The term “construct” as used herein refers to a recombinant binding protein comprising one or more designed ankyrin repeat domains and optionally a peptide linker and / or tag sequence. An example of a peptide linker is provided in SEQ ID NO: 164 and an example of a tag sequence is provided in SEQ ID NO: 163. For clarity, the term “ankyrin repeat domain” includes at least one N-terminal and at least one C- terminal capping modules.

[0427] The term "designed" as used in “designed repeat protein”, “designed repeat domain” and the like refers to the property that such repeat proteins and repeat domains, respectively, are man-made and do not occur in nature.

[0428] The term "target interaction residue(s)" refers to amino acid residue(s) of a repeat module, which contribute to the direct interaction with a target. The term "framework residue(s)" refers to amino acid residue(s) of a repeat module, which contribute to the folding topology, i.e. which contribute to the fold of said repeat module or which contribute to the interaction with a neighboring module. Such contribution may be the interaction with other residues in the repeat module, or the influence on the polypeptide backbone conformation as found in a-helices or p-sheets, or the participation in amino acid stretches forming linear polypeptides or loops. Such framework and target interaction residues may be identified by analysis of the structural data obtained by physicochemical methods, such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known and related structural information.

[0429] The term "repeat modules" refers to the repeated amino acid sequence and structural units of the designed repeat domains, which are originally derived from the repeat units of naturally occurring repeat proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, preferably the family of ankyrin repeat proteins. Furthermore, each repeat module comprised in a repeat domain may comprise a “repeat sequence motif’ deduced from homologous repeat modules obtained from repeat domains selected on a target and having the same target specificity. A “repeat module” as used herein encompasses internal repeat modules and capping modules such as N-terminal and C-terminal capping modules. An “internal repeat module” refers to a repeat module that is flanked by two repeat modules. In other words, an internal repeat module is N- terminally flanked by one repeat module and C-terminally flanked by another repeat module. Examples of internal repeat modules are provided with SEQ ID NOs: 43 to 78, 101 to 112, 132 to 151 , and 203 to 205. Examples of N-terminal capping modules are provided with SEQ ID NOs: 79 to 90, 113 to 115, 121 to 131 , and 201 to 202. Examples of C-terminal capping modules are provided with SEQ ID NOs: 91 to 100, 116 to 120, and 152 to 160.

[0430] Accordingly, the term "ankyrin repeat module" refers to a repeat module, which is originally derived from the repeat units of occurring ankyrin repeat proteins. Ankyrin repeat proteins are known to the person skilled in the art.

[0431] The term "repeat sequence motif refers to an amino acid sequence, which is deduced from one or more repeat modules. Preferably, said repeat modules are from repeat domains with binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. Said framework residue positions correspond to the positions of framework residues of the repeat modules. Likewise, said target interaction residue positions correspond to the positions of target interaction residues of the repeat modules. Repeat sequence motifs comprise non-randomized positions and randomized positions.

[0432] The term “repeat unit” refers to amino acid sequences comprising sequence motifs of one or more naturally occurring proteins, wherein said "repeat units" are found in multiple copies, and exhibit a defined folding topology common to all said motifs determining the fold of the protein. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units. Repeat modules may comprise positions with amino acid residues which have not been randomized in a library for the purpose of selecting target-specific repeat domains ("non-randomized positions") and positions with amino acid residues which have been randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). The non-randomized positions comprise framework residues. The randomized positions comprise target interaction residues. “Have been randomized” means that two or more amino acids were allowed at an amino acid position of a repeat module, for example, wherein any of the usual twenty naturally occurring amino acids were allowed, or wherein most of the twenty naturally occurring amino acids were allowed, such as amino acids other than cysteine, or amino acids otherthan glycine, cysteine and proline. Forthe purpose of the present disclosure, amino acid residues 3, 4, 6, 11 , 14 and 15 of SEQ ID NOs: 43 to 78, 101 to 112, 132 to 151 , and 203 to 205 are randomized positions of the internal repeat modules; amino acid residues 4, 8, 11 and 12 of SEQ ID NOs: 79 to 90, 113 to 115, 121 to 131 , and 201 to 202 are randomized positions of the N-terminal capping repeat modules; and amino acid residues 3, 4, 6, 14 and 15 of SEQ ID NOs: 91 to 100, 116 to 120, and 152 to 160 are randomized positions of the C-terminal capping repeat modules. Amino acid residues 1 to 3, 5 to 7, 9, 10, 13 to 30 of SEQ ID NOs: 79 to 90, 113 to 115, 121 to 131 , and 201 to 202 are framework residues comprised in the N-terminal capping repeat modules, amino acid residues 1 , 2, 5, 7 to 10, 12, 13, 16 to 33 of SEQ ID NOs: 43 to 78, 101 to 112, 132 to 151 , and 203 to 205 are framework residues comprised in the internal repeat modules, and amino acid residues of 1 , 2, 5, 7 to 13, 16 to 28 of SEQ ID NOs: 91 to 100, 116 to 120, and 152 to 160 are framework residues comprised in the C-capping repeat modules. For the purposes of the present disclosure, a substitution in framework positions shall apply to all embodiments irrespective of whether such substitution is explicitly described.

[0433] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0434] Exemplary and conservative amino acid substitutions are shown in Table 1.

[0435] Table 1 : Exemplary and conservative Amino Acid Substitutions

[0436] Table 2 shows typical properties of common amino acids as considered in the present specification.

[0437] Table 2: Properties of common amino acids.

[0438] Categories of charge-modifying amino acid residue substitutions considered in this specification and their impact on charge are shown in Table 3.

[0439] Table 3: Amino acid residue substitutions and their impact on charge

[0440] The term "isoelectric point" or “pl” refers to the pH value at which a macromolecule such as a protein carries no net electrical charge. In proteins there may be many charged groups, and at the isoelectric point the sum of all these charges is zero. At a pH above the isoelectric point the overall net charge of the polypeptide will be negative, whereas at pH values below the isoelectric point the overall net charge of the polypeptide will be positive. Isoelectric points can be determined experimentally or can be calculated for polypeptides based on the primary sequence. The skilled person is aware of methods to determine the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is computed based on the amino acid sequence of the protein. Numerous tools are available online that allow computing the isoelectric point of a protein. One such preferred tool is “ExPASy Compute pl / Mw” (https: / / web.expasy.org / compute_pi / ); see Protein Identification and Analysis Tools on the ExPASy Server; Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607. This “ExPASy Compute pl / Mw” tool is preferably used for the determination of the pl of engineered ankyrin repeat domain disclosed herein. Any N-terminal or C- terminal tags comprising one or more amino acids which may be fused to a repeat domain for production or other purposes, as well as any N-terminal or C-terminal peptide linkers are not considered for computing the pl.

[0441] In addition, (a) the second last position of any C-capping module described herein can be “A” or“L”, and / or the last position can be “A” or “N” and / or (b) the third last position can be a “Q”. Furthermore, each ankyrin repeat domain described herein may optionally comprise a “G,” an “S,” or a “GS” sequence at its N- terminus.

[0442] For determining the percent identity between two sequences (e.g., polynucleotide or polypeptide) the sequences are aligned for optimal comparison. A position in the first sequence is considered identical to the corresponding position in the second sequence when they have the same nucleotide or amino acid. The percent identity is calculated by dividing the number of identical positions by the total number of positions in the reference sequence and multiplying by 100. To assess similarity, the alignment is typically performed over the length of the reference sequence. For example, to determine if a test sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), the alignment is carried out against SEQ ID NO: 1 , and the number of identical positions is compared. If at least 80% of the positions are identical, the test sequence is considered at least 80% identical to SEQ ID NO: 1. Gaps or missing positions in a shorter sequence are considered non-identical positions. Various computer programs are available to determine sequence homology. The Needleman and Wunsch algorithm can be used with specific parameters such as using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6 to calculate percent identity between amino acid or nucleic acid sequences. One example of suitable parameters is a Blosum 62 scoring matrix, a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0443] Individual binding domains may be linked covalently with a peptide linker. A suitable linker may comprise from 1 to 50 amino acids, suitably from 6 to 38 amino acids. A peptide linker may be a proline-threonine (PT) rich peptide linker. An example of a suitable PT-rich linker in the context of the present disclosure comprises the amino acid sequence of SEQ ID NO: 164. Another example of a suitable linker in the context of the present disclosure comprises the amino acid sequence of SEQ ID NO: 206.

[0444] The terms “binding specificity”, “has binding specificity for a target”, “specifically binding to a target”, “binding to a target with high specificity”, “specific for a target”, “target specificity”, or “specifically binds” and the like means that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e. it binds with higher affinity) than it binds to an unrelated protein such as the E. coli maltose binding protein (MBP). Preferably, the dissociation constant (“KD”) for the target is at least 102; more preferably, at least 103; more preferably, at least 104; or more preferably, at least 105times lower than the corresponding dissociation constant for MBP. Methods to determine dissociation constants of protein-protein interactions, such as surface plasmon resonance (SPR) based technologies (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC) are known to the person skilled in the art. The measured KD values of a particular protein-protein interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of KD values are preferably made with standardized solutions of protein and a standardized buffer, such as PBS. The term “PBS” means a phosphate buffered water solution comprising 137 mM NaCI, 10 mM phosphate and 2.7 mM KCI and having a pH of 7.4.

[0445] Binding of any molecule to another is governed by two forces, namely the association rate (kon) and the dissociation rate (kOff). The affinity of any binder [B] to a target [T] can then be expressed by the equilibrium dissociation constant KD, which is the quotient of koir / kon. kon is a second-order rate constant of the binding reaction, with the unit whereas the dissociation reaction kotr is a first-order rate constant with the unit s~1. From this it becomes clear that the association reaction depends on the concentration of the reactants, whereas the dissociation is independent of the concentration, following a simple exponential decay function.

[0446] A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. For example, as exemplified herein, the binding affinity of a particular binding moiety to a drug molecule target can be expressed as KD value, which refers to the dissociation constant of the binding moiety and the drug molecule target. KD is the ratio of the rate of dissociation, also called the “off-rate (kotr)”, to the association rate, or “on-rate (kon)”. Thus, KD equals kotr / kon and is expressed as a molar concentration (M), and the smaller the KD, the stronger the affinity of binding.

[0447] KD values can be determined using any suitable method. One exemplary method for measuring KD is surface plasmon resonance (SPR) (see, e.g., Nguyen et al. Sensors (Basel). 2015 May 5; 15(5):10481- 510). KD value ...

Claims

CLAIMS1. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for membrane-bound mesothelin (mbMSLN).

2. The recombinant binding protein of claim 1 , wherein said ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid(s) in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids.

3. The recombinant binding protein of any one of claims 1 or 2, wherein said ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module, and optionally a third ankyrin repeat module, optionally wherein said first ankyrin repeat module, said second ankyrin repeat module, and said optional third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NOs: 62 to 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 62 to 78 are substituted with other amino acids.

4. The recombinant binding protein of any one of the preceding claims, wherein i. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 65, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids; ii. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids;Hi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 69 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 70 are substituted with other amino acids;iv. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 71 , or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids and a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids; v. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 62 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 63, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 63 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 64 are substituted with other amino acids; vi. said ankyrin repeat domain comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 73 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 75 are substituted with other amino acids; or vii. comprises a first ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 76 are substituted with other amino acids, a second ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 77 are substituted with other amino acids, and a third ankyrin repeat module comprising an amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids.

5. The recombinant binding protein of any one of the preceding claims, further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 79, 87 to 90, or 201 , or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 79, 87 to 90, or 201 are substituted by other amino acids; and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NOs: 91 , 98 to100, or an amino acid sequence wherein to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acids in any one of SEQ ID NOs: 91 , 98 to 100 are substituted by other amino acids.

6. The recombinant binding protein of any one of the preceding claims, 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 NO: 9 to 35 or 195 to 200.

7. The recombinant binding protein of any one of the preceding claims, wherein said recombinant binding protein binds human membrane-bound mesothelin in PBS with a dissociation constant (KD) 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, or of or below about 10-11M and / or wherein said recombinant binding protein binds human mesothelin with an EC50 of about 0.1 to about 10 nM.

8. The recombinant binding protein of any one of the preceding claims, further comprising at least one binding moiety with binding specificity for a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell), optionally wherein said binding moiety comprises a ankyrin repeat domain with binding specificity for CD3, optionally 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: 36 to 40.

9. The recombinant binding protein of any one of the preceding claims, further comprising at least one halflife extending moiety, optionally wherein said half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin, optionally comprising an amino acid sequence 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: 41 to 42.

10. The recombinant binding protein of any one of the preceding claims, further comprising a drug moiety, optionally wherein said drug moiety is a radionuclide or a cytotoxin.11 . A nucleic acid encoding the recombinant binding protein of any one of the preceding claims.

12. A vector comprising the nucleic acid of claim 11 , optionally wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

13. A cell comprising the nucleic acid of claim 11 or the vector of claim 12.

14. A method of producing a recombinant binding protein, the method comprising culturing the cell of claim 13 and collecting the recombinant binding protein from the cell and / or the culture medium.

15. A pharmaceutical composition comprising the recombinant binding protein of any one of claims 1 to 10, the nucleic acid of claim 11 , the vector of claim 12, or the cell of claim 13, and a pharmaceutically acceptable carrier and / or diluent.

16. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant binding protein of any one of claims 1 to 10, the nucleic acid of claim 11 , the vector of claim 12, the cell of claim 13, or the pharmaceutical composition of claim 15.