Recombinant mesothelin-binding proteins and uses thereof

Abstract

The present invention relates to recombinant binding proteins comprising ankyrin repeat domains with binding specificity for soluble mesothelin or membrane-bound mesothelin. Furthermore, the present 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 mammals, including humans.

Description

Technical Field

[0001] This invention relates to recombinant binding proteins comprising a designed ankyrin repeating domain that is specific for binding to mesothelin. Furthermore, this 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 subjects of need. Background Technology

[0002] Mesothelin (MSLN) is primarily expressed in the mesothelial lining of specific types of epithelial cells, such as the pleura, pericardium, and peritoneum, but it is significantly upregulated in several solid tumors, including mesothelioma, ovarian cancer, pancreatic adenocarcinoma, and some gastric and lung cancers. Abnormal MSLN expression on tumor cells plays a role in tumor progression, metastasis, drug resistance, and immune evasion through interaction with MUC16 / CA125, 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). In clinical observations, elevated MSLN expression was associated with increased tumor burden and poorer overall survival (Kachala SS et al., Clin Cancer Res. 2014;20(4):1020–8; Tozbikian G et al., PLoS One.2014; 9(12):e114900.).

[0003] The MSLN gene encoding mesothelin encodes a 622-residue (69 kDa) precursor protein called pre-pro mesothelin. After insertion into the cell membrane, the N-terminal signal peptide is cleaved, followed by truncation of several C-terminal residues, allowing for the addition of a GPI anchor. The proteolytic processing site is recognized by furin and other proteases, promoting the cleavage of the precursor protein. This proteolytic cleavage produces a 303-amino acid residue (40 kDa) GPI-anchored fragment (“membrane-bound mesothelin”) and a 31 kDa secreted fragment, also known as “megakaryocytic enhancer factor” (MPF). Similar to other GPI-anchored proteins, mesothelin detaches in a protease-rich extracellular environment. At several cleavage sites on the membrane anchor near the mesothelin-expressing cell, alternative splicing by various enzymes and protease cleavage lead to the production of soluble mesothelin (sMSLN) and soluble mesothelin-related peptide (SMRP). Soluble forms of mesothelin, also known as exfoliated mesothelin, have gained particular attention as biomarkers for cancer detection and monitoring. Elevated levels of soluble MSLN in blood or other bodily fluids are 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 diagnosis, providing a potential pathway to improve patient outcomes through early intervention. Beyond its utility as a diagnostic biomarker, mesothelin expression is also associated with prognosis in cancer patients. In many cases, higher levels of mesothelin expression are associated with more aggressive disease and poorer clinical outcomes.

[0004] Mesothelin's unique properties, particularly its restricted expression in cancer cells and limited presence in normal tissues, make it an attractive target for cancer therapy. Various approaches have been explored to leverage mesothelin's potential 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. On the other hand, targeting membrane-bound mesothelin, in particular, is not suitable for all therapeutic approaches. Therefore, antibody-based approaches that bind mesothelin by targeting epitopes near the membrane may be beneficial. In conclusion, given the role of mesothelin in diagnostic and therapeutic treatments, there is a need for novel mesothelin-specific binding proteins, including binding proteins specific for membrane-bound mesothelin but not for soluble mesothelin, as well as binding proteins specific for soluble mesothelin. Summary of the Invention

[0005] Designed ankyrin repeat proteins (DARPin) are uniquely suited as cancer therapeutic agents because their small size, specificity, and high affinity can lead to high tumor uptake and tumor retention. Current approaches that rely on activating the immune system to target tumors can be circumvented using radioligand therapy. Furthermore, the systemic clearance of DARPin can be fine-tuned. DARPin is also readily linked and surface-engineered, making it an ideal molecule for radiotherapy and radiodiagnostic purposes. Such binding proteins can be used in therapeutic and diagnostic approaches to treat and characterize diseases, including cancer. This disclosure relates to recombinant binding proteins comprising ankyrin repeat domains with binding specificity to mesothelin (MSLN), such as those expressed by tumor cells. More specifically, this disclosure provides recombinant binding proteins comprising ankyrin repeat domains with binding specificity to membrane-bound mesothelin (mbMSLN). This disclosure also relates to recombinant binding proteins comprising ankyrin repeat domains with binding specificity to soluble mesothelin (sMSLN). Such binding proteins can serve as tools or building blocks for generating novel therapeutics or diagnostics with enhanced tumor targeting and / or reduced off-target effects to improve therapeutic efficacy. This paper also discloses recombinant binding proteins in which an MSLN-specific ankyrin repeat domain is combined with one or more other functional motifs within a molecule. These other functional motifs include half-life extensions and / or binding portions, such as ankyrin repeat domains with binding specificity to targets present on immune cells.

[0006] Based on the disclosure provided herein, those skilled in the art will recognize, or will be able to determine, many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the following embodiments (E).

[0007] Specifically, this disclosure provides, individually or in combination, the following aspects, advantageous features, and specific implementation methods:

[0008] E1. A recombinant binding protein comprising an ankyrin repeat domain that is specific for binding to mesothelin (MSLN).

[0009] E2. A recombinant binding protein comprising an ankyrin repeat domain that is specific for binding to soluble mesothelin (sMSLN).

[0010] E3. The recombinant binding protein according to E1 or E2, wherein the ankyrin repeat domain comprises an ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which 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 in any one of SEQ ID NO: 43 to 61 is replaced by other amino acids.

[0011] E4. The recombinant binding protein according to any one of E1 to E3, wherein the ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module.

[0012] E5. The recombinant binding protein according to E4, wherein the first ankyrin repeat module and the second ankyrin repeat module each independently comprise an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which 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 in any one of SEQ ID NO: 43 to 61 are replaced by other amino acids.

[0013] E6. The recombinant binding protein according to any one of E1 to E5, wherein

[0014] i. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence in which 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 of SEQ ID NO: 46 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence in which 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 of SEQ ID NO: 47 is replaced by other amino acids;

[0015] ii. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence in which 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 of SEQ ID NO: 48 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence in which 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 of SEQ ID NO: 49 is replaced by other amino acids;

[0016] iii. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence in which 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 of SEQ ID NO: 50 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence in which 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 of SEQ ID NO: 51 is replaced by other amino acids;

[0017] iv. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 58 are substituted by other amino acids; and a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 59 are substituted by other amino acids; or

[0018] v. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence in which 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 of SEQ ID NO: 60 is replaced by other amino acids; and a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence in which 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 of SEQ ID NO: 61 is replaced by other amino acids.

[0019] E7. The recombinant binding protein according to any one of E1 to E3, wherein the ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module, and a third ankyrin repeat module.

[0020] E8. The recombinant binding protein according to E7, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module each independently comprise an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which 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 in any one of SEQ ID NO: 43 to 61 is replaced by other amino acids.

[0021] E9. The recombinant binding protein according to any one of E1 to E3 or E7 to E8, wherein

[0022] i. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the 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 by other amino acids; a second ankyrin repeating module, the second ankyrin repeating module comprising the 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 by other amino acids; and a third ankyrin repeating module, the third ankyrin repeating module comprising the 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: 44 are substituted by other amino acids; and a third ankyrin repeating module, the third ankyrin repeating module comprising the 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 by other amino acids; 45 amino acid sequences in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids;

[0023] ii. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 52 are substituted by other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 53 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 53 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 54 ... 54, an amino acid sequence in which at most 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids; or

[0024] iii. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 55 are substituted by other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 56 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 57 are substituted by other amino acids; 57 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids.

[0025] E10. The recombinant binding protein according to any one of E3 to E9, the recombinant binding protein further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79 to 86, or an amino acid sequence in which 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 in any one of SEQ ID NO: 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 NO: 91 to 97, or an amino acid sequence in which 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 in any one of SEQ ID NO: 91 to 97 are substituted by other amino acids.

[0026] E11. The recombinant binding protein according to any one of E1 to E10, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8.

[0027] E12. The recombinant binding protein according to any one of E1 to E11, wherein the mesothelin is human mesothelin.

[0028] E13. The recombinant binding protein according to any one of E1 to E12, wherein the ankyrin repeating domain is at or below about 10 in PBS. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant (KD) of M binds to soluble human dermal linoleate.

[0029] E14. A recombinant binding protein comprising an ankyrin repeating domain that is specific for binding to membrane-bound mesothelin (mbMSLN).

[0030] E15. The recombinant binding protein according to E1 and E14, wherein the ankyrin repeat domain comprises ankyrin repeat modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which 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 in any one of SEQ ID NO: 62 to 78 are replaced by other amino acids.

[0031] E16. The recombinant binding protein according to any one of E1, E14 to E15, wherein the ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module.

[0032] E17. The recombinant binding protein according to any one of E1, E14 to E16, wherein the first ankyrin repeat module and the second ankyrin repeat module each independently comprise the amino acid sequence of any one of SEQ ID NO: 62 to 78, or wherein at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 or at most 1 amino acids of any one of SEQ ID NO: 62 to 78 are replaced by other amino acids, or wherein the first ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 101 to 105, and wherein the second ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 106 to 109.

[0033] E18. The recombinant binding protein according to any one of E1, E14 to E17, wherein

[0034] i. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence in which 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 of SEQ ID NO: 65 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence in which 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 of SEQ ID NO: 66 is replaced by other amino acids;

[0035] ii. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence in which 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 of SEQ ID NO: 67 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence in which 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 of SEQ ID NO: 68 is replaced by other amino acids;

[0036] iii. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the 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 acid of SEQ ID NO: 69 is replaced by other amino acids; and a second ankyrin repeating module, the second ankyrin repeating module comprising the 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 acid of SEQ ID NO: 70 is replaced by other amino acids; or

[0037] iv. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 71 are substituted by other amino acids; and a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 72 are substituted by other amino acids; or

[0038] v. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence in which 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 of SEQ ID NO: 67 is replaced by other amino acids; and a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence in which 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 of SEQ ID NO: 74 is replaced by other amino acids.

[0039] E19. The recombinant binding protein according to any one of E1, E14 to E15, wherein the ankyrin repeat domain comprises a first ankyrin repeat module, a second ankyrin repeat module and a third ankyrin repeat module.

[0040] E20. The recombinant binding protein according to E19, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module each independently comprise the amino acid sequence of any one of SEQ ID NO: 62 to 78, or wherein any one of SEQ ID NO: 62 to 78 comprises an amino acid sequence in which 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 is replaced by other amino acids, or wherein the first ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 101 to 105, the second ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 106 to 109, and the third ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 110 to 112.

[0041] E21. The recombinant binding protein according to any one of E1, E14 to E15, or E19 to E20, wherein...

[0042] i. The ankyrin repeating domain comprises a first ankyrin repeating module, the first ankyrin repeating module comprising the 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 by other amino acids; a second ankyrin repeating module, the second ankyrin repeating module comprising the 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 by other amino acids; and a third ankyrin repeating module, the third ankyrin repeating module comprising the 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 by other amino acids; 64 amino acid sequences in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids;

[0043] ii. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 73 are substituted by other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 74 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 74 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 74 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 75 ... 75 amino acid sequences in which at most 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids; or

[0044] iii. The ankyrin repeating domain comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 76 are substituted by other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 77 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 77 are substituted by other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 78 ... 78 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids.

[0045] E22. The recombinant binding protein according to any one of E15 to E21, the recombinant binding protein further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90 or 201, 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 of any one of SEQ ID NO: 79, 87 to 90 or 201 is replaced by other amino acids; and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100, 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 of any one of SEQ ID NO: 91, 98 to 100 is replaced by other amino acids.

[0046] E23. The recombinant binding protein according to any one of E1, E14 to E22, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as any one of SEQ ID NO: 9 to 15, 195, 198, or 199.

[0047] E24. The recombinant binding protein according to any one of E14 to E23, wherein the mesothelin is human mesothelin.

[0048] E25. The recombinant binding protein according to any one of E1, E14 to E24, wherein the recombinant binding protein contains at or below about 10 mg / L in PBS. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant (KD) of M binds to human dermal tincture, and / or the recombinant binding protein binds to human dermal tincture at an EC50 of about 0.1 nM to about 10 nM.

[0049] E26. The recombinant binding protein according to any one of E1, E14 to E24, wherein the ankyrin repeat domain comprises an engineered ankyrin repeat module.

[0050] E27. The recombinant binding protein according to E26, wherein the engineered ankyrin repeat module contains fewer basic amino acids and / or a lower isoelectric point (pI) compared to the non-engineered ankyrin repeat module.

[0051] E28. The recombinant binding protein according to E26 or E27, wherein the ankyrin repeating domain comprises an N-terminal capping module, the N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 121 to 131 or 202, 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 of any one of SEQ ID NO: 121 to 131 or 202 is substituted by other amino acids; at least one, at least two, or at least three internal repeating modules, each module independently comprising an amino acid sequence of any one of SEQ ID NO: 75, 132 to 151, or 203 to 205, or wherein SEQ ID NO: An amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any of SEQ ID NO: 75, 132 to 151, or 203 to 205 are replaced by other amino acids; and / or a C-terminal capping module comprising an amino acid sequence in any of SEQ ID NO: 91, 152 to 160, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any of SEQ ID NO: 91, 152 to 160 are replaced by other amino acids.

[0052] E29. The recombinant binding protein according to any one of E26 to E28, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as any one of SEQ ID NO: 16 to 35, 196, 197, or 200.

[0053] E30. The recombinant binding protein according to any one of E1 to E29, wherein the recombinant binding protein comprises at least one ankyrin repeat domain having binding specificity to soluble mesothelin according to any one of E2 to E13 and at least one ankyrin repeat domain having binding specificity to membrane-bound mesothelin according to any one of E14 to E29.

[0054] E31. The recombinant binding protein according to any one of E1 to E30, wherein the recombinant binding protein further comprises at least one binding portion having binding specificity to a protein expressed on the surface of an immune cell, suitably a T lymphocyte (T cell).

[0055] E32. The recombinant binding protein according to E31, wherein the binding moiety comprises an ankyrin repeating domain that is specific for binding to CD3.

[0056] E33. The recombinant binding protein according to E32, wherein the ankyrin repeating domain having binding specificity to CD3 comprises 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% of the same amino acid sequence as any of SEQ ID NO: 36 to 40.

[0057] E34. The recombinant binding protein according to any one of E1 to E33, wherein the recombinant binding protein further comprises at least one half-life extension portion.

[0058] E35. The recombinant binding protein according to E34, wherein the extended half-life portion comprises an ankyrin repeat domain that has binding specificity to human serum albumin.

[0059] E36. The recombinant binding protein according to E35, wherein the ankyrin repeating domain having binding specificity to human serum albumin comprises 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% of the same amino acid sequence as any of SEQ ID NO: 41 to 42.

[0060] E37. The recombinant binding protein according to any one of E1 to E36, wherein the recombinant binding protein further comprises a pharmaceutical portion.

[0061] E38. The recombinant binding protein according to E37, wherein the drug portion is a radionuclide or cytotoxin.

[0062] E39. A nucleic acid that encodes a recombinant binding protein according to any one of E1 to E38.

[0063] E40. A vector comprising the nucleic acid as described in E39.

[0064] E41. The vector according to E40, wherein the vector is a DNA vector, RNA vector, plasmid, granule or viral vector.

[0065] E42. A cell comprising a nucleic acid as described in E39 or a vector as described in any one of E40 to E41.

[0066] E43. A pharmaceutical composition comprising a recombinant binding protein according to any one of E1 to E38, a nucleic acid according to E39, a carrier according to any one of E40 to E41 or a cell according to E42, and a pharmaceutically acceptable carrier and / or diluent.

[0067] E44. A method for producing a recombinant binding protein, the method comprising culturing cells according to E42 and collecting the recombinant binding protein from the cells and / or culture medium.

[0068] E45. The recombinant binding protein according to any one of E1 to E38, the nucleic acid according to E39, the vector according to any one of E40 to E41, the cell according to E42, or the pharmaceutical composition according to E43, used as a pharmaceutical.

[0069] E46. Use in the manufacture of a medicament the recombinant binding protein according to any one of E1 to E38, the nucleic acid according to E39, the vector according to any one of E40 to E41, the cell according to E42, or the pharmaceutical composition according to E43.

[0070] E47. The recombinant binding protein according to E45, the nucleic acid according to E45, the vector according to E45, the cell according to E45, or the pharmaceutical composition according to E45, or the use according to E46, wherein the pharmaceutical is a drug for treating cancer.

[0071] E48. A method of treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of a recombinant binding protein according to any one of E1 to E38, a nucleic acid according to E39, a vector according to any one of E40 to E41, a cell according to E42, or a pharmaceutical composition according to E43.

[0072] E49. The method according to E48, wherein the treatment is a treatment for cancer.

[0073] E50. A method for detecting the presence of mesothelin-expressing cells, and suitable cancer cells, in a subject, said method comprising:

[0074] (a) Contacting a sample containing one or more cells obtained from the subject with a recombinant binding protein according to any one of E1 to E38, a nucleic acid according to E39, a vector according to any one of E40 to E41, cells according to E42, or a pharmaceutical composition according to E43, and

[0075] (b) Detect the binding of the recombinant binding protein to one or more of the cells.

[0076] E51. The method according to E50, wherein the detection of the binding of the recombinant binding protein to the one or more cells indicates the presence of cancer cells in the sample. Attached Figure Description

[0077] Figure 1 This section outlines the binding profile of recombinant binding proteins containing ankyrin repeat domains with binding specificity to membrane-bound mesothelin on OVCAR-3 cancer cells. Serial dilutions of His-tagged ankyrin repeat single domains with binding specificity to membrane-bound mesothelin were incubated with target cells, and the binding of the constructs to target cells was determined by flow cytometry using the His tag. Median fluorescence intensity (MFI) values ​​of the ankyrin repeat single domains mbMSLN DARPin #1 to mbMSLN DARPin #7 bound to membrane-bound mesothelin (mbMSLN) are shown. Median fluorescence intensity (MFI) values ​​obtained by fitting a standard nonlinear regression three-parameter logistic model are also shown.

[0078] Figure 2: Binding of mbMSLN DARPin #6 and reference antibody to biotinylated mesothelin targets. Plates were coated with biotinylated human, cynomolgus monkey, and mouse mesothelin and serially diluted with ankyrin repeat domain mbMSLN DARPin #6, which specifically binds to membrane-bound mesothelin, and reference antibody 15B06. (A) Ankyrin repeat domain detected with anti-DARPin 1.1.1 antibody. MbMSLN DARPin #6 cross-reacted with human, cynomolgus monkey, and mouse mesothelin. (B) Reference antibody 15B06 detected with goat anti-mouse IgG HRP. 15B06 cross-reacted with human and cynomolgus monkey mesothelin but not with mouse mesothelin.

[0079] Figure 3Amino acid sequence alignment of membrane-bound mesothelin from humans (residues 296-596), cynomolgus monkeys (residues 296-596), and mice (residues 298-600). C-terminal peptide epitopes are marked in boxes.

[0080] Figure 4 Renal uptake of indium-labeled DARPin. Radiolabeled DARPin was injected into wild-type mice. Data are presented as mean injection activity % / tissue mass g (% IA / g). Measurements were taken 4 hours post-injection. Error bars show standard deviation.

[0081] Figure 5: Binding of proximal MSLN peptide fragments to the membrane by ELISA. The binding of mbMSLN DARPin #2 (A), #3 (B), #4 (C), #5 (D), and #6 (E), sMSLN DARPin #1 (F), and comparative antibody 15B6 (G) to proximal MSLN peptide fragments (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 (“soluble ECD”), and residues 296-597 of SEQ ID NO: 167 (“mbECD”) was assessed by ELISA. Binding with the carrier (Fc-KiH) and a control sample containing no target or binding agent were also shown.

[0082] Figure 6: Binding competition of selected DARPins in the presence of soluble MSLN mimics. The binding of mbMSLN DARPins #6 and #2.1 and sMSLN DARPin #7 to immobilized full-length MSLN was determined in the presence of sMSLN mimics, which are generated by different protease cleavage sites (short (amino acid residues 296-580 of SEQ ID NO: 166), intermediate (amino acid residues 296-586 of SEQ ID NO: 167), long (amino acid residues 296-591 of SEQ ID NO: 167), or full-length (amino acid residues 296-597 of SEQ ID NO: 167)) within the C-terminal peptide region of MSLN.

[0083] Figure 7: Cell binding competition assay. The selected DARPin and control antibody anetumab were pre-incubated with recombinant MSLN versions (full-length (SEQ ID NO: 167 residues 296-597), long (SEQ ID NO: 167 residues 296-591), intermediate (SEQ ID NO: 167 residues 296-586), and short (SEQ ID NO: 166 residues 296-580)) and subsequently incubated with OVCAR-8 cells. Cell binding was assessed by flow cytometry. The figure shows the mean fluorescence intensity of the viable cell population.

[0084] Figure 8 The selected mbMSLN DARPin binds to hMSLN on OVCAR-3 cells. DARPin was detected using anti-DARPin antibody 1.4.8. MFI: median fluorescence intensity. Detailed Implementation

[0085] Mesothelin is an attractive therapeutic target for treating certain cancers, particularly those that overexpress mesothelin, such as ovarian cancer, mesothelioma, or pancreatic ductal adenocarcinoma. The recombinant binding protein described herein comprises an ankyrin repeat domain of at least one ankyrin repeat module that specifically binds to mesothelin. More specifically, in some embodiments, the recombinant binding protein described herein comprises an ankyrin repeat domain containing at least two or at least three ankyrin repeat modules in a soluble form that specifically bind to mesothelin. In other embodiments, the recombinant binding protein described herein comprises an ankyrin repeat domain containing at least two or at least three ankyrin repeat modules that specifically bind to membrane-bound mesothelin. Optionally, the ankyrin repeat domain containing at least two or at least three engineered ankyrin repeat modules that specifically bind to membrane-bound mesothelin does not bind soluble mesothelin.

[0086] Ankylosing spondylase domain

[0087] Designed ankyrin repeat protein libraries (WO2002 / 020565; Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701, 2008) can be used to select target-specific ankyrin repeat domains that bind to their targets with high affinity. These target-specific ankyrin repeat domains can then serve as valuable components of recombinant binding proteins for the treatment of diseases. Ankyrin repeat proteins are a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies, thereby allowing for 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; US Patent No. 9,458,211). Ankyrin repeat proteins, containing only a single designed ankyrin repeat domain, are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These characteristics, along with the possibility of combining two or more ankyrin repeat domains in a single protein, make ankyrin repeat proteins ideal candidates for agonistic, antagonistic, and / or inhibitory drugs. Furthermore, such ankyrin repeat proteins can be engineered to possess various effector functions, such as cytotoxic agents, radionuclides, half-life extenders, and / or immune cell conjugate molecules, thereby achieving novel drug formats. By simultaneously targeting antigens on tumor cells and activating receptors on immune cells, such immune cell conjugate molecules enhance the interaction between the two cell types and increase immune cell effector functions. Therefore, tumor-localized activation of immune cells can be achieved.

[0088] Ankylosing spicules with binding specificity to soluble mesothelin

[0089] In one aspect, a recombinant binding protein comprising an ankyrin repeating domain having binding specificity to soluble mesothelin is provided. Specifically, the ankyrin repeating domain comprises an ankyrin repeating module comprising the amino acid sequence of any one of SEQ ID NO: 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 from any one of SEQ ID NO: 43 to 61 is substituted with other amino acids. Thus, in one embodiment, the ankyrin repeating module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence wherein up to 9 amino acids from any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankyrin repeating module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence wherein up to 8 amino acids from any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 7 amino acids of any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 6 amino acids of any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 5 amino acids of any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 4 amino acids of any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In one embodiment, the ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to three amino acids in any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. In another embodiment, the ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to two amino acids in any one of SEQ ID NO: 43 to 61 are substituted with other amino acids.In one embodiment, the ankyrin repeat module comprises the amino acid sequence of any of SEQ ID NO: 43 to 61, or an amino acid sequence in which at most one amino acid of any of SEQ ID NO: 43 to 61 is substituted with another amino acid. In one embodiment, all of the 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions occur at a frame position of the ankyrin repeat module. In one embodiment, the ankyrin repeat domain comprises the amino acid sequence of any of SEQ ID NO: 43 to 61.

[0090] In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 43 or a sequence wherein one or two amino acids of SEQ ID NO: 43 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 44 or a sequence wherein one or two amino acids of SEQ ID NO: 44 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 45 or a sequence wherein one or two amino acids of SEQ ID NO: 45 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 46 or a sequence wherein one or two amino acids of SEQ ID NO: 46 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 47 or a sequence wherein one or two amino acids of SEQ ID NO: 47 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 48 or a sequence wherein one or two amino acids of SEQ ID NO: 48 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 49 or a sequence wherein one or two amino acids of SEQ ID NO: 49 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 50 or a sequence wherein one or two amino acids of SEQ ID NO: 50 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 51 or a sequence wherein one or two amino acids of SEQ ID NO: 51 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 52 or a sequence wherein one or two amino acids of SEQ ID NO: 52 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 53 or a sequence wherein one or two amino acids of SEQ ID NO: 53 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 54 or a sequence wherein one or two amino acids of SEQ ID NO: 54 are substituted with other amino acids.In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 55 or a sequence in which one or two amino acids of SEQ ID NO: 55 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 56 or a sequence in which one or two amino acids of SEQ ID NO: 56 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 57 or a sequence in which one or two amino acids of SEQ ID NO: 57 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 58 or a sequence in which one or two amino acids of SEQ ID NO: 58 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 59 or a sequence in which one or two amino acids of SEQ ID NO: 59 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 60 or a sequence in which one or two amino acids of SEQ ID NO: 60 are substituted with other amino acids. In one embodiment, the ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 61 or a sequence in which one or two amino acids of SEQ ID NO: 61 are substituted with other amino acids.

[0091] In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 43. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 44. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 45. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 46. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 47. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 48. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 49. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 50. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 51. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 52. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 53. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 54. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 55. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 56. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 57. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 58. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 59. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 60. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 61.

[0092] In one embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module and a second ankyrin repeating module. In one embodiment, the first ankyrin repeating module is located at the N-terminus of the second ankyrin repeating module within the ankyrin repeating domain. In one embodiment, the first ankyrin repeating module and the second ankyrin repeating module each independently comprise an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which 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 from any one of SEQ ID NO: 43 to 61 are substituted with other amino acids. Therefore, in one embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 43 to 61 is substituted with other amino acids, and further comprises a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 43 to 61 is substituted with other amino acids.

[0093] In one specific embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence in which 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 of SEQ ID NO: 46 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence in which 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 of SEQ ID NO: 47 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 46 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 47 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 46, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 47, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0094] In one specific embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 48 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 49 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 48 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 49 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 48, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 49, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0095] In one specific embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 50 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 51 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 50 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 51 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 50, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 51, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0096] In one specific embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 58 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 59 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 58 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 59 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 58, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 59, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0097] In one specific embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 60 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 61 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 60 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 61 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 60, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 61, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0098] In one embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module, a second ankyrin repeating module, and a third ankyrin repeating module. In a preferred embodiment, the first ankyrin repeating module is located at the N-terminus of the second ankyrin repeating module within the ankyrin repeating domain, and the second ankyrin repeating module is located at the N-terminus of the third ankyrin repeating module within the ankyrin repeating domain.

[0099] In one embodiment, the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module each independently comprise an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which 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 in any one of SEQ ID NO: 43 to 61 is replaced by other amino acids. Therefore, in one embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any one of SEQ ID NO: 43 to 61 is substituted with other amino acids; a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any one of SEQ ID NO: 43 to 61 is substituted with other amino acids; and a third ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61. The amino acid sequence of any one of SEQ ID NO: 43 to 61, or the amino acid sequence in which 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 in any one of SEQ ID NO: 43 to 61 are replaced by other amino acids.

[0100] In one specific embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 43 are substituted with other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 44 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 45 ...5 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence wherein up to 9, 8, 7, 6, 3, 2, or 1 amino acids of SEQ ID NO: 45 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 45, or an amino 45. An amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 43 is replaced by other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 44 is replaced by other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 45 is replaced by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 43, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 45, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0101] In one specific embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 52 are substituted with other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 53 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 54 ...4 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence wherein up to 9, 8, 7, 6, 3, 4, 3, 2, or 1 amino acids of SEQ ID NO: 54 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 54 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 52 is replaced by other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 53 is replaced by other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 54 is replaced by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 52, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 53, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 54, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0102] In one specific embodiment, the ankyrin repeating domain that specifically binds to soluble mesothelin comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 55 are substituted with other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 56 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 57 ...3, 2, or 1 amino acids of SEQ ID NO: 57 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 57, or an amino 57 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 55 is substituted with other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 56 is substituted with other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 57 is substituted with other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 55, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 56, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 57, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0103] In one embodiment, all the amino acid substitutions of the ankyrin repeat module, as described and mentioned herein, occur in the frame position of the ankyrin repeat module, wherein the overall structure of the module is generally unaffected by the substitutions.

[0104] As disclosed herein, the ankyrin repeat domain that specifically binds to soluble mesothelin preferably comprises an N-terminal and / or C-terminal capping module (hereinafter also referred to as a “capped repeat sequence”). The capping module is located at the N-terminus and / or C-terminus of the ankyrin repeat domain and typically forms a tight tertiary interaction (i.e., a tertiary structural interaction) with the ankyrin repeat module therebetween, thereby providing a cap that protects the hydrophobic core of the ankyrin repeat domain on one side from exposure to solvents. Examples of capping sequences are described in International Patent Publications WO 2002 / 020565 and WO 2012 / 069655, US Patent Publication US20130296221, and Interlandi et al., J Mol Biol. 18 January 2008; 375(3):837-54. Examples of amino acid sequences (i.e., N-terminal capped repeat sequences) of the N-terminal capped module are provided in SEQ ID NO: 79 to 86, and examples of amino acid sequences (i.e., C-terminal capped repeat sequences) of the C-terminal capped module are provided in SEQ ID NO: 91 to 97.

[0105] Therefore, in some embodiments, a recombinant binding protein is provided comprising an ankyrin repeating domain as described herein that is specific for binding to soluble mesothelin, wherein the ankyrin repeating domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79 to 86, or an amino acid sequence in which 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 in any one of SEQ ID NO: 79 to 86 is replaced by other amino acids. Alternatively or additionally, the ankyrin repeating domain includes a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91 to 97, or an amino acid sequence in which 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 in any one of SEQ ID NO: 91 to 97 is replaced by other amino acids.

[0106] In a preferred embodiment, the ankyrin repeat domain that specifically binds to soluble mesothelin comprises, from the N-terminus to the C-terminus: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat modules as described more specifically herein; and a C-terminal capping module.

[0107] In one embodiment, the ankyrin repeat domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79 to 86, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 79 to 86 is substituted with other amino acids; at least one, at least two, or at least three ankyrin repeat modules independently comprising an amino acid sequence of any one of SEQ ID NO: 43 to 61, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 43 to 61 is substituted with other amino acids; and a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91 to 97, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 91 to 97 is substituted with other amino acids; at least one, at least two, or at least three ankyrin repeat modules ...7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 91 to 97 is substituted with other amino acids; at least one, at NO: An amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids in any one of NO: 91 to 97 are replaced by other amino acids.

[0108] On the other hand, a recombinant binding protein comprising an ankyrin repeating domain having binding specificity to soluble mesothelin is provided, wherein the ankyrin repeating domain comprises 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 amino acid sequences to any of SEQ ID NO: 1 to 8. Optionally, the A at the penultimate position of SEQ ID NO: 1 to 8 is replaced by L, and / or optionally, the A at the last position of SEQ ID NO: 1 to 8 is replaced by N.

[0109] Therefore, in one embodiment, the ankyrin repeat domain having binding specificity to soluble mesothelin comprises at least about 80% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8. In one embodiment, the ankyrin repeat domain having binding specificity to soluble mesothelin comprises at least about 90% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8. In another embodiment, the ankyrin repeat domain having binding specificity to soluble mesothelin comprises at least about 93% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8. In another embodiment, the ankyrin repeat domain having binding specificity to soluble mesothelin comprises at least about 95% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8. In one embodiment, the ankyrin repeat domain having binding specificity to soluble mesothelin comprises at least about 98% of the same amino acid sequence as any one of SEQ ID NO: 1 to 8. In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises the amino acid sequence of any one of SEQ ID NO: 1 to 8.

[0110] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 1. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0111] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 2.

[0112] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 3.

[0113] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 4.

[0114] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 5. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 5.

[0115] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 6.

[0116] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 7.

[0117] In one embodiment, the ankyrin repeating domain having binding specificity to soluble mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 8.

[0118] A recombinant binding protein with binding specificity to soluble mesothelin is also provided, which 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 NO: 1 to 8. In one embodiment, the competitive recombinant binding protein with binding specificity to soluble mesothelin comprises an ankyrin repeat domain. A recombinant binding protein with binding specificity to soluble mesothelin is also provided, which 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 NO: 1 to 8. In one embodiment, the competitive recombinant binding protein with binding specificity to soluble mesothelin comprises an ankyrin repeat domain.

[0119] In one embodiment, the recombinant binding protein comprising an ankyrin repeating domain with binding specificity to soluble mesothelin is 10 or less. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant of M (K) D It binds to human soluble mesothelin. Therefore, in one embodiment, the recombinant binding protein is at or below about 10... -7 M of K D It binds to soluble mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -8 M of K D It binds to soluble mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -9 M of K D It binds to soluble mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -10 M of K D It binds to soluble mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -11 M of K D Binding to soluble mesothelin. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain having binding specificity to soluble human mesothelin also binds to soluble cynomolgus mesothelin. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain having binding specificity to mesothelin is at or below about 10 -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant of M (K) D It binds to soluble mesothelin from cynomolgus monkeys. Suitably, the dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR), for example as described in Examples 1 and 2.

[0120] Therefore, in one embodiment, the recombinant binding protein comprises an ankyrin repeating domain, wherein the binding protein in PBS is at or below about 10 -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 M of K D The binding is to human soluble mesothelin, and the ankyrin repeating domain therein comprises 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% of the same amino acid sequence as any of SEQ ID NO: 1 to 8.

[0121] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K D The protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 1. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 1.

[0122] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K D The protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises an amino acid sequence that is 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 that of SEQ ID NO: 2. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 2.

[0123] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K D The protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises an amino acid sequence that is 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 that of SEQ ID NO: 3. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 3.

[0124] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K DThe protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises an amino acid sequence that is 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 that of SEQ ID NO: 4. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 4.

[0125] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K D The protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises an amino acid sequence that is 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 that of SEQ ID NO: 5. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 5.

[0126] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K DThe protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 6. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 6.

[0127] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K D The protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises an amino acid sequence that is 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 that of SEQ ID NO: 7. In one embodiment, the recombinant binding protein comprising the ankyrin repeat sequence is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 7.

[0128] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain, wherein the recombinant binding protein is present in PBS at or below approximately 10 -8 M of K DThe protein binds to soluble human dermal tannin, and the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 8. In one embodiment, the recombinant binding protein comprising the ankyrin repeat domain is in PBS at or below about 10 -8 M of K D The binding soluble human dermal tannin, wherein the ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 8.

[0129] In one embodiment, a recombinant binding protein is provided comprising an ankyrin repeating domain having binding specificity to soluble mesothelin, wherein the ankyrin repeating domain has a melting temperature (Tm) above about 65°C, above about 70°C, above about 72°C, above about 75°C, above about 78°C, above about 80°C, about 75°C, about 80°C, about 82°C, about 85°C, between about 65°C and about 95°C, between about 70°C and about 90°C, or between about 72°C and about 88°C. Therefore, in one embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 65°C. In another embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 70°C. In another embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 75°C. In another embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 80°C. In another embodiment, the ankylosing spicule repeating domain has a melting temperature (Tm) between about 65°C and about 95°C. In another embodiment, the ankylosing spicule repeating domain has a melting temperature (Tm) between about 72°C and about 88°C.

[0130] Ankylosing spondylase domains that are specific for binding to membrane-bound mesothelin

[0131] In one aspect, a recombinant binding protein comprising an ankyrin repeating domain having binding specificity to membrane-bound mesothelin is provided. In one embodiment, the recombinant binding protein comprising the ankyrin repeating domain binds membrane-bound mesothelin, wherein membrane-bound mesothelin refers to amino acid sequence 296-597 (SEQ ID NO: 167) of UniProt ID Q13421-3. In a preferred embodiment, the recombinant binding protein comprising the ankyrin repeating domain, referring to UniProt ID Q13421-3 (SEQ ID NO: 167), binds to amino acid residues 580-598 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein comprising the ankyrin repeating domain, referring to UniProt ID Q13421-3 (SEQ ID NO: 167), binds to an epitope contained within amino acid residues 580-598 of membrane-bound mesothelin. In one preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to amino acid residues 596-598 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to an epitope contained within amino acid residues 596-598 of membrane-bound mesothelin. In one preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to amino acid residues 593-598 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to an epitope contained within amino acid residues 593-598 of membrane-bound mesothelin. In one preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to amino acid residues 580-595 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to an epitope contained within amino acid residues 580-595 of membrane-bound mesothelin. In one preferred embodiment, the recombinant binding protein reference UniProt ID Q13421-3 (SEQ ID NO: 167) comprising an ankyrin repeat domain binds to amino acid residues 593-595 of membrane-bound mesothelin.In another preferred embodiment, the recombinant binding protein comprising an ankyrin repeat domain, reference UniProt ID Q13421-3 (SEQ ID NO: 167), binds to an epitope contained within amino acid residues 593-595 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein comprising an ankyrin repeat domain, reference UniProt ID Q13421-3 (SEQ ID NO: 167), binds to amino acid residues 590-592 of membrane-bound mesothelin. In another preferred embodiment, the recombinant binding protein comprising an ankyrin repeat domain, reference UniProt ID Q13421-3 (SEQ ID NO: 167), binds to an epitope contained within amino acid residues 590-592 of membrane-bound mesothelin. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain having binding specificity to membrane-bound mesothelin does not bind to soluble mesothelin (sMSLN). Recombinant binding proteins comprising an ankyrin repeating domain with binding specificity to membrane-bound mesothelin are not inhibited by the presence of sMSLN. Specifically, the ankyrin repeating domain comprises an ankyrin repeating module comprising the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. Therefore, in one embodiment, the ankyrin repeating module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In another embodiment, the ankyrin repeating module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 8 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In one embodiment, the ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 7 amino acids in any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In another embodiment, the ankyrin repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 6 amino acids in any one of SEQ ID NO: 62 to 78 are substituted with other amino acids.In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 5 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 4 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 3 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In one embodiment, the ankylosing spicule repeat module comprises the amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 2 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of any of SEQ ID NO: 62 to 78, or an amino acid sequence in which at most one amino acid in any of SEQ ID NO: 62 to 78 is substituted with another amino acid. In one embodiment, all of the 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions occur at a frame position of the ankylosing spicule module. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of any of SEQ ID NO: 62 to 78.

[0132] In one embodiment, the ankylosing spicule module comprises the amino acid sequence of SEQ ID NO: 62 or a sequence wherein one or two amino acids of SEQ ID NO: 62 are substituted with other amino acids. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of SEQ ID NO: 63 or a sequence wherein one or two amino acids of SEQ ID NO: 63 are substituted with other amino acids. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of SEQ ID NO: 65 or a sequence wherein one or two amino acids of SEQ ID NO: 65 are substituted with other amino acids. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of SEQ ID NO: 66 or a sequence wherein one or two amino acids of SEQ ID NO: 66 are substituted with other amino acids. In one embodiment, the ankylosing spicule module comprises the amino acid sequence of SEQ ID NO: 67 or a sequence wherein one or two amino acids of SEQ ID NO: 67 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 68 or a sequence wherein one or two amino acids of SEQ ID NO: 68 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 69 or a sequence wherein one or two amino acids of SEQ ID NO: 69 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 70 or a sequence wherein one or two amino acids of SEQ ID NO: 70 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 71 or a sequence wherein one or two amino acids of SEQ ID NO: 71 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 72 or a sequence wherein one or two amino acids of SEQ ID NO: 72 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 73 or a sequence wherein one or two amino acids of SEQ ID NO: 73 are substituted with other amino acids.In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 74 or a sequence wherein one or two amino acids of SEQ ID NO: 74 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 75 or a sequence wherein one or two amino acids of SEQ ID NO: 75 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 76 or a sequence wherein one or two amino acids of SEQ ID NO: 76 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 77 or a sequence wherein one or two amino acids of SEQ ID NO: 77 are substituted with other amino acids. In one embodiment, the ankylin repeat module comprises the amino acid sequence of SEQ ID NO: 78 or a sequence wherein one or two amino acids of SEQ ID NO: 78 are substituted with other amino acids.

[0133] In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 62. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 63. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 64. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 65. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 66. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 67. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 68. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 69. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 70. In one embodiment, the ankylosing spicule repeat module comprises or is composed of the amino acid sequence of SEQ ID NO: 71. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 72. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 73. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 74. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 75. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 76. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 77. In one embodiment, the ankylosing spicule module comprises or is composed of the amino acid sequence of SEQ ID NO: 78.

[0134] In one embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module and a second ankyrin repeating module. In one embodiment, the first ankyrin repeating module is located at the N-terminus of the second ankyrin repeating module within the ankyrin repeating domain. In one embodiment, the first ankyrin repeating module and the second ankyrin repeating module each independently comprise an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which 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 from any one of SEQ ID NO: 62 to 78 are substituted with other amino acids. Therefore, in one embodiment, the ankyrin repeating domain comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 62 to 78 is substituted by other amino acids, and further comprises a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 62 to 78 is substituted by other amino acids.

[0135] In one specific embodiment, the ankyrin repeat domain that specifically binds to membrane-bound mesothelin includes a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 65 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 66 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 65, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 66, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0136] In one specific embodiment, the ankyrin repeat domain that specifically binds to membrane-bound mesothelin includes a first ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids; and a second ankyrin repeat module comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 67 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 68 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 67, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 68, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0137] In one specific embodiment, the ankyrin repeat domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeat module containing the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence in which 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 of SEQ ID NO: 69 is substituted with other amino acids; and a second ankyrin repeat module containing the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence in which 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 of SEQ ID NO: 70 is substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 69 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 70 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 69, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 70, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0138] In one specific embodiment, the ankyrin repeat domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeat module containing the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids; and a second ankyrin repeat module containing the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids. In one embodiment, the first ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 71 are substituted with other amino acids, and the second ankylosing spicule repeat module comprises the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence in which at most 3, 2, or 1 amino acids of SEQ ID NO: 72 are substituted with other amino acids, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module. In another embodiment, the first ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 71, and the second ankylosing spicule repeat module comprises or consists of the amino acid sequence of SEQ ID NO: 72, wherein the first ankylosing spicule repeat module is located at the N-terminus of the second ankylosing spicule repeat module.

[0139] In another embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 101 to 105, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 101 to 105 is substituted with other amino acids; and a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 106 to 109, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of any one of SEQ ID NO: 106 to 109 is substituted with other amino acids. In one embodiment, the first ankyrin repeat module comprises an amino acid sequence of any of SEQ ID NO: 106 to 109, or an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of any of SEQ ID NO: 106 to 109 is substituted with other amino acids, and the second ankyrin repeat module comprises an amino acid sequence of any of SEQ ID NO: 106 to 109, or an amino acid sequence in which up to 3, up to 2, or up to 1 amino acid of any of SEQ ID NO: 106 to 109 is substituted with other amino acids, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module.

[0140] In one embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin includes a first ankyrin repeating module, a second ankyrin repeating module, and a third ankyrin repeating module. In a preferred embodiment, the first ankyrin repeating module is located at the N-terminus of the second ankyrin repeating module, and the second ankyrin repeating module is located at the N-terminus of the third ankyrin repeating module within the ankyrin repeating domain.

[0141] In one embodiment, the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module each independently comprise an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which 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 in any one of SEQ ID NO: 62 to 78 is replaced by other amino acids. Therefore, in one embodiment, the ankyrin repeating domain comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids; a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of any one of SEQ ID NO: 62 to 78 are substituted with other amino acids; and a third ... An amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids in any of 62 to 78 are replaced by other amino acids.

[0142] In one specific embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 62 are substituted with other amino acids; a second ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 63 are substituted with other amino acids; and a third ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 64 ...4 are substituted with other amino acids; and a third ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence in which up to 9, 8, 7, 6, 6, 6, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 64 are substituted with other amino acids; and a 64 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 62 is substituted by other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 63 is substituted by other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 64 is substituted by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 62, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 63, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 64, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0143] In one specific embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 73 are substituted with other amino acids; a second ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 74 are substituted with other amino acids; and a third ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 75 ...5 are substituted with other amino acids; and a third ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence in which up to 9, 8, 7, 6, 3, 2, or 1 amino acids of SEQ ID NO: 75 are substituted with other amino acids; and a third ankyrin repeating module containing the amino acid sequence of SEQ ID NO: 75 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 73 is replaced by other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 74 is replaced by other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 75 is replaced by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 73, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 74, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 75, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0144] In one specific embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 76 are substituted with other amino acids; a second ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence wherein up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of SEQ ID NO: 77 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78 ...8 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 9, 8, 7, 6, 3, 2, or 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 9, 8, 7, 6, 3, 2, or 1 amino acids of SEQ ID NO: 78 are substituted with other amino acids; and a third ankyrin repeating module comprising the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence wherein up to 78 is an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid is replaced by other amino acids. In one embodiment, the first ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 76 is substituted by other amino acids; the second ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 77 is substituted by other amino acids; and the third ankyrin repeat module comprises the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence in which at most 3, at most 2, or at most 1 amino acid of SEQ ID NO: 78 is substituted by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module, and the third ankyrin repeat module are arranged in a first module-second module-third module direction within the ankyrin repeat domain in the direction from the N-terminus to the C-terminus. In one embodiment, the ankyrin repeating domain comprises (i) a first ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 76, (ii) a second ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 77, and (iii) a third ankyrin repeating module comprising or consisting of the amino acid sequence of SEQ ID NO: 78, wherein the first ankyrin repeating module, the second ankyrin repeating module, and the third ankyrin repeating module are arranged in a first module-second module-third module in the direction from the N-terminus to the C-terminus within the ankyrin repeating domain.

[0145] In another embodiment, the ankyrin repeating domain that specifically binds to membrane-bound mesothelin comprises a first ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 101 to 105, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any one of SEQ ID NO: 101 to 105 is substituted with other amino acids; a second ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 106 to 109, or an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from any one of SEQ ID NO: 106 to 109 is substituted with other amino acids; and a third ankyrin repeating module comprising an amino acid sequence of any one of SEQ ID NO: 106 to 109; The amino acid sequence of any one of SEQ ID NO: 110 to 112, or the amino acid sequence in which 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 in any one of SEQ ID NO: 110 to 112 are replaced by other amino acids, wherein the first ankyrin repeat module, the second ankyrin repeat module and the third ankyrin repeat module are arranged in the direction from the N-terminus to the C-terminus within the ankyrin repeat domain as first module-second module-third module.

[0146] In another embodiment, the ankyrin repeat domain that specifically binds membrane-bound mesothelin to the first ankyrin repeat module, optionally the second ankyrin repeat module, and optionally the third ankyrin repeat module independently comprises an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, 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 1 framework residue is replaced by another amino acid.

[0147] The ankyrin repeat domain that specifically binds to membrane-bound mesothelin preferably comprises an N-terminal and / or C-terminal capped module (hereinafter also referred to as a “capped repeat sequence”). Examples of the amino acid sequences of the N-terminal capped module (i.e., N-terminal capped repeat sequences) are provided in SEQ ID NO: 79, 87 to 90, 113 to 115 and 201, and examples of the amino acid sequences of the C-terminal capped module (i.e., C-terminal capped repeat sequences) are provided in SEQ ID NO: 91, 98 to 100 and 116 to 120.

[0148] Therefore, in some embodiments, a recombinant binding protein is provided comprising an ankyrin repeating domain as described herein that is specific for binding to membrane-bound mesothelin, wherein the ankyrin repeating domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, 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 of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201 is replaced by other amino acids. Alternatively or additionally, the ankyrin repeating domain includes a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100, or 116 to 120, or an amino acid sequence in which 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 in any one of SEQ ID NO: 91, 98 to 100, or 116 to 120 are replaced by other amino acids.

[0149] In a preferred embodiment, the ankyrin repeat domain that specifically binds to membrane-bound mesothelin comprises, from the N-terminus to the C-terminus: an N-terminal capping module; at least one, at least two, at least three or more ankyrin repeat modules that specifically bind to membrane-bound mesothelin as described herein; and a C-terminal capping module.

[0150] Therefore, in some embodiments, an ankyrin repeating domain is provided, wherein the ankyrin repeating domain comprises an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201, or an amino acid sequence wherein at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201 is substituted with other amino acids; at least one, at least two, or at least three internal ankyrin repeating modules, each internal ankyrin repeating module independently comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 and 101 to 112, or wherein SEQ ID NO: An amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any of SEQ ID NO: 91, 98 to 100, or 116 to 120 are replaced by other amino acids; a C-terminal capping module comprising an amino acid sequence in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in any of SEQ ID NO: 91, 98 to 100, or 116 to 120 are replaced by other amino acids.

[0151] In one embodiment, all the amino acid substitutions of the ankyrin repeat module as described and mentioned herein occur at frame positions, for example, in positions other than randomized positions 3, 4, 6, 11, 14, and 15 of the ankyrin repeat modules of SEQ ID NOs: 62 to 78 and 101 to 112, for example, in positions other than positions 4, 8, 11, and 12 of SEQ ID NOs: 79, 87 to 90, 113 to 115, and 201, and for example, in positions 3, 4, 6, 14, and 15 of SEQ ID NOs: 91, 98 to 100, and 116 to 120, wherein the overall structure of the module is generally unaffected by the substitutions. Whether such substitutions are explicitly described or not, embodiments of such substitutions at frame positions should be applicable to all embodiments.

[0152] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises (i) Tyr or Trp at position 4, (ii) His at position 6, (iii) Leu or Val at position 11, and / or (iv) Tyr at position 14, relative to the amino acid residue numbered SEQ ID NO: 101.

[0153] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises (i) Tyr or Trp at position 4, (ii) His at position 6, (iii) Leu at position 11, and / or (iv) Tyr at position 14, relative to the amino acid residue numbered SEQ ID NO: 101.

[0154] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises amino acid residues (i) Trp at position 4, (ii) His at position 6, (iii) Leu at position 11, and / or (iv) Tyr at position 14, relative to the SEQ ID NO: 101.

[0155] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises Tyr at position (i) 4, His at position (ii) 6, Val at position (iii) 11, and / or Tyr at position (iv) 14, relative to the amino acid residue numbered SEQ ID NO: 101.

[0156] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises the amino acid residues (i) Tyr at position 4, (ii) His at position 6, (iii) Leu at position 11, and / or (iv) Tyr at position 14, relative to the SEQ ID NO: 101.

[0157] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises His at position (i) 6 and / or Trp or Tyr at position (ii) 14 of amino acid residues corresponding to the number of SEQ ID NO: 101.

[0158] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises His at position 6 and Tyr at position 14 of amino acid residues corresponding to the SEQ ID NO: 101.

[0159] In some embodiments, the internal ankyrin repeat module, suitably the first internal ankyrin repeat module, comprises His at position (i) 6 and Trp at position (ii) 14 of amino acid residues corresponding to the SEQ ID NO: 101.

[0160] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises the amino acid residues (i) Phe, Tyr or Trp at position 4, (ii) Ile at position 6, (iii) Val at position 11, (iv) Tyr at position 14 and / or (v) Arg at position 15, relative to the amino acid residue numbered in SEQ ID NO: 106.

[0161] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises amino acid residues (i) Phe or Tyr at position 4, (ii) Ile at position 6, (iii) Val at position 11, (iv) Tyr at position 14, and / or (v) Arg at position 15, relative to the amino acid residue numbered in SEQ ID NO: 106.

[0162] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises amino acid residues (i) Phe at position 4, (ii) Ile at position 6, (iii) Val at position 11, (iv) Tyr at position 14 and / or (v) Arg at position 15, relative to the SEQ ID NO: 106.

[0163] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises amino acid residues (i) Tyr at position 4, (ii) Ile at position 6, (iii) Val at position 11, (iv) Tyr at position 14, and / or (v) Arg at position 15, relative to the SEQ ID NO: 106.

[0164] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises amino acid residues (i) Trp at position 4, (ii) Ile at position 6, (iii) Val at position 11, (iv) Tyr at position 14 and / or (v) Arg at position 15, relative to the SEQ ID NO: 106.

[0165] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises Phe or Tyr at position (i) 4, Tyr at position 14, and / or Arg or Lys at position 15, relative to the amino acid residue numbered SEQ ID NO: 106.

[0166] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises Tyr at position (i) 4, Tyr at position 14, and / or Lys at position 15, relative to the amino acid residue numbered SEQ ID NO: 106.

[0167] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises Phe at position 4, Tyr at position 14, and / or Lys at position 15, relative to the amino acid residue numbered in SEQ ID NO: 106.

[0168] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises Tyr at position (i) 4, Tyr at position (ii) 14, and / or Arg at position (iii) 15, relative to the amino acid residue numbered SEQ ID NO: 106.

[0169] In some embodiments, the internal ankyrin repeat module, suitably a second internal ankyrin repeat module, comprises Phe at position 4, Tyr at position 14, and / or Arg at position 15, relative to the amino acid residue numbered SEQ ID NO: 106.

[0170] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises the amino acid residues corresponding to the numbers in SEQ ID NO: 110: (i) Val or Arg at position 3, (ii) Glu or Tyr at position 4, (iii) Trp at position 6, (iv) Ile at position 11, (v) Gln or Glu at position 14, and / or (vi) Ser or His at position 15.

[0171] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises the amino acid residues corresponding to the numbers in SEQ ID NO: 110: (i) Val or Arg at position 3, (ii) Glu at position 4, (iii) Trp at position 6, (iv) Ile at position 11, (v) Gln at position 14, and / or (vi) Ser or His at position 15.

[0172] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises the amino acid residues corresponding to the numbers in SEQ ID NO: 110: (i) Val at position 3, (ii) Glu or Tyr at position 4, (iii) Trp at position 6, (iv) Ile at position 11, (v) Gln or Glu at position 14, and / or (vi) His at position 15.

[0173] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises Tyr at position 4 and His at position 15 of amino acid residues corresponding to the SEQ ID NO: 110.

[0174] In some embodiments, the internal ankyrin repeat module, suitably the third internal ankyrin repeat module, comprises Trp at position 6, Gln or Glu at position 14, and / or His at position 15, relative to the amino acid residue numbered in SEQ ID NO: 110.

[0175] In some embodiments, the N-terminal capping module comprises Ala at position 11 (i) and / or Arg, Gln, or Lys at position 12 (ii) of the amino acid residues numbered relative to SEQ ID NO: 115.

[0176] In some embodiments, the N-terminal capping module includes the amino acid residue Ala at position 11 relative to SEQ ID NO: 115.

[0177] In some embodiments, the C-terminal capping module comprises amino acid residues corresponding to the following positions: (i) Glu at position 3, (ii) Ala at position 4, (iii) Leu at position 6, (iv) Arg or Ala at position 14, (v) Leu at position 15, and / or (vi) Glu or Asp at position 18.

[0178] In some embodiments, the C-terminal capping module includes amino acid residues (i) Ala at position 4, (ii) Leu at position 6, and / or (iii) Leu at position 15, relative to the amino acid residue numbered in SEQ ID NO: 119.

[0179] In some embodiments, the C-terminal capping module comprises the amino acid residues (i) Lys at position 3, (ii) Ser at position 4, (iii) Lys at position 6, (iv) Arg at position 14, (v) Ala at position 15, and / or (vi) Gln at position 18, relative to the amino acid residues numbered in SEQ ID NO: 119.

[0180] On the other hand, a recombinant binding protein comprising an ankyrin repeating domain having binding specificity to human membrane-bound mesothelin is provided, wherein the ankyrin repeating domain comprises 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% of the amino acid sequence of any of SEQ ID NO: 9 to 15, 195, 198, or 199. Therefore, in one embodiment, the ankyrin repeating domain comprises at least about 80% of the amino acid sequence of any of SEQ ID NO: 9 to 15, 195, 198, or 199. In one embodiment, the ankylosing spicule domain comprises at least about 90% of the amino acid sequence identical to any one of SEQ ID NO: 9 to 15, 195, 198, or 199. In another embodiment, the ankylosing spicule domain comprises at least about 93% of the amino acid sequence identical to any one of SEQ ID NO: 9 to 15, 195, 198, or 199. In yet another embodiment, the ankylosing spicule domain comprises at least about 95% of the amino acid sequence identical to any one of SEQ ID NO: 9 to 15, 195, 198, or 199. In one embodiment, the ankylosing spicule domain comprises an amino acid sequence having any one of SEQ ID NO: 9 to 15, 195, 198, or 199.

[0181] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 9. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 9.

[0182] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 10. In one embodiment, the ankyrin repeating domain comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0183] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 11.

[0184] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0185] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 13.

[0186] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 14. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 14.

[0187] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 15. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0188] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 195. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 195.

[0189] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 198. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 198.

[0190] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 199. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 199.

[0191] A recombinant binding protein specific for binding to membrane-bound mesothelin is also provided, which competes with a reference binding protein comprising an ankyrin repeat domain containing the amino acid sequence of any one of SEQ ID NO: 9 to 15, 195, 198, or 199. In one embodiment, the recombinant binding protein specific for binding to membrane-bound mesothelin comprises an ankyrin repeat domain. A recombinant binding protein specific for binding to membrane-bound mesothelin is also provided, which binds to the same epitope as the reference binding protein comprising an ankyrin repeat domain containing the amino acid sequence of any one of SEQ ID NO: 9 to 15, 195, 198, or 199. In one embodiment, the recombinant binding protein specific for binding to membrane-bound mesothelin comprises an ankyrin repeat domain.

[0192] In some implementations, the ankyrin repeat modules described herein are further engineered to reduce the number of basic amino acids (positively charged) and / or lower the isoelectric point (pI) compared to unengineered ankyrin repeat modules. This method is also described in PCT / EP2023 / 071188. Recombinant binding proteins containing such engineered ankyrin repeat modules have been found to reduce renal accumulation of linked drug moieties (such as radionuclides) after administration.

[0193] The charge characteristics of the engineered ankyrin repeating domain can be alternatively or additionally defined by the ratio (also known as the KR / DE ratio) between the sum of Arg(R) and Lys(K) residues contained in the engineered ankyrin repeating domain and the sum of Asp(D) and Glu(E) residues. The KR / DE ratio can be calculated based on the amino acid sequence of the ankyrin repeating domain described herein.

[0194] Therefore, in some embodiments, the engineered ankyrin repeating domain has a KR / DE ratio equal to or less than 0.44, equal to or less than 0.42, equal to or less than 0.40, equal to or less than 0.38, equal to or less than 0.36, equal to or less than 0.34, equal to or less than 0.32, equal to or less than 0.30, equal to or less than 0.28, equal to or less than 0.26, equal to or less than 0.24, equal to or less than 0.22, equal to or less than 0.20, equal to or less than 0.18, equal to or less than 0.16, equal to or less than 0.14, equal to or less than 0.12, equal to or less than 0.10, or equal to or less than 0.08. Suitably, the KR / DE ratio is equal to or less than 0.3. Unless otherwise stated, the KR / DE ratio is determined by taking into account all residues contained in the engineered ankyrin repeating domain. In other embodiments, the KR / DE ratio is calculated only in the framework residues of the ankyrin repeating domain. The locations of the framework residues are described herein. Therefore, in some embodiments, the engineered ankyrin repeating domain has a KR / DE ratio equal to or less than 0.36, 0.34, 0.32, 0.30, 0.28, 0.26, 0.24, 0.22, 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, or 0.08 in the frame residues. Suitably, the KR / DE ratio in the frame residues is equal to or less than 0.25.

[0195] In some embodiments, the engineered ankylosing spicule has an isoelectric point (pI) that is at least 0.1 pH points lower than that of the parental (non-engineered) ankylosing spicule. In some embodiments, the pI 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 that of the parental (non-engineered) ankylosing spicule.

[0196] In some implementations, the engineered ankyrin repeating domain has a percentage of Arg and Lys residues equal to or less than 8.0%, equal to or less than 7.5%, equal to or less than 7.0%, equal to or less than 6.5%, or equal to or less than 6.0%, equal to or less than 5.5%, equal to or less than 5.0%, equal to or less than 4.5%, equal to or less than 4.0%, or equal to or less than 3.5%.

[0197] In some implementations, the engineered ankyrin repeating domain has a percentage of Arg and Lys residues equal to or less than 7.5%, equal to or less than 7.0%, equal to or less than 6.5%, or equal to or less than 6.0%, equal to or less than 5.5%, equal to or less than 5.0%, equal to or less than 4.5%, equal to or less than 4.0%, or equal to or less than 3.5% in the framework residues.

[0198] In some implementations, the engineered ankyrin repeating domain includes one or more internal repeating modules, each internal repeating module containing one or more of the following amino acid residues: (i) Ser or Glu at position 1, (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27.

[0199] In some implementations, the engineered ankyrin repeating domain includes one or more internal repeating modules, each internal repeating module containing one or more amino acid residues of the following: (i) Ser or Glu at position 1 and / or (ii) Glu at position 26.

[0200] In some embodiments, the engineered ankyrin repeating domain includes an N-terminal capping module containing one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gln at position 23.

[0201] In some embodiments, the engineered ankyrin repeating domain includes an N-terminal capping module containing one or more of the following amino acid residues: (i) Gln at position 5, (ii) Thr at position 17, and / or (iii) Gln at position 23.

[0202] In some embodiments, the engineered ankyrin repeating domain includes a C-terminal capping module containing one or more of the following amino acid residues: (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) Gln at position 26.

[0203] In some embodiments, the engineered ankyrin repeating domain includes a C-terminal capping module containing one or more of the following amino acid residues: (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gln at position 26.

[0204] In some implementations, the engineered ankyrin repeating domain contains

[0205] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23, and / or

[0206] (2) One or more internal repeating modules, wherein each internal repeating module independently contains one or more of the following amino acid residues: (i) Ser or Glu at position 1, (ii) Leu at position 25, (iii) Glu at position 26 and (iv) Ala at position 27, and / or

[0207] (3) C-terminal capping module comprising one or more of the following amino acid residues: (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 (vi) Gln at position 26.

[0208] In some implementations, the engineered ankyrin repeating domain contains

[0209] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23, and / or

[0210] (2) One or more internal repeating modules, wherein each internal repeating module independently contains one or more of the following amino acid residues: (i) Ser or Glu at position 1, (ii) Leu at position 25, (iii) Glu at position 26 and (iv) Ala at position 27, and / or

[0211] (3) A C-terminal capping module comprising one or more of the following amino acid residues: (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0212] In some implementations, the engineered ankyrin repeating domain contains

[0213] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23; and / or

[0214] (2) One or more internal repeating modules, wherein each internal repeating module independently comprises one or more of the following amino acid residues: (i) Gln at position 5, (ii) Thr at position 17 and / or (iii) Gln at position 23; and / or

[0215] (3) C-terminal capping module comprising one or more of the following amino acid residues: (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 (vi) Gln at position 26.

[0216] In some implementations, the engineered ankyrin repeating domain contains

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

[0218] (2) One or more internal repeating modules, wherein each internal repeating module independently comprises one or more of the following amino acid residues: (i) Gln at position 5, (ii) Thr at position 17 and / or (iii) Gln at position 23; and / or

[0219] (3) A C-terminal capping module comprising one or more of the following amino acid residues: (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0220] In some implementations, the engineered ankyrin repeating domain contains

[0221] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23, and / or

[0222] (2) One or more internal repeating modules, wherein each internal repeating module independently contains one or more of the following amino acid residues: (i) Ser or Glu at position 1 and / or (ii) Glu at position 26, and / or

[0223] (3) C-terminal capping module comprising one or more of the following amino acid residues: (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 (vi) Gln at position 26.

[0224] In some implementations, the engineered ankyrin repeating domain contains

[0225] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23, and / or

[0226] (2) One or more internal repeating modules, wherein each internal repeating module independently contains one or more of the following amino acid residues: (i) Ser or Glu at position 1 and / or (ii) Glu at position 26, and / or

[0227] (3) A C-terminal capping module comprising one or more of the following amino acid residues: (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0228] In some implementations, the engineered ankyrin repeating domain contains

[0229] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23; and / or

[0230] (2) One or more internal repeating modules, wherein each internal repeating module independently comprises one or more of the following amino acid residues: (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0231] (3) C-terminal capping module comprising one or more of the following amino acid residues: (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 (vi) Gln at position 26.

[0232] In some implementations, the engineered ankyrin repeating domain contains

[0233] (1) An N-terminal capping module comprising one or more of the following amino acid residues: (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and (v) Gln at position 23; and / or

[0234] (2) One or more internal repeating modules, wherein each internal repeating module independently comprises one or more of the following amino acid residues: (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0235] (3) A C-terminal capping module comprising one or more of the following amino acid residues: (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0236] In some embodiments, the engineered ankyrin repeating domain includes one or more internal repeating modules containing an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, and the one or more internal repeating modules further include one or more amino acid substitutions at positions 1, 25, 26 and / or 27, wherein the amino acid substitutions are (i) Ser or Glu at position 1, (ii) Leu at position 25, (iii) Glu at position 26 and / or (iv) Ala at position 27.

[0237] In some embodiments, the engineered ankyrin repeating domain includes one or more internal repeating modules containing an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, and the one or more internal repeating modules further include one or more amino acid substitutions at position 1 and / or position 26, wherein the amino acid substitution is (i) Ser or Glu at position 1 and / or (ii) Glu at position 26.

[0238] In some embodiments, the engineered ankyrin repeating domain includes an N-terminal capping module containing an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201, the N-terminal capping module further containing one or more amino acid substitutions at positions 4, 5, 8, 17, and / or 23, wherein the amino acid substitutions are (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17, and / or (v) Gln at position 23.

[0239] In some embodiments, the engineered ankyrin repeating domain includes an N-terminal capping module containing an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115, or 201, and the N-terminal capping module further includes one or more amino acid substitutions at position 5, position 17, and / or position 23, wherein the amino acid substitutions are (i) Gln at position 5, (ii) Thr at position 17, and / or (iii) Gln at position 23.

[0240] In some embodiments, the engineered ankyrin repeating domain includes a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100, or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 3, 6, 14, 17, 18, and / or 26, wherein the amino acid substitutions are (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 Gln at position 26.

[0241] In some embodiments, the engineered ankyrin repeating domain includes a C-terminal capping module containing an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100, or 116 to 120, the C-terminal capping module further containing one or more amino acid substitutions at positions 17, 18, and / or 26, wherein the amino acid substitutions are (i) Asn at position 17, (ii) Glu at position 18, and (iii) Gln at position 26.

[0242] In some implementations, the engineered ankyrin repeating domain contains

[0243] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at positions 4, 5, 8, 17 and / or 23, wherein the amino acid substitutions are (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17 and / or (v) Gln at position 23; and / or

[0244] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at positions 1, 25, 26 and / or 27, wherein the amino acid substitutions are (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

[0245] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 3, 6, 14, 17, 18 and / or 26, wherein the amino acid substitutions are (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 Gln at position 26.

[0246] In some implementations, the engineered ankyrin repeating domain contains

[0247] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at positions 4, 5, 8, 17 and / or 23, wherein the amino acid substitutions are (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17 and / or (v) Gln at position 23; and / or

[0248] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at positions 1, 25, 26 and / or 27, wherein the amino acid substitutions are (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

[0249] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 17, 18 and / or 26, wherein the amino acid substitutions are (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0250] In some implementations, the engineered ankyrin repeating domain contains

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

[0252] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at positions 1, 25, 26 and / or 27, wherein the amino acid substitutions are (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

[0253] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 3, 6, 14, 17, 18 and / or 26, wherein the amino acid substitutions are (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 Gln at position 26.

[0254] In some implementations, the engineered ankyrin repeating domain contains

[0255] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at position 5, position 17 and / or position 23, wherein the amino acid substitution is (i) Gln at position 5, (ii) Thr at position 17 and / or (iii) Gln at position 23; and / or

[0256] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at positions 1, 25, 26 and / or 27, wherein the amino acid substitutions are (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

[0257] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 17, 18 and / or 26, wherein the amino acid substitutions are (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0258] In some implementations, the engineered ankyrin repeating domain contains

[0259] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at positions 4, 5, 8, 17 and / or 23, wherein the amino acid substitutions are (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17 and / or (v) Gln at position 23; and / or

[0260] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at position 1 and / or position 26, wherein the amino acid substitution is (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0261] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 3, 6, 14, 17, 18 and / or 26, wherein the amino acid substitutions are (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 Gln at position 26.

[0262] In some implementations, the engineered ankyrin repeating domain contains

[0263] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at positions 4, 5, 8, 17 and / or 23, wherein the amino acid substitutions are (i) Thr at position 4, (ii) Gln at position 5, (iii) Glu at position 8, (iv) Thr at position 17 and / or (v) Gln at position 23; and / or

[0264] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at position 1 and / or position 26, wherein the amino acid substitution is (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0265] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 17, 18 and / or 26, wherein the amino acid substitutions are (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0266] In some implementations, the engineered ankyrin repeating domain contains

[0267] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at position 5, position 17 and / or position 23, wherein the amino acid substitution is (i) Gln at position 5, (ii) Thr at position 17 and / or (iii) Gln at position 23; and / or

[0268] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at position 1 and / or position 26, wherein the amino acid substitution is (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0269] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 3, 6, 14, 17, 18 and / or 26, wherein the amino acid substitutions are (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 Gln at position 26.

[0270] In some implementations, the engineered ankyrin repeating domain contains

[0271] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90, 113 to 115 or 201, the N-terminal capping module further comprising one or more amino acid substitutions at position 5, position 17 and / or position 23, wherein the amino acid substitution is (i) Gln at position 5, (ii) Thr at position 17 and / or (iii) Gln at position 23; and / or

[0272] (2) One or more internal repeating modules comprising an amino acid sequence of any one of SEQ ID NO: 62 to 78 or 101 to 112, the one or more internal repeating modules further comprising one or more amino acid substitutions at position 1 and / or position 26, wherein the amino acid substitution is (i) Ser or Glu at position 1 and / or (ii) Glu at position 26; and / or

[0273] (3) A C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100 or 116 to 120, the C-terminal capping module further comprising one or more amino acid substitutions at positions 17, 18 and / or 26, wherein the amino acid substitutions are (i) Asn at position 17, (ii) Glu at position 18 and (iii) Gln at position 26.

[0274] In some embodiments, the engineered ankyrin repeating domain comprises one or more internal repeating modules, wherein each internal repeating module independently comprises an amino acid sequence of any one of SEQ ID NO: 132 to 151 or 203 to 205, or wherein an amino acid sequence in which up to 9 frame residues, up to 8 frame residues, up to 7 frame residues, up to 6 frame residues, up to 5 frame residues, up to 4 frame residues, up to 3 frame residues, up to 2 frame residues, or up to 1 frame residue in any one of SEQ ID NO: 132 to 151 or 203 to 205 are replaced by other amino acids.

[0275] Alternatively or additionally, in some embodiments, the engineered ankyrin repeating domain includes an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 121 to 131 or 202, or an amino acid sequence in which 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 framework residue of any one of SEQ ID NO: 121 to 131 or 202 is replaced by an amino acid.

[0276] Alternatively or additionally, in some embodiments, the engineered ankyrin repeating domain includes a C-terminal capping module comprising an amino acid sequence of any of SEQ ID NO: 152 to 160, or an amino acid sequence in which 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 framework residue of any of SEQ ID NO: 152 to 160 is replaced by an amino acid.

[0277] Therefore, in some implementations, the engineered ankyrin repeating domain contains

[0278] (1) An N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 121 to 131 or 202, or an amino acid sequence wherein up to 9 frame residues, up to 8 frame residues, up to 7 frame residues, up to 6 frame residues, up to 5 frame residues, up to 4 frame residues, up to 3 frame residues, up to 2 frame residues, or up to 1 frame residue in any one of SEQ ID NO: 121 to 131 or 202 are replaced by an amino acid; and / or

[0279] (2) One or more internal repeating modules, wherein each internal repeating module independently comprises an amino acid sequence of any one of SEQ ID NO: 132 to 151 or 203 to 205, or wherein an amino acid sequence in any one of SEQ ID NO: 132 to 151 or 203 to 205 has at most 9 frame residues, at most 8 frame residues, at most 7 frame residues, at most 6 frame residues, at most 5 frame residues, at most 4 frame residues, at most 3 frame residues, at most 2 frame residues, or at most 1 frame residue substituted by other amino acids; and / or

[0280] (3) A C-terminal capping module comprising an amino acid sequence of any of SEQ ID NO: 152 to 160, or an amino acid sequence in which 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 framework residue in any of SEQ ID NO: 152 to 160 are replaced by an amino acid.

[0281] On the other hand, a recombinant binding protein comprising an engineered ankyrin repeating domain having binding specificity to human membrane-bound mesothelin is provided, wherein the ankyrin repeating domain comprises 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% of the amino acid sequence identical to any of SEQ ID NO: 16 to 35, 196, 197, or 200. Therefore, in one embodiment, the engineered ankyrin repeating domain comprises at least about 80% of the amino acid sequence identical to any of SEQ ID NO: 16 to 35, 196, 197, or 200. In one embodiment, the engineered ankylosing spicule repeating domain comprises at least about 90% of the amino acid sequence identical to any one of SEQ ID NO: 16 to 35, 196, 197, or 200. In another embodiment, the engineered ankylosing spicule repeating domain comprises at least about 93% of the amino acid sequence identical to any one of SEQ ID NO: 16 to 35, 196, 197, or 200. In another embodiment, the engineered ankylosing spicule repeating domain comprises at least about 95% of the amino acid sequence identical to any one of SEQ ID NO: 16 to 35, 196, 197, or 200. In one embodiment, the engineered ankylosing spicule repeating domain comprises an amino acid sequence having any one of SEQ ID NO: 16 to 35, 196, 197, or 200. In another embodiment, the engineered ankyrin repeating domain comprises or is composed of an amino acid sequence having SEQ ID NO: 161 or 162.

[0282] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 16. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 16.

[0283] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 17. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 17.

[0284] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 18. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 18.

[0285] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 19. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 19.

[0286] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 20. In one embodiment, the ankyrin repeating domain comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0287] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 21.

[0288] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 22. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 22.

[0289] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 23. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 23.

[0290] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 24. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 24.

[0291] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 25. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 25.

[0292] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 26. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 26.

[0293] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 27. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 27.

[0294] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 28. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 28.

[0295] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 29. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 29.

[0296] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 30. In one embodiment, the ankyrin repeating domain comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0297] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 31. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 31.

[0298] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 32. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 32.

[0299] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 33. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 33.

[0300] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 34. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 34.

[0301] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 35. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 35.

[0302] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 196. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 196.

[0303] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises 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% of the amino acid sequence of SEQ ID NO: 197. In one embodiment, the ankyrin repeating domain comprises or is composed of the amino acid sequence of SEQ ID NO: 197.

[0304] In one embodiment, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin comprises an amino acid sequence that is 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 that of SEQ ID NO: 200. In one embodiment, the ankyrin repeating domain comprises or consists of the amino acid sequence of SEQ ID NO: 200.

[0305] In one embodiment, the recombinant binding protein comprising an ankyrin repeating domain with binding specificity to membrane-bound mesothelin is 10 or less. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant of M (K) D It binds to human membrane-bound mesothelin. Therefore, in one embodiment, the recombinant binding protein is at or below about 10... -7 M of K D It binds to membrane-bound mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -8 M of K D It binds to membrane-bound mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -9 M of K D It binds to membrane-bound mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -10 M of K DIt binds to membrane-bound mesothelin. In another embodiment, the recombinant binding protein is at or below about 10. -11 M of K D Binding to membrane-bound mesothelin. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain having binding specificity to membrane-bound mesothelin also binds to cynomolgus mesothelin. In one embodiment, the recombinant binding protein comprising an ankyrin repeat domain having binding specificity to membrane-bound mesothelin is 10 or less. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 The dissociation constant of M (K) D It binds to mesothelin in the cynomolgus monkey membrane. Suitably, the dissociation constant is determined in PBS, suitably using surface plasmon resonance (SPR), for example as described in Examples 1 to 4.

[0306] Therefore, in one embodiment, the recombinant binding protein comprises an ankyrin repeating domain that is specific for binding to membrane-bound mesothelin, wherein the recombinant binding protein is present in PBS at or below about 10 -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10- 9 M, or is or is less than approximately 10- 10 M, or is or is less than approximately 10- 11 M of K D The membrane-bound human cortisol, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as any of SEQ ID NO: 9 to 35 or 195 to 200.

[0307] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding membrane binds to human dermal ...

[0308] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 10.

[0309] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 11.

[0310] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 12.

[0311] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 13.

[0312] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 14.

[0313] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 15.

[0314] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 16.

[0315] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 17.

[0316] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 18.

[0317] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 19.

[0318] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 20.

[0319] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 21.

[0320] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 22.

[0321] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 23.

[0322] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 24.

[0323] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 25.

[0324] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 26.

[0325] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 27.

[0326] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 28.

[0327] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 29.

[0328] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 30.

[0329] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 31.

[0330] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 32.

[0331] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 33.

[0332] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 34.

[0333] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 35.

[0334] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 195.

[0335] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 196.

[0336] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 197.

[0337] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K DThe binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 198.

[0338] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 199.

[0339] In one embodiment, the recombinant binding protein is present in PBS at or below approximately 10. -8 M of K D The binding of human membrane-bound mesothelin, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 200.

[0340] In one embodiment, the recombinant binding protein comprises an ankyrin repeating domain that is specific for binding to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at EC50 values ​​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, and less than about 0.2 nM.

[0341] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain that is specific for binding to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM. In another embodiment, the ankyrin repeat domain that is specific for binding to membrane-bound mesothelin binds human MSLN at an EC50 of about 0.1 nM to about 1 nM. In yet another embodiment, the ankyrin repeat domain that is specific for binding to membrane-bound mesothelin binds human MSLN at an EC50 of about 0.1 nM to about 0.5 nM.

[0342] Suitablely, binding of ankyrin repeat domains that are specific for binding to membrane-bound human cortisol is performed on OVCAR-3 cells, for example as described in Example 4.

[0343] In one embodiment, the recombinant binding protein comprises an ankyrin repeating domain that is specific for binding to membrane-bound mesothelin, wherein the recombinant binding protein binds to human mesothelin at EC50 values ​​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, and less than about 0.2 nM, wherein the ankyrin repeating domain comprises an ankyrin repeating domain that is specific for binding to membrane-bound mesothelin. The amino acid sequence is 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.

[0344] In one embodiment, the recombinant binding protein comprises an ankyrin repeating domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeating domain comprises 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 amino acid sequences to any of SEQ ID NO: 9 to 35 or 195 to 200.

[0345] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0346] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 10.

[0347] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 11.

[0348] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0349] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 13.

[0350] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 14.

[0351] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 15.

[0352] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0353] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 17.

[0354] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 18.

[0355] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 19.

[0356] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 20.

[0357] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 21.

[0358] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 22.

[0359] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 23.

[0360] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 24.

[0361] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 25.

[0362] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0363] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 27.

[0364] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 28.

[0365] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 29.

[0366] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 30.

[0367] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 31.

[0368] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 32.

[0369] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 33.

[0370] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0371] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to those in SEQ ID NO: 35.

[0372] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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 amino acid sequences to SEQ ID NO: 195.

[0373] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises an amino acid sequence that is 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.

[0374] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 197.

[0375] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 198.

[0376] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeat domain comprises 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% of the same amino acid sequence as SEQ ID NO: 199.

[0377] In one embodiment, the recombinant binding protein comprises an ankyrin repeating domain having binding specificity to membrane-bound mesothelin, wherein the recombinant binding protein binds human mesothelin at an EC50 of about 0.1 nM to about 10 nM, and wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as SEQ ID NO: 200.

[0378] In another embodiment, a recombinant binding protein comprising an ankyrin repeating domain having binding specificity for membrane-bound mesothelin binds to membrane-bound mesothelin as described herein, and substantially does not bind to soluble mesothelin. Therefore, in a preferred embodiment, the recombinant binding protein comprising an ankyrin repeating domain having binding specificity for membrane-bound mesothelin has a higher binding affinity for membrane-bound mesothelin than for soluble mesothelin. Therefore, in one embodiment, the recombinant binding protein comprising an ankyrin repeating domain having binding specificity for membrane-bound mesothelin has a binding affinity for soluble mesothelin that is at least 2, at least 5, at least 10, at least 20, at least 50, or at least 10 times lower than that of the recombinant binding protein for soluble mesothelin. 2 times, at least 10 3 times, at least 10 4 times or at least 10 5 K times D It binds to membrane-bound mesothelin.

[0379] In one embodiment, a recombinant binding protein comprising an ankyrin repeating domain comprising at least about 80% identical amino acid sequence to any one of SEQ ID NO: 9 to 35 or 195 to 200, the ankyrin repeating domain having binding specificity to membrane-bound mesothelin, wherein the ankyrin repeating domain has a melting temperature above about 65°C, above about 70°C, above about 72°C, above about 75°C, above about 78°C, above about 80°C, about 75°C, about 80°C, about 82°C, about 85°C, between about 65°C and about 95°C, between about 70°C and about 90°C, or between about 72°C and about 88°C. Therefore, in one embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 65°C. In another embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 70°C. In yet another embodiment, the ankyrin repeating domain has a melting temperature (Tm) above about 75°C. In another embodiment, the ankylosing spicule has a melting temperature (Tm) above about 80°C. In another embodiment, the ankylosing spicule has a melting temperature (Tm) between about 65°C and about 95°C. In yet another embodiment, the ankylosing spicule has a melting temperature (Tm) between about 72°C and about 88°C.

[0380] In one implementation, a recombinant binding protein comprising an ankyrin repeat domain with binding specificity to membrane-bound mesothelin binds to mesothelin in humans, mice, and cynomolgus monkeys. In this case, the recombinant binding protein comprising an ankyrin repeat domain with binding specificity to membrane-bound mesothelin is considered "cross-reactive".

[0381] Multivalent and multicomplementary ankyrin repeat domains with binding specificity to human cortisol

[0382] Recombinant binding proteins with multiple complementary sites are also provided, i.e., recombinant binding domains containing two, three, four or more epitopes recognizing the same target protein (suitably human mesothelin). Thus, in another aspect, a recombinant bicomponent binding protein with binding specificity to mesothelin is provided, wherein the recombinant binding protein comprises at least one ankyrin repeat domain as described herein with binding specificity to membrane-bound mesothelin and at least one ankyrin repeat domain as described herein with binding specificity to soluble mesothelin. In another aspect, a recombinant bivalent binding protein with binding specificity to mesothelin is provided, wherein the recombinant binding protein comprises at least two ankyrin repeat domains as described herein with binding specificity to membrane-bound mesothelin. In yet another aspect, a recombinant bivalent binding protein with binding specificity to mesothelin is provided, wherein the recombinant binding protein comprises at least two ankyrin repeat domains as described herein with binding specificity to soluble mesothelin.

[0383] immune cell conjugate

[0384] Mesothelin-specific binding proteins can be used as components of bispecific or multispecific binding molecules targeting disease-associated cells. These mesothelin-specific binding proteins can be used in bispecific or multispecific binding proteins that also contain at least one binding moiety specific to immune cells (e.g., CD3). Such bispecific or multispecific binding proteins can recruit immune cells (such as T cells) to cancer cells.

[0385] On the other hand, a recombinant binding protein with mesothelin binding specificity as described herein is provided, the recombinant binding protein further comprising at least one binding moiety with binding specificity to a protein expressed on the surface of an immune cell (suitably a T lymphocyte (T cell)). Suitably, the protein expressed on the surface of an immune cell (e.g., a T cell) is an activation receptor of the immune cell. Examples of such activation receptors of immune cells include CD3. Thus, in one specific embodiment, a recombinant binding protein is provided comprising an ankyrin repeat domain with mesothelin binding specificity as described herein, the recombinant binding protein further comprising a binding moiety with CD3 binding specificity. Suitably, the recombinant binding protein comprises a CD3 binding moiety and an ankyrin repeat domain with mesothelin binding specificity, wherein the CD3 binding moiety is located at the C-terminus of the ankyrin repeat domain with mesothelin binding specificity. In one embodiment, the recombinant binding protein comprises a CD3 binding moiety and an ankyrin repeat domain with mesothelin binding specificity, wherein the CD3 binding moiety is located at the N-terminus of the ankyrin repeat domain with mesothelin binding specificity.

[0386] In one embodiment, the binding agent having CD3 binding specificity is an ankyrin repeat domain. In one embodiment, the ankyrin repeat domain having CD3 binding specificity comprises 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% of the same amino acid sequence as any of SEQ ID NO: 36 to 40. In one embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises 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% of the same amino acid sequence as SEQ ID NO: 36. In one embodiment, the ankyrin repeat domain having CD3 binding specificity comprises an amino acid sequence that is 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, the ankyrin repeat domain having CD3 binding specificity comprises 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% of the same amino acid sequence as SEQ ID NO: 38.In one embodiment, the ankyrin repeat domain having CD3 binding specificity comprises an amino acid sequence that is 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, the ankyrin repeat domain having CD3 binding specificity comprises 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% of the same amino acid sequence as SEQ ID NO: 40.

[0387] Extended half-life portion

[0388] Compared to the same protein without the extended half-life portion, the "extended half-life portion" prolongs the in vivo serum half-life of the recombinant binding protein described herein. Examples of extended half-life portions include, but are not limited to, multihistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin domain, maltose-binding protein (MBP), human serum albumin (HSA) binding domain, or polyethylene glycol (PEG).

[0389] In one embodiment, the recombinant binding protein provided herein further comprises one or more extended half-life moieties. Thus, in one specific embodiment, the recombinant binding protein comprises an ankyrin repeat domain as described herein that is specific for binding to mesothelin, and further comprises one or more extended half-life moieties. Preferably, the extended half-life moieties bind to human serum albumin. In one embodiment, the extended half-life moieties binding to human serum albumin are an ankyrin repeat domain.

[0390] In one embodiment, the half-life extension portion comprises an ankyrin repeat domain that binds to human serum albumin, said ankyrin repeat domain comprising 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% of the amino acid sequence identical to any of SEQ ID NO: 41 to 42. In another embodiment, the half-life extension portion comprises an ankyrin repeat domain comprising at least about 90% of the amino acid sequence identical to any of SEQ ID NO: 41 to 42. In one embodiment, the half-life extension portion comprises an ankyrin repeat domain containing 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% of the same amino acid sequence as SEQ ID NO: 41. In one embodiment, the half-life extension portion comprises an ankyrin repeat domain containing 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% of the same amino acid sequence as SEQ ID NO: 42.

[0391] Suitably, the ankyrin repeat domain binding to human serum albumin is located at the C-terminus of the ankyrin repeat domain that is specific for binding to mesothelin. Suitably, the ankyrin repeat domain binding to human serum albumin is located at the N-terminus of the ankyrin repeat domain that is specific for binding to 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.

[0392] In some embodiments, the extended half-life portion comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an Fc domain. In some embodiments, the Fc domain is derived from any of the following known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In some embodiments, the Fc domain is derived from any of the following known heavy chain isotypes or subtypes: IgG1 (γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), or IgA2 (α2). In some embodiments, the Fc domain is the Fc domain of human IgG1.

[0393] In some embodiments, the Fc domain comprises an uninterrupted natural sequence (i.e., wild-type sequence) of the Fc domain. In some embodiments, the immunoglobulin Fc domain comprises a variant Fc domain that results in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain to reduce or eliminate effector activity (e.g., WO 2005 / 063815), and / or increase homogeneity during recombinant-binding protein production. In some embodiments, the Fc domain is the Fc domain of human IgG1 and contains one or more of the following effector empty substitutions: L234A, L235A, and G237A (Eu number). In some embodiments, the Fc domain does not contain a lysine residue at the C-terminal position of human IgG1 (i.e., K447, Eu number). The absence of the lysine residue increases homogeneity during recombinant-binding protein production. In some embodiments, the Fc domain contains a lysine residue at the C-terminal position (K447, Eu number).

[0394] Conjugate

[0395] On the other hand, recombinant binding proteins that are specific for binding to soluble or membrane-bound mesothelin and are fused to or chemically conjugated (including both covalent and non-covalent conjugations) with a pharmaceutical moiety are also provided. The pharmaceutical moiety may be used for diagnostic or therapeutic purposes. Such a pharmaceutical moiety may be a heterologous protein, polypeptide, or peptide having the desired biological activity for producing the fusion protein. Such proteins may include, for example, toxins; antibiotics; proteins; or biological response modulators, such as, for example, lymphokines. Methods for fusing or conjugating proteins, polypeptides, or peptides are known in the art.

[0396] Furthermore, the recombinant binding protein can be conjugated with radionuclides, such as radioactive metal ions, for example, alpha or beta emitters. In a more specific embodiment, the radionuclide is conjugated with the recombinant binding protein via a chelating agent. Appropriate chelating agents can be selected based on the purpose of the application, such as diagnostic or therapeutic purposes.

[0397] In one embodiment, the recombinant binding protein described herein is conjugated to a pharmaceutical portion that serves as a diagnostic or detectable part. Such conjugates can be used to monitor or predict the onset, development, progression, and / or severity of a disease or condition as part of clinical trial procedures, such as determining the efficacy of a particular therapy and / or for diagnosing a disease. Such diagnostics and detections can be achieved by conjugating the recombinant binding protein described herein to a detectable substance, 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 tomography (PET) imaging techniques, as well as non-radioactive paramagnetic metal ions. In a more specific embodiment, the diagnostic part is a fluorophore, chromophore, imaging agent, or radionuclide.

[0398] Nucleic acid and vector

[0399] This document also provides nucleic acids encoding the recombinant binding proteins or ankyrin repeat domains described herein. Such nucleic acids may encode polypeptides comprising segments or domains of the recombinant binding proteins or ankyrin repeat domains described above. Due to the degeneracy of the code, multiple nucleic acid sequences will encode each ankyrin repeat domain sequence in the ankyrin repeat domain sequences described herein. Nucleic acids may comprise nucleotide sequences as shown in any of SEQ ID NO: 175 to 189, or sequences substantially identical thereto (e.g., sequences having at least about 85%, 90%, 95%, or 99% sequence identity, or sequences differing from any of SEQ ID NO: 175 to 189 by no more than 3, 6, 15, 30, or 45 nucleotides).

[0400] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 1 to 35 or 195 to 200. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 4. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 5. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 6. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 7. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 8. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 9. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 10. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding an 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 of the ankyrin repeat-binding domains or any of the ankyrin repeat-binding modules disclosed herein.

[0401] Also provided are nucleic acid molecules derived from any of SEQ ID NO: 175 to 189, which have been targeted to suitable host cells (such as prokaryotic host cells, for example, Escherichia coli). E. coli Protein expression in the vector has been optimized. Nucleic acids can be present in vectors (such as phage display vectors) or in recombinant plasmid vectors. Therefore, a cloning or expression vector comprising one or more nucleic acid sequences comprising any one of SEQ ID NO: 175 to 189 is also provided, wherein the vector is suitable for recombinant production of recombinant binding proteins as described herein.

[0402] To express the recombinant binding protein described herein, standard techniques can be used to transfect host cells with an expression vector that suitably contains a nucleic acid sequence encoding any of SEQ ID NO: 175 to 189 as described herein. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0403] In one implementation, the cloning or expression vector as described herein comprises a nucleic acid sequence of any of SEQ ID NO: 175 to 189 operatively linked to a suitable promoter sequence.

[0404] A vector comprising a nucleic acid encoding any of the recombinant binding proteins described herein is also provided, wherein the vector is a DNA vector, RNA vector, plasmid, granule, or viral vector.

[0405] When a recombinant expression vector encoding the recombinant binding protein disclosed herein is introduced into a suitable host cell (e.g., a prokaryotic host cell), the recombinant binding protein is generated by culturing the host cell for a period sufficient to allow the recombinant binding protein to be expressed in the host cell or secreted into the culture medium in which the host cell grows. The recombinant binding protein can be recovered from the host cell and / or culture medium using standard protein purification methods. In one embodiment, the method includes culturing a suitable host cell, such as a prokaryotic host cell as described herein, and collecting the recombinant binding protein from the host cell and / or culture medium.

[0406] Compositions, uses and treatments

[0407] In one aspect, a pharmaceutical composition is also provided comprising a recombinant binding protein as described herein and / or a nucleic acid encoding a recombinant binding protein as described herein, and pharmaceutically acceptable loaders, stabilizers, and / or diluents. Pharmaceutically acceptable loaders, excipients, and stabilizers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed., 1980.

[0408] Therefore, in one aspect, a pharmaceutical composition is provided comprising a recombinant binding protein and a pharmaceutically acceptable carrier, stabilizer, and / or diluent, wherein the recombinant binding protein comprises an ankyrin repeating domain having binding specificity to mesothelin, wherein the ankyrin repeating domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 1 to 35 or 195 to 200. Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art and will be described in more detail below.

[0409] Suitable carriers, diluents, excipients, or stabilizers include, for example, saline, Ringer's solution, dextran solution, Hank's solution, fixed oil, ethyl oleate, 5% dextran saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Pharmaceutically acceptable excipients generally do not have a significant adverse effect on patients receiving the composition; these excipients include proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination formulation containing additional active agents, such as anticancer agents or antiangiogenic agents, or other bioactive compounds. The composition intended for in vivo administration must be sterile or sterile. This is easily achieved through filtration using a sterile filter membrane.

[0410] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein, as well as a detergent (such as a nonionic 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.

[0411] The recombinant binding proteins with mesothelin binding specificity described herein have numerous in vitro and in vivo diagnostic and therapeutic uses in the diagnosis and treatment of conditions involving mesothelin-dependent pathophysiology. For example, these molecules can be administered in vitro or ex vivo to cells in cultures, or to human subjects, such as in vivo, to treat, prevent, and diagnose a variety of conditions involving mesothelin-dependent pathophysiology. Therefore, in one aspect, this document provides a method for treating a medical condition (e.g., a disease, such as cancer) in a subject of need, comprising administering a therapeutically effective amount of the recombinant binding protein described herein, a nucleic acid encoding such recombinant binding protein, a vector containing the nucleic acid encoding such recombinant binding protein, a cell containing such nucleic acid or the vector, or a pharmaceutical composition containing such recombinant binding protein, nucleic acid, vector, or cell.

[0412] On the other hand, a method is provided for treating a subject, for example, reducing or improving symptoms or conditions of excessive proliferation (e.g., cancer), such as solid tumors, hematologic cancers, soft tissue tumors, or metastatic lesions. Therefore, in one embodiment, a method is provided for inhibiting tumor cell growth, comprising administering, alone, a therapeutically effective amount of a recombinant mesothelin-binding protein as described herein, or in combination with other agents (e.g., therapeutic agents or modes of treatment) to a subject in need. In some embodiments, such a method may further include the step of determining the expression level of mesothelin (suitably soluble mesothelin) in a sample obtained from the subject (e.g., a tissue biopsy of the subject).

[0413] Also provided are the recombinant binding protein described herein, the nucleic acid encoding such recombinant binding protein, the vector containing the nucleic acid encoding such recombinant binding protein, the cell containing such nucleic acid or the vector, or the pharmaceutical composition containing such recombinant binding protein, nucleic acid, vector or cell, for use in treating medical conditions, such as diseases, such as cancer.

[0414] In one aspect, the use of the recombinant binding protein described herein, the nucleic acid encoding such recombinant binding protein, the vector comprising the nucleic acid encoding such recombinant binding protein, the cell comprising such nucleic acid or the vector, or the pharmaceutical composition comprising such recombinant binding protein, nucleic acid, vector or cell in the manufacture of a medicament, for example, for treating cancer.

[0415] In one embodiment, the recombinant binding protein with mesothelin binding specificity as described herein further comprises an immune cell activation portion, suitably a CD3-binding ankyrin repeat domain as described herein, a nucleic acid encoding the recombinant binding protein, or a pharmaceutical composition comprising the recombinant binding protein. In one embodiment, the activation is tumor-localized. In one embodiment, the immune cell is an adaptive immune cell. In one embodiment, the adaptive immune cell is a T cell.

[0416] Administration can include topical, oral, or parenteral administration. Parenteral administration is the most typical route. In parenteral administration, the pharmaceutical composition is formulated in combination with a suitable pharmaceutically acceptable excipient, such as those described above, into a unit-dose injectable form, such as a solution, suspension, or emulsion. The dosage and method of administration depend on the individual being treated and the disease.

[0417] The recombinant binding protein with mesothelin binding specificity, the nucleic acid encoding such recombinant binding protein, or the carrier or cell containing the nucleic acid encoding such recombinant binding protein, or the pharmaceutical composition containing such recombinant binding protein, nucleic acid, carrier, or cell described herein, can be administered to a subject extraintestinally, for example via intravenous, intratumoral, or subcutaneous routes. In some embodiments, such recombinant binding protein, nucleic acid, carrier, cell, or pharmaceutical composition is administered intravenously.

[0418] Dosing regimens are adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. It is particularly advantageous to formulate the parenteral composition in unit dosage form to facilitate administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of the active compound, which is calculated to bind with the desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form disclosed herein are determined by and directly depend on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the field of formulating such active compounds for treating individual sensitivities.

[0419] A "medical condition" may be a condition characterized by inappropriate cell proliferation. A medical symptom may be a condition of excessive proliferation. In one embodiment, the medical condition is a neoplastic disease. As used herein, the term "neoplastic disease" refers to an abnormal state or symptom of cells or tissues characterized by cell growth or rapid tumor proliferation. In one embodiment, the medical condition is a malignant neoplastic disease. Suitably, the medical condition or disease is cancer. In a preferred embodiment, the cancer is a cancer expressing mesothelin. In one embodiment, the cancer is ovarian cancer, fallopian tube cancer, peritoneal cancer, mesothelioma, or pancreatic ductal adenocarcinoma (PDAC). In one embodiment, the cancer is ovarian cancer. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0420] The recombinant binding protein, nucleic acid encoding such recombinant binding protein, vector containing nucleic acid encoding such recombinant binding protein, cell containing such nucleic acid or vector, or pharmaceutical composition containing such recombinant binding protein, nucleic acid, vector, or cell may also be used in combination with one or more other therapies known in the art. As used herein, the term "in combination with" means co-administration under a given regimen. This includes simultaneous administration of different compounds and staggered administration of different compounds (e.g., administering compound A once, followed by multiple administrations of compound B, or vice versa, or administering two compounds simultaneously and one of the compounds in a subsequent phase).

[0421] In one aspect, a kit is also provided comprising a recombinant binding protein comprising an ankyrin repeat domain having mesothelin binding specificity as described herein. In one aspect, a kit is provided comprising a nucleic acid encoding the recombinant binding protein disclosed herein. In one aspect, a kit is provided comprising a pharmaceutical composition as described herein. In one aspect, a kit is provided comprising the recombinant protein as described herein, and / or the nucleic acid as described herein, and / or the pharmaceutical composition as described herein. In one aspect, a kit is provided comprising a recombinant protein comprising an ankyrin repeat domain having binding specificity to mesothelin, such as a recombinant binding protein comprising an ankyrin repeat domain containing any of the amino acids in SEQ ID NO: 1 to 35 or 195 to 200; and / or a nucleic acid encoding a recombinant protein comprising an ankyrin repeat domain having binding specificity to mesothelin, such as a recombinant binding protein comprising an ankyrin repeat domain containing any of the amino acids in SEQ ID NO: 1 to 35 or 195 to 200; and / or a pharmaceutical composition comprising a recombinant protein comprising an ankyrin repeat domain having binding specificity to mesothelin, such as a recombinant binding protein comprising an ankyrin repeat domain containing any of the amino acids in SEQ ID NO: 1 to 35 or 195 to 200. In one aspect, a kit is provided comprising a recombinant protein as described herein, such as a recombinant binding protein comprising an ankyrin repeat domain containing an amino acid sequence of any one of SEQ ID NO: 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.

[0422] definition

[0423] The selected terms are defined below and throughout the specification. It should be understood that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention.

[0424] All publications, patents and registration numbers mentioned in this article are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually cited.

[0425] Unless otherwise claimed, any and all examples or exemplary language (such as "such as") provided herein are intended merely to better illustrate this disclosure and not to limit its scope.

[0426] As used in this article, the articles “a” and “a kind” can mean “one”, but their usage is also consistent with the meanings of “one or more”, “at least one” and “one or more”.

[0427] Unless the context clearly indicates otherwise, the term “or” is used herein to mean the term “and / or” and is used interchangeably with that term, and should be interpreted as meaning an inclusive “or” of any one or any combination thereof.

[0428] Throughout the specification and the following claims, and unless the context otherwise requires, the terms “comprising,” “having,” and “including” are used interchangeably. The terms “comprising” and “including” should be interpreted as specifying the presence of the stated features or components mentioned, but do not exclude the presence or addition of one or more features or components or groups thereof. Furthermore, the terms “comprising” and “including” are intended to include examples covered by the term “consisting of.” Therefore, the term “consisting of” may be used in place of the terms “comprising” and “including” to provide more specific embodiments of the invention.

[0429] The terms “about” and “approximately” should generally be intended to indicate the acceptable degree of error for a quantity measured given the nature or precision of the measurement. An exemplary degree of error is within 10% of a given value or a range of values. For example, when a dose is mentioned as “about” a specific value, it is intended to include a range of approximately 10% plus or minus the specified value. It should be understood that, although not always explicitly stated, all numerical names are preceded by the term “about”.

[0430] It should also be understood that, although not always explicitly stated, the reagents described herein are merely examples, and equivalents of these reagents are known in the art.

[0431] As used herein, the term "peptide" refers to a molecule comprising a chain of multiple amino acids linked by peptide bonds. Preferably, a polypeptide consists of eight or more amino acids linked by peptide bonds. Furthermore, the term also encompasses peptides modified by, for example, glycosylation, and proteins comprising two or more polypeptide chains cross-linked by, for example, disulfide bonds.

[0432] As used herein, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or is capable of acquiring a defined three-dimensional arrangement by forming secondary, tertiary, and / or quaternary structures within and / or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be non-covalently or covalently linked, for example, by disulfide bonds between the two polypeptides. A portion of a protein that individually has or is capable of acquiring a defined three-dimensional arrangement by forming secondary and / or tertiary structures is referred to as a "protein domain".

[0433] The term "recombinant" as used in "recombinant protein," "recombinant polypeptide," etc., refers to the protein or polypeptide being produced using recombinant DNA techniques known to those skilled in the art. For example, a "recombinant DNA molecule" encoding a polypeptide (e.g., produced through gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or into DNA capable of in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., *Escherichia coli*), the recombinant expression plasmid can be effectively expressed. Escherichia coli In this process, these bacteria can produce polypeptides encoded by the recombinant DNA. The corresponding polypeptides or proteins produced are called "recombinant polypeptides" or "recombinant proteins".

[0434] As used herein, the term "binding protein" refers to a protein containing at least one binding domain. Binding proteins may also contain two, three, four, five, or more binding domains. Preferably, the binding protein is a recombinant binding protein. The recombinant binding proteins of this disclosure contain an ankyrin repeating domain that is specific for binding to human mesothelin (e.g., membrane-bound mesothelin or soluble mesothelin). Furthermore, any such binding protein may contain additional polypeptides (such as, for example, polypeptide tags, peptide linkers, fusions with other protein domains having binding specificity, cytokines, hormones, or antagonists), or chemical modifications known to those skilled in the art (such as coupling to polyethylene glycol, toxins (e.g., DM1), small molecules, antibiotics, etc.).

[0435] The term "binding domain" refers to a protein domain that has target-specific binding properties. Preferably, the binding domain is a recombinant binding domain.

[0436] As used herein, the term "target" refers to a single molecule, such as a nucleic acid molecule, polypeptide or protein, carbohydrate, or any other naturally occurring molecule, including any part of such a single molecule, or a complex of two or more such molecules, or a whole cell or tissue sample, or any non-natural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein containing chemical modifications (e.g., natural or non-natural phosphorylation, acetylation, or methylation). In the context of this disclosure, mesothelin protein expressed on tumor cells is a target of mesothelin-specific binding proteins. Preferably, the target is human mesothelin.

[0437] Mesothelin can be derived from multiple different isoforms of the mesothelin precursor protein. The recombinant binding protein described herein can bind to mesothelin derived from any precursor isoform. The MSLN gene encodes a 622-residue (69 kDa) precursor protein identified as pre-pro mesothelin (e.g., major isoform of UniProt ID Q13421-3; SEQ ID NO: 167). Upon insertion into the cell membrane, the N-terminal signal peptide containing residues 1-33 of SEQ ID NO: 167 undergoes cleavage. Truncation of C-terminal residues 599-622 suggests the addition of a GPI anchor at S598. Cleavage of the precursor protein yields a 303-amino acid residue (40 kDa) GPI-anchored fragment, termed "membrane-bound mesothelin," and a 31 kDa secreted fragment, also known as megakaryocyte enhancer factor (MPF). Mesothelin can detach from the cell membrane via proteolytic cleavage at one of several cleavage sites in the proximal region of the mesothelin membrane, producing truncated mesothelin protein and soluble mesothelin retained on the cell surface. As used herein, the term "soluble mesothelin" refers to amino acid sequence 296-580 of mesothelin, while "membrane-bound mesothelin" refers to amino acid sequence 296-597, both referring to UniProt ID Q13421-3.

[0438] The term "CD3" or "differentiation cluster 3" refers to a multimeric protein complex composed of four distinct chains. In mammals, the complex contains a CD3γ (γ) chain, a CD3δ (δ) chain, and two CD3ε (ε) chains. These chains associate with the T cell receptor (TCR) and the CD3ζ (ζ) chain to generate activation signals in T cells. CD3 is essential 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 coordinating adaptive immune responses. The amino acid sequences of the human CD3 γ, δ, ε, and ζ chains are shown in NCBI Ref. Seq. NP_000064.1, NP_000723.1, NP_000724.1, and NP_932170.1, respectively. The ankyrin repeat domain that specifically binds to CD3 is disclosed in WO 2022 / 129428 (incorporated by reference).

[0439] Patent application WO 2002 / 020565 and Forrer et al., ( FEBS Letters 539, 2-6, 2003) contains a general description of the features, techniques, and applications of repeating proteins and repeating domains. The term "repeating protein" refers to a protein containing one or more repeating domains. Preferably, a repeating protein contains one, two, three, four, five, or six repeating domains. Furthermore, the repeating protein may contain additional non-repeating protein domains, peptide tags, and / or peptide linkers. Repeating domains may be binding domains.

[0440] The term "repetitive domain" refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein the repeating modules have structural and sequence homology. Preferably, the repetitive domain further comprises an N-terminal and / or C-terminal capping module. For clarity, the capping module may be a repeating module. Such repetitive domains, repeating modules and capping modules, sequence motifs, and structural and sequence homology are known to those skilled in the art based on the following examples: ankyrin repeating domain (WO 2002 / 020565), leucine-rich repeating domain (WO 2002 / 020565), 34-peptide repeating domain (Main, ER et al., Structure 11(5), 497-508, 2003) and armadillo repeat domain (WO 2009 / 040338). Those skilled in the art also know that such repeat domains differ from proteins containing repeating amino acid sequences, where each repeating amino acid sequence is capable of forming a single domain (e.g., the FN3 domain of fibronectin).

[0441] The term "ankylin repeat domain" refers to a repeat domain that contains two or more consecutive ankylin repeat modules as structural units. Standard recombinant DNA techniques can be used (see, for example, Forrer, P. et al., FEBS letters 539, 2-6, 2003; WO 2002 / 020565, WO 2016 / 156596; WO 2018 / 054971).

[0442] As used herein, the term "construct" refers to a recombinant binding protein comprising one or more designed ankyrin repeat domains and optionally a peptide linker and / or a tag sequence. Examples of peptide linkers are provided in SEQ ID NO: 164, and examples of tag sequences are provided in SEQ ID NO: 163. For clarity, the term "ankyrin repeat domain" includes at least one N-terminal capping module and at least one C-terminal capping module.

[0443] As used in terms such as "designed repeating proteins" and "designed repeating domains," the term "designed" refers to the fact that such repeating proteins and repeating domains are artificial and do not exist in nature.

[0444] The term "target-interacting residue" refers to amino acid residues in repeating modules that facilitate direct interaction with a target.

[0445] The term "framework residue" refers to an amino acid residue in a repeating module that contributes to the folding topology, i.e., facilitates the folding of the repeating module or facilitates interaction with adjacent modules. Such contributions can be interactions with other residues in the repeating module, influences on the conformation of the polypeptide backbone, such as in α-helices or β-sheets, or involve amino acid elongation in the formation of straight-chain polypeptides or rings. These framework-target interaction residues can be identified by analyzing structural data obtained using physicochemical methods such as X-ray crystallography, NMR, and / or CD spectroscopy, or by comparison with known and relevant structural information.

[0446] The term "repetitive module" refers to a repetitive amino acid sequence and structural unit of a designed repetitive domain, originally derived from a repetitive unit of a naturally occurring repetitive protein. Each repetitive module contained within a repetitive domain is derived from one or more repetitive units of a naturally occurring family or subfamily of repetitive proteins (preferably the ankylosing repetitive protein family). Furthermore, each repetitive module contained within a repetitive domain may contain a "repetitive sequence motif" derived from a homologous repetitive module that is obtained from a target-selected repetitive domain and has the same target specificity. As used herein, "repetitive module" encompasses internal repetitive modules and capped modules, such as N-terminal and C-terminal capped modules. An "internal repetitive module" refers to a repetitive module side-joined with two repetitive modules. In other words, an internal repetitive module side-joins one repetitive module at the N-terminus and another repetitive module at the C-terminus. Examples of internal repetitive modules are provided in SEQ ID NOs: 43 to 78, 101 to 112, 132 to 151, and 203 to 205. Examples of N-end sealing modules are provided in SEQ ID NO: 79 to 90, 113 to 115, 121 to 131 and 201 to 202. Examples of C-end sealing modules are provided in SEQ ID NO: 91 to 100, 116 to 120 and 152 to 160.

[0447] Therefore, the term "ankylin repeat module" refers to a repeat module that originates from repeating units of an existing ankylin repeat protein. Ankylin repeat proteins are known to those skilled in the art.

[0448] The term "repetitive sequence motif" refers to an amino acid sequence derived from one or more repeating modules. Preferably, the repeating modules originate from repeating domains that have binding specificity to the same target. Such repetitive sequence motifs include framework residue positions and target-interacting residue positions. The framework residue positions correspond to the framework residue positions of the repeating module. Similarly, the target-interacting residue positions correspond to the target-interacting residue positions of the repeating module. Repetitive sequence motifs include non-randomized positions and randomized positions.

[0449] The term "repetitive unit" refers to an amino acid sequence comprising one or more naturally occurring protein sequence motifs, wherein the "repetitive unit" exists in multiple copies and exhibits a defined folding topology common to all said motifs that define protein folding. Examples of such repetitive units include leucine-rich repetitive units, ankyrin repetitive units, armadillo repetitive units, 34-peptide repetitive units, HEAT repetitive units, and leucine-rich variant repetitive units.

[0450] The repeat module may contain positions of amino acid residues in the library that are not randomized for the purpose of selecting target-specific repeat domains (“non-randomized positions”) and positions of amino acid residues in the library that are randomized for the purpose of selecting target-specific repeat domains (“randomized positions”). Non-randomized positions contain framework residues. Randomized positions contain target-interacting residues. “Randomized” means that two or more amino acids are allowed at the amino acid position of the repeat module, for example, allowing any one of the typically twenty naturally occurring amino acids, or allowing most of the twenty naturally occurring amino acids, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline. For the purposes of this disclosure, amino acid residues 3, 4, 6, 11, 14, and 15 of SEQ ID NO: 43 to 78, 101 to 112, 132 to 151, and 203 to 205 are randomized positions of the internal repeating module; amino acid residues 4, 8, 11, and 12 of SEQ ID NO: 79 to 90, 113 to 115, 121 to 131, and 201 to 202 are randomized positions of the N-terminal capped repeating module; and amino acid residues 3, 4, 6, 14, and 15 of SEQ ID NO: 91 to 100, 116 to 120, and 152 to 160 are randomized positions of the C-terminal capped repeating module. Amino acid residues 1 to 3, 5 to 7, 9, 10, and 13 to 30 of SEQ ID NO: 79 to 90, 113 to 115, 121 to 131, and 201 to 202 are framework residues contained in the N-terminal capped repeat module; amino acid residues 1, 2, 5, 7 to 10, 12, 13, and 16 to 33 of SEQ ID NO: 43 to 78, 101 to 112, 132 to 151, and 203 to 205 are framework residues contained in the inner repeat module; and amino acid residues 1, 2, 5, 7 to 13, and 16 to 28 of SEQ ID NO: 91 to 100, 116 to 120, and 152 to 160 are framework residues contained in the C-terminal capped repeat module. For the purposes of this disclosure, substitutions in the framework positions should apply to all embodiments, regardless of whether such substitutions are explicitly described.

[0451] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by 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 having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0452] Exemplary and conserved amino aci...

Claims

1. A recombinant binding protein comprising an ankyrin repeat domain having binding specificity to membrane-bound mesothelin (mbMSLN).

2. The recombinant binding protein according to claim 1, wherein the ankyrin repeat domain comprises an ankyrin repeat module, the ankyrin repeat module comprising an amino acid sequence of any one of SEQ ID NO: 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 in any one of SEQ ID NO: 62 to 78 is replaced by other amino acids.

3. The recombinant binding protein according to any one of claims 1 or 2, wherein the ankyrin repeat domain comprises a first ankyrin repeat module and a second ankyrin repeat module, and optionally a third ankyrin repeat module, wherein optionally the first ankyrin repeat module, the second ankyrin repeat module and the optional third ankyrin repeat module each independently comprises an amino acid sequence of any one of SEQ ID NO: 62 to 78, or 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 sequence of any one of SEQ ID NO: 62 to 78 is replaced by other amino acids.

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

5. The recombinant binding protein according to any one of the preceding claims, further comprising an N-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 79, 87 to 90 or 201, or an amino acid sequence in which 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 in any one of SEQ ID NO: 79, 87 to 90 or 201 are substituted with other amino acids; and / or a C-terminal capping module comprising an amino acid sequence of any one of SEQ ID NO: 91, 98 to 100, or an amino acid sequence in which 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 in any one of SEQ ID NO: 91, 98 to 100 are substituted with other amino acids.

6. The recombinant binding protein according to any one of the preceding claims, wherein the ankyrin repeating domain comprises 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% of the same amino acid sequence as any one of SEQ ID NO: 9 to 35 or 195 to 200.

7. The recombinant binding protein according to any one of the preceding claims, wherein the recombinant binding protein contains at least 10 mg / L or less in PBS. -7 M, or is or is less than approximately 10 -8 M, or is or is less than approximately 10 -9 M, or is or is less than approximately 10 -10 M, or is or is less than approximately 10 -11 The dissociation constant (KD) of M binds to human membrane-bound mesothelin, and / or the recombinant binding protein binds to human mesothelin at an EC50 of about 0.1 nM to about 10 nM.

8. The recombinant binding protein according to any one of the preceding claims, the recombinant binding protein further comprising at least one binding moiety having binding specificity to a protein expressed on an immune cell, suitably a T lymphocyte (T cell), optionally wherein the binding moiety comprises an ankyrin repeat domain having binding specificity to CD3, the ankyrin repeat domain optionally comprising 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% of the same amino acid sequence as any of SEQ ID NO: 36 to 40.

9. The recombinant binding protein according to any one of the preceding claims, the recombinant binding protein further comprising at least one half-life extension portion, optionally wherein the half-life extension portion comprises an ankyrin repeat domain that binds to human serum albumin, the ankyrin repeat domain optionally comprising at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 41 to 42.

10. The recombinant binding protein according to any one of the preceding claims, wherein the recombinant binding protein further comprises a pharmaceutical portion, optionally wherein the pharmaceutical portion is a radionuclide or a cytotoxin.

11. A nucleic acid that encodes a recombinant binding protein according to any one of the preceding claims.

12. A vector comprising the nucleic acid according to claim 11, optionally wherein the vector is a DNA vector, RNA vector, plasmid, granule or viral vector.

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

14. A method for producing a recombinant binding protein, the method comprising culturing cells according to claim 13 and collecting the recombinant binding protein from the cells and / or culture medium.

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

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

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