Metherin-binding protein and its use

Abstract

Disclosed are single domain antibodies that specifically bind to mesothelin, mesothelin-binding proteins, anti-mesothelin antibodies and antibody fragments thereof, antibody-drug conjugates, synthetic immunoreceptors, and diagnostic agents containing the same. Also disclosed are pharmaceutical compositions containing any of the foregoing, and the use of any of the foregoing in the treatment and / or diagnosis and / or monitoring of diseases associated with mesothelin expression.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 392,581, filed July 27, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing The computer-readable amino acid sequence listing submitted herewith is incorporated by reference in its entirety and is identified as follows: 84,748 byte XML file entitled 10189-WO01-SEC_SequenceListing_24July2023; created July 24, 2023.

[0003] Disclosed herein are single-domain antibodies and mesothelin-binding proteins that specifically bind to mesothelin (MSLN), anti-mesothelin antibodies and antibody fragments thereof, antibody-drug conjugates, synthetic immunoreceptors, and diagnostic agents containing the same. Also disclosed are pharmaceutical compositions containing any of the foregoing, and uses of any of the foregoing in the treatment and / or diagnosis and / or monitoring of diseases associated with MSLN expression. [Background technology]

[0004] Mesothelin (MSLN; also known as CAK1; UniProt Q13421; HGNC ID 7371) is a tumor-associated antigen widely expressed as a cell surface glycoprotein on various malignant tumor cells. Mesothelin (MSLN) was first identified in 1992 by Pastan et al. using the monoclonal antibody (mAb) K1, generated by immunization of mice with human ovarian carcinoma (OVCAR-3) cells (Chang, et al., Int J. Cancer. (1992) 50:373-81). MSLN was purified from the human pancreatic cancer cell line HPC-Y5 and shown to have megakaryocyte-enhancing activity. The MSLN gene encodes a 71-kDa precursor protein that is cleaved by furin into two products: an amino-terminal shed fragment called megakaryocyte potentiating factor (MPF; 31 kDa) and the glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein MSLN (40 kDa), which remains tethered to the plasma membrane via a GPI linkage. The MSLN protein is organized into a coiled coil with ARM-type repeats.

[0005] Although both MPF and MDLN exhibit physiological activity, their precise biological functions remain unclear. Knockout mice in which the mesothelin gene was disrupted by homologous recombination have been generated (Bera, T. K., and Pastan, I. (2000) Mol. Cell. Biol. 20:2902-2906). No anatomical, hematological, or reproductive abnormalities were detected, indicating that mesothelin function is not essential for growth or reproduction, at least in these knockout mice. MSLN specifically interacts with MUC16 (CA125), a mucin-like glycoprotein present on the surface of tumor cells that was previously identified as an ovarian cancer antigen. MSLN has been associated with tumor cell proliferation and migration (Rump A et al., J. Biol. Chem. 2004 Mar. 5;279(10):9190-8), and mesothelin-MUC16 interaction has been proposed to function in cell adhesion, invasion, and metastasis. For example, mesothelin expression in the peritoneal lining correlates with preferential sites of ovarian cancer metastasis formation, and mesothelin-MUC16 binding is thought to promote peritoneal metastasis of ovarian tumors (Gubbels, JA et al. (2006) Mol. Cancer. 5:50).

[0006] MSLN is highly expressed in several tumor types, including mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer (Hassan et al., Eur J Cancer (2008) 44:46-53; Ordonez, Am J Surg Pathol (2003) 27:1418-28; Ho et al., Clin Cancer Res (2007) 13:1571-5). However, normal tissue expression of MSLN is limited to mesothelial cells in the pleura, pericardium, and peritoneum, as well as the surface epithelium of normal ovaries, fallopian tubes, and tonsils, making MSLN a promising target for antibody-drug conjugates, monoclonal antibodies, and CAR-T cell therapy (Hassan R et al., J Clin Oncol. 2016 Dec;34(34):4171-4179). Furthermore, MSLN-binding agents can be used as markers for the diagnosis and prognosis of certain types of cancer, because trace amounts of mesothelin can be detected in the blood of some patients with mesothelin-positive cancers (Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007). Overexpression of MSLN is associated with poor prognosis in, for example, lung adenocarcinoma and triple-negative breast cancer.

[0007] In addition to its cell surface expression, mesothelin is also shed into the serum through the action of ADAM17 / TACE. Serum levels of shed mesothelin are elevated in patients with ovarian and other cancers. MESOMARK®, an ELISA test for shed serum mesothelin, has been approved by the FDA for compassionate use and may aid in the diagnosis or monitoring of mesothelioma. Shed mesothelin has also been used, alone or in combination with other markers, to aid in the diagnosis or prognosis of other cancer types. Correlation of serum levels of shed mesothelin with disease has suggested a potential role for the mesothelin protein in cancer progression.

[0008] Therefore, there is a need for additional and effective therapeutic and diagnostic agents for diseases associated with mesothelin expression. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Chang, et al., Int J. Cancer. (1992) 50:373-81 [Non-patent document 2] Bera, TKand Pastan, I. (2000)Mol.Cell.Biol.20:2902-2906 [Non-patent document 3] Rump A et al., J Biol Chem.2004 Mar 5;279(10):9190-8 [Non-patent document 4] Gubbels,JAet al.(2006)Mol.Cancer.5:50 [Non-patent document 5] Hassan et al.,Eur J Cancer(2008)44:46-53 [Non-patent document 6] Ordonez, Am J Surg Pathol (2003) 27:1418-28 [Non-Patent Document 7] Ho et al., Clin Cancer Res (2007) 13:1571-5 [Non-patent document 8] Hassan R et al.,J Clin Oncol.2016 Dec;34(34):4171-4179 [Non-Patent Document 9] Cristaudo et al.,Clin.Cancer Res.13:5076-5081,2007 Summary of the Invention [Means for solving the problem]

[0010] Disclosed herein are single domain antibodies that specifically bind to mesothelin (MSLN), for example, human MSLN.

[0011] In some embodiments, the single domain antibody comprises: (i) Sequence A VH complementarity determining region 1 (CDR1) comprising GFX1FX2SSYX3 (SEQ ID NO: 80), a VH complementarity determining region 1 (CDR1) in which X1 is T or S, X2 is S or T, and X3 is D, P, or A; (ii) Sequence IX4X5X6GX7X8X9 (SEQ ID NO: 81), X4 is S or T, X5 is Y, G, or S, X6 is D or S, X7 is S, G, or D, X8 is K, G, or S, and X9 is K or T; and (iii) Sequence AX 10 PPSYYDFLSDPDY (SEQ ID NO: 82), In the formula, X 10 is R or K; and a VH CDR3,

[0012] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35.

[0013] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence having up to two (e.g., zero, one, two) amino acid modifications compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and (ii) a VH CDR2 comprising a sequence having up to two (e.g., zero, one, two) amino acid modifications compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; and (iii) a VH CDR3 comprising a sequence having up to two (e.g., 0, 1, 2) amino acid modifications compared to SEQ ID NO: 13 or SEQ ID NO: 14.

[0014] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution. In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution listed in Table A1.

[0015] In some embodiments, the VH CDR1 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the VH CDR3 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:13 or SEQ ID NO:14. In some embodiments, the at most one amino acid modification is an amino acid substitution. In some embodiments, the at most one amino acid modification is a conservative amino acid substitution. In some embodiments, the at most one amino acid modification is an amino acid deletion. In some embodiments, the at most one amino acid modification is an amino acid addition.

[0016] In some embodiments, VH CDR1 comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, VH CDR2 comprises a sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, VH CDR3 comprises a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0017] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (ii) a VH CDR2 comprising a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; and (iii) a heavy chain variable (VH) region comprising: a VH CDR3 comprising a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0018] In some embodiments, the single domain antibody comprises: (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively; (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively; (k) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively; (l) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively; or (m) a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively.

[0019] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 15-35.

[0020] In some embodiments, the VH CDR1, VH CDR2 and VH CDR3 sequences are present in a human VH framework.

[0021] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 95% sequence identity to any one of SEQ ID NOs: 15-35.

[0022] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region selected from SEQ ID NOs: 15-35.

[0023] In some embodiments, the single domain antibody specifically binds to human MSLN. In some embodiments, the single domain antibody specifically binds to human MSLN. -9 M~10 -6 K of M D It binds to human MSLN.

[0024] In some embodiments, the single domain antibody is an isolated single domain antibody.

[0025] Also disclosed herein are mesothelin binding proteins, including single domain antibodies that specifically bind to mesothelin as described herein.

[0026] In some embodiments, the mesothelin-binding protein specifically binds to human MSLN. In some embodiments, the mesothelin-binding protein is about 10 -9 M~10 -6 K of M D It binds to human MSLN.

[0027] In some embodiments, the mesothelin-binding protein further binds to one or more target antigens other than mesothelin. In some embodiments, the mesothelin-binding protein is multispecific. In some embodiments, the mesothelin-binding protein is bispecific.

[0028] In some embodiments, the mesothelin binding protein further specifically binds to CD3. In some embodiments, the mesothelin binding protein further specifically binds to human CD3. In some embodiments, the mesothelin binding protein further specifically binds to human CD3 epsilon. In some embodiments, the mesothelin binding protein binds to an epitope on CD3 that includes at least one residue selected from CD3 epsilon (SEQ ID NO: 86): K73 and S83; and CD3 delta (SEQ ID NO: 87) K82 and C93. In some embodiments, the epitope on CD3 includes a region of CD3 epsilon defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. In some embodiments, the epitope on CD3 comprises a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, S83. In some embodiments, the epitope comprises a conformational epitope having residues of both CD3 delta and CD3 epsilon. In some embodiments, the conformational epitope comprises residues CD3ε K73 and S83; CD3δ K82 and C93, respectively.

[0029] In some embodiments, the mesothelin-binding protein further comprises a CD3-binding VH region. In some embodiments, the mesothelin-binding protein further comprises a CD3-binding VH region paired with a light chain (VL) region.

[0030] In some embodiments, the CD3-binding VH region is (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having up to two (e.g., 0, 1, or 2) amino acid modifications compared to any one of SEQ ID NOs: 36 to 41; and (ii) a VH CDR2 comprising a sequence having up to two (e.g., zero, one, or two) amino acid modifications relative to SEQ ID NO: 42; and (iii) a VH CDR3 comprising a sequence having up to two (e.g., 0, 1, or 2) amino acid modifications compared to any one of SEQ ID NOs: 43 to 46.

[0031] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution. In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution listed in Table A1.

[0032] In some embodiments, the CD3-binding VH CDR1 comprises a sequence with at most one amino acid modification compared to any one of SEQ ID NOs: 36-41. In some embodiments, the CD3-binding VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO: 42. In some embodiments, the CD3-binding VH CDR3 comprises a sequence with at most one amino acid modification compared to any one of SEQ ID NOs: 43-46. In some embodiments, the at most one amino acid modification is an amino acid substitution. In some embodiments, the at most one amino acid modification is a conservative amino acid substitution. In some embodiments, the at most one amino acid modification is an amino acid deletion. In some embodiments, the at most one amino acid modification is an amino acid addition.

[0033] In some embodiments, the CD3-binding VH CDR1 comprises a sequence selected from SEQ ID NOs: 36-41. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42. In some embodiments, the CD3-binding VH CDR3 comprises a sequence selected from SEQ ID NOs: 43-46.

[0034] In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64.

[0035] In some embodiments, the CDR3-binding VH region comprises the CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 47-64.

[0036] In some embodiments, the CD3-binding VH region is (i) Array GFTFX 11 X 12 A VH complementarity determining region 1 (CDR1) comprising YA (SEQ ID NO: 83), In the formula, X 11 is D, A, or H, and X 12is D or N; and (ii) a VH CDR2 comprising the sequence ISWNSGSI (SEQ ID NO: 42); and (iii) sequence AKDSRGYGX 13 YX 14 X 15 a VH CDR3 comprising GGAY (SEQ ID NO: 84), In the formula, X 13 is D or S, and X 14 is R or S, and X 15 is L or R.

[0037] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are present within a human VH framework.

[0038] In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 95% sequence identity to any one of SEQ ID NOs: 47-64.

[0039] In some embodiments, the CD3-binding VH region is (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 43, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 44, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 45, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 46, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 37, 42, and 44, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 38, 42, and 44, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 39, 42, and 44, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 44, respectively; (i) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 45, respectively; or (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 41, 42, and 45, respectively.

[0040] In some embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 68. In some embodiments, the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 comprising the sequences of SEQ ID NOs: 65, 66, and 67, respectively. In some embodiments, the VL CDR1, VL CDR2, and VL CDR3 sequences are present in a human VH framework.

[0041] In some embodiments, the light chain variable region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 95% sequence identity to SEQ ID NO: 68.

[0042] In some embodiments, the mesothelin binding protein is an anti-mesothelin antibody or a fragment thereof. In some embodiments, the anti-mesothelin antibody is a monoclonal antibody or a fragment thereof. In some embodiments, the anti-mesothelin antibody is an isolated monoclonal antibody or a fragment thereof.

[0043] In some embodiments, the anti-mesothelin antibody is an anti-IgG1 antibody. In some embodiments, the anti-mesothelin antibody is an anti-IgG2 antibody. In some embodiments, the anti-mesothelin antibody is an anti-IgG4 antibody.

[0044] In some embodiments, the mesothelin-binding protein is an antibody fragment. In some embodiments, the mesothelin-binding protein is a heavy-chain-only antibody. In some embodiments, the mesothelin-binding protein is a three-chain antibody-like molecule.

[0045] In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises an Fc region. In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises a variant Fc region. In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the Fc region is a silenced Fc region.

[0046] Also disclosed herein are polynucleotides that encode single domain antibodies that specifically bind to mesothelin described herein.

[0047] Also disclosed herein are compositions comprising one or more polynucleotides encoding the mesothelin-binding proteins described herein. In some embodiments, the mesothelin-binding protein is an anti-mesothelin antibody or fragment thereof.

[0048] Also disclosed herein are recombinant expression vectors comprising the single domain antibodies that specifically bind to mesothelin described herein, as well as host cells comprising the recombinant expression vectors.

[0049] Also disclosed herein are one or more recombinant expression vectors comprising one or more polynucleotides encoding the mesothelin-binding proteins described herein, as well as host cells comprising one or more recombinant expression vectors.

[0050] Also disclosed herein are synthetic immunoreceptors comprising single domain antibodies that specifically bind to mesothelin described herein, as well as cells comprising the synthetic immunoreceptors.

[0051] Also disclosed herein are antibody-drug conjugates comprising the single domain antibodies that specifically bind to mesothelin described herein. In some embodiments, the antibody-drug conjugates are for use in diagnostic applications, such as, for example, the detection or monitoring of a disease associated with expression of mesothelin, such as, for example, a proliferative disease or cancer.

[0052] Also disclosed herein are pharmaceutical compositions comprising a mesothelin binding protein, an antibody-drug conjugate, or an anti-mesothelin antibody or fragment thereof, and a pharmaceutically acceptable excipient.

[0053] Also disclosed herein are methods of treating a disease associated with mesothelin expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease and cancer. In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0054] Also disclosed herein are mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein for use in treating a disease associated with mesothelin expression. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease and a cancer. In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0055] Also disclosed herein is the use of a mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein in the manufacture of a medicament for the treatment of a disease associated with mesothelin expression. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease and a cancer. In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. [Brief explanation of the drawings]

[0056] [Figure 1A] 1 shows a representative CHO cell binding dose curve for an exemplary single domain antibody of the disclosure, where the CHO cells express human MSLN. [Figure 1B] 1 shows a representative HeLa cell binding dose curve for an exemplary single domain antibody of the disclosure. [Figure 1C]Representative CHO cell binding dose curves for an exemplary single domain antibody of the present disclosure are shown, where the CHO cells do not express the MSLN protein. In Figures 1A-1C, the mean fluorescence intensity of PE is plotted as fold over background (i.e., cells incubated with the secondary detection antibody only). [Figure 2A] FIG. 1 is a schematic diagram of a CAR-T structure comprising an anti-MSLN extracellular binding domain comprising an antibody sequence described herein. [Figure 2B] Shown is T cell activity of Jurkat cells transfected with anti-MSLN 394940 CAR with CHO-huMSLN (***p=0.0006) and HeLa (*p=0.016). DETAILED DESCRIPTION OF THE INVENTION

[0057] Definition: In some embodiments, "about," when used in connection with a measurable numerical variable, refers to the stated value of the variable and all values ​​of the variable that are within experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or ±10% of the stated value, whichever is greater. In some embodiments, numerical ranges are inclusive of the numbers (i.e., endpoints) that define the range.

[0058] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0059] As used herein, the terms "a" and "an" mean "one or more" unless specifically indicated otherwise. Furthermore, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context otherwise requires, singular terms include plurals and plural terms include the singular.

[0060] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (e.g., light chain polypeptides each approximately 25 kDa) and two heavy chain polypeptides (e.g., heavy chain polypeptides each approximately 50-70 kDa). The terms "light chain" or "immunoglobulin light chain" as used herein refer to a polypeptide comprising, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG-class and IgA-class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains. In some embodiments, antibodies of the present disclosure are human or humanized antibodies of the IgG type. In some embodiments, antibodies of the present disclosure are human or humanized antibodies and can be of the IgG1, IgG2, IgG3, or IgG4 type.

[0061] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) joined by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein (e.g., MSLN or CD3). From N- to C-terminus, both naturally occurring light and heavy chain variable regions typically have the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDRs and FRs of a given antibody can be identified using this system. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). In some embodiments of the present disclosure, "CDR" refers to the complementarity determining region of an antibody as defined in Lefranc, MP et al., IMGT, the International ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999).

[0062] "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as herein defined.

[0063] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1 to 113 of the heavy chain) (e.g., according to Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domains of antibodies, single domain antibodies, antibody fragments, etc. refer to the numbering of residues according to the EU numbering system.

[0064] As used herein, an "anti-mesothelin antibody" is an antibody that specifically binds to mesothelin (MSLN).

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), a monoclonal antibody generally is directed against a single determinant on an antigen. As a non-limiting example, monoclonal antibodies according to the present disclosure can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be made by recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0066] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0067] As used herein, an "antibody fragment" generally refers to a fragment of a full-length antibody, such as, for example, a VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "r IgG" (a "half antibody" consisting of a heavy and light chain), or a modified fragment of a full-length antibody, such as, for example, a three-chain antibody-like molecule, a heavy-chain-only antibody, a single-chain variable fragment (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, a single-chain Fab (scFab), Fab2, Fab3, a diabody, a single-chain diabody, a tandem diabody (Tandabs), a tandem di-scFv, a tandem tri-scFv, or the following: (VH-V L-CH3), (scFv-CH3), ((scFv)-CH3) or (scFv-CH3-scFv), multibodies (e.g., triabodies or tetrabodies) and single domain antibodies (e.g., nanobodies or single variable domain antibodies comprising only one variable region which may be a VHH, VH or VL, which specifically binds to an antigen or target independent of other variable regions or domains).

[0068] As used herein, the term "heavy chain-only antibody" refers to a dimeric immunoglobulin protein composed of two heavy chain polypeptides (e.g., heavy chain polypeptides each approximately 50-70 kDa). A "heavy chain-only antibody" is an antibody fragment that lacks the two light chain polypeptides found in conventional antibodies. In some embodiments, a "heavy chain-only antibody" is a homodimeric antibody comprising a VH antigen-binding domain and CH2 and CH3 constant domains, in the absence of a CH1 domain. In some embodiments, a heavy chain-only antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3, and framework 4. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Also encompassed herein are heavy chain-only antibodies in which the CH2 and / or CH3 domains have been truncated. The heavy chain-only antibodies described herein may belong to the IgG subclass, although heavy chain-only antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also encompassed herein. In some embodiments, the heavy chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, e.g., the IgG1 or IgG4 subtype. In some embodiments, the heavy chain-only antibody is of the IgG1 or IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibody is of the IgG1 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. Modifications of CH domains to alter effector function are further described herein.Non-limiting examples of heavy chain-only antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0069] As used herein, a "single-domain antibody" refers to a single-chain polypeptide that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (i.e., a single-domain antibody contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of a human antibody).

[0070] As used herein, the term "three-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody or an antigen-binding fragment of such an antibody chain comprising an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion comprising a CH2, CH3, and / or CH4 domain in the absence of a CH1 domain, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, wherein such binding domains comprise, consist essentially of, or consist of heavy chain sequences derived from or having sequence identity with the variable regions of antibody heavy or light chains. Portions of such variable regions are V H and / or V L Gene segments, D and J H Gene segment or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L or V LJ L It can be encoded by a gene segment.

[0071] As used herein, an "antigen-binding fragment" refers to a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chains, but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy chain antibody; VHH fragments, see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and CDR fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel.

[0072] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments (each of which has a single antigen-binding site) and a residual "Fc" fragment (containing all but the first domain of the immunoglobulin heavy chain constant region). The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fd fragment" contains the VH domain and CH1 domain from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0073] An "Fc fragment" or "Fc region" of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. In some embodiments of the present disclosure, a mesothelin-binding protein (e.g., an anti-mesothelin antibody fragment (e.g., a TCA or heavy-chain-only antibody)) contains an Fc region derived from an immunoglobulin. The Fc region can be an Fc region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region contains the CH2 and CH3 domains derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0074] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC; Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with a receptor, such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays known in the art.

[0075] A "dead" or "silenced" Fc is one that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

[0076] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Examples of native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0077] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, e.g., one or more (e.g., two or more, three or more, four or more) amino acid substitutions. Illustratively, in some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, e.g., about one to about five amino acid substitutions in a native-sequence Fc region or the Fc region of a parent polypeptide, compared to the native-sequence Fc region or the Fc region of a parent polypeptide. In some embodiments, a variant Fc region herein has at least about 80% homology to a native-sequence Fc region and / or the Fc region of a parent polypeptide, e.g., at least about 85% homology thereto, e.g., at least about 90% homology thereto, e.g., at least about 95% homology thereto, e.g., at least about 99% homology thereto.

[0078] As used herein, "heterodimerization alterations" refer to alterations in the A and B chains of an F region (i.e., the two chains comprising an Fc region, where one chain is referred to herein as the "A" chain and the other as the "B" chain) that promote the formation of heterodimeric Fc regions, i.e., Fc regions in which the A and B chains of the Fc region do not have identical amino acid sequences. In some embodiments, heterodimerization alterations can be asymmetric, i.e., an A chain with a particular alteration can pair with a B chain with a different alteration. These alterations promote heterodimerization and disfavor homodimerization. Whether hetero- or homodimers are formed can be assessed by size difference, as determined by polyacrylamide gel electrophoresis, for example, in a situation where one polyethylene chain is a dummy Fc and the other is an scFv-Fc. One non-limiting example of such paired heterodimerization alterations is the so-called "knob and hole" substitution. See, e.g., U.S. Patent No. 7,695,936 and U.S. Patent Application Publication No. 2003 / 0078385. As used herein, an Fc region comprising a pair of knob and hole substitutions comprises one substitution in the A chain and another substitution in the B chain. For example, the following knob and hole substitutions in the A and B chains of an IgG1 Fc region have been found to increase heterodimer formation compared to that found in the unmodified A and B chains and may be employed in non-limiting embodiments of the present disclosure: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394W in the other chain; 94S; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; and 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other polypeptide.Alternatively or additionally, substitutions that create new disulfide bridges can promote heterodimer formation. See, for example, U.S. Patent Application Publication No. 2003 / 0078385. Such changes in the IgG1 Fc region include, but are not limited to, the following substitutions: Y349C in one Fc polypeptide chain and S354C in the other; Y349C in one Fc polypeptide chain and E356C in the other; Y349C in one Fc polypeptide chain and E357C in the other; L351C in one Fc polypeptide chain and S354C in the other; T394C in one Fc polypeptide chain and E397C in the other; or D399C in one Fc polypeptide chain and K392C in the other. Additionally or alternatively, substitutions that alter the charge of one or more residues in the CH3-CH3 interface can enhance heterodimer formation, for example, as described in WO 2009 / 089004, which is incorporated herein by reference. Such substitutions are referred to herein as "charge-pair substitutions," and an Fc region containing one pair of charge-pair substitutions contains one substitution in the A chain and a different substitution in the B chain. Non-limiting examples of charge pair substitutions include: 1) K409D or K409E in one chain plus D399K or D399R in the other chain; 2) K392D or K392E in one chain plus D399K or D399R in the other chain; 3) K439D or K439E in one chain plus E356K or E356R in the other chain; and 4) K370D or K370E in one chain plus E357K or E357R in the other chain. Additionally, substitutions R355D, R355E, K360D, or K360R in both chains can stabilize heterodimers when used in conjunction with other heterodimerization changes. Specific charge pair substitutions can be used alone or in conjunction with other charge pair substitutions.Specific examples of single pairs of charge pair substitutions and their combinations include: 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E in the other chain. 356K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K in the other chain. and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain. Any of these heterodimerization changes can be used in polypeptides comprising the variant Fc regions described herein.

[0079] In some non-limiting embodiments, the variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 in human IgG1 or IgG2 and at positions 327, 330, and 331 in IgG4 significantly reduce ADCC and CDC (e.g., Armour KL et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL et al., 2001 J Biol Chem. 276(9):6591-604). The Fc amino acid sequence of human IgG4 (UniProtKB number P01861) is shown herein as SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4):915-27, the disclosures of which are incorporated herein by reference in their entireties.

[0080] Other Fc variants are possible, including, but not limited to, those in which regions capable of forming disulfide bonds have been deleted, or specific amino acid residues have been removed at the N-terminus of the native Fc, or a methionine residue has been added. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, an antibody may comprise an Fc variant.

[0081] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting, or adding amino acid residues to confer complement binding or Fc receptor binding. For example, and without limitation, deletions can occur in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0082] Antibodies and antibody fragments with reduced effector function include, but are not limited to, those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (see, e.g., U.S. Pat. No. 6,737,056). In some embodiments, variant Fc regions with reduced effector function comprise substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine according to EU numbering (i.e., D265A and N297A according to EU numbering) (see, e.g., U.S. Pat. No. 7,332,581). In some embodiments, variant Fc regions with reduced effector function comprise the following two amino acid substitutions: D265A and N297A.

[0083] In some embodiments, effector function is reduced by a mutation in the constant region that eliminates glycosylation, e.g., an "effectorless mutation." In some embodiments, the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). In some embodiments, the effectorless mutation is an N297G or DANG mutation (D265A+N297G) in the CH2 region. In some embodiments, the variant Fc region lacks glycosylation at N297, e.g., the variant Fc region is a variant Fc region lacking glycosylation at N297 as described in WO 2014 / 153063, which is incorporated herein by reference. Additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in a non-glycosylating host cell (e.g., E. coli) or in a host cell that results in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0084] In some embodiments, the proline at position 329 (EU numbering) of the wild-type human Fc region (P329) is substituted with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between P329 of the Fc and tryptophan residues W87 and W110 of FcgRIII (Sondermann et al., Nature 406, 267-273 (20 Jul. 2000)). In some further embodiments, the at least one additional amino acid substitution in the Fc variant region is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, the at least one additional amino acid substitution is L234A and L235A in the human IgG1 Fc region or S228P and L235E in the human IgG4 Fc region, all according to EU numbering (see, e.g., U.S. Pat. No. 8,969,526, incorporated by reference in its entirety).

[0085] In some embodiments, the variant Fc region has P329 of the human IgG Fc region substituted with glycine, and the variant Fc region comprises at least two additional amino acid substitutions at L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, where residues are numbered according to EU numbering (see, e.g., U.S. Patent No. 8,969,526). In some embodiments, variant Fc regions comprising the P329G, L234A, and L235A (EU numbering) substitutions exhibit reduced affinity for human FcγRIIIA and FcγRIIA.

[0086] In some embodiments, the variant Fc region comprises a triple mutation: amino acid substitutions at positions P329, L234A, and L235A according to EU numbering (P329 / LALA) (see, e.g., U.S. Patent No. 8,969,526). In some embodiments, the variant Fc region comprises the following amino acid substitutions: P329G, L234A, and L235A according to EU numbering.

[0087] In some embodiments, the antibody or antibody fragment comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, the antibody or antibody fragment comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein may be utilized in any suitable way to place a "knob" in the first heavy chain constant region of the first monomer in the antibody dimer and a "hole" in the second heavy chain constant region of the second monomer in the antibody dimer, thereby promoting proper pairing of the desired pair of heavy chain polypeptide subunits in the antibody (heterodimerization).

[0088] In some embodiments, the antibody or antibody fragment comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising a S228P mutation, a F234A mutation, a L235A mutation, and a T366W mutation (knob). In some embodiments, the antibody or antibody fragment comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising a S228P mutation, a F234A mutation, a L235A mutation, a T366S mutation, a L368A mutation, and a Y407V mutation (hole).

[0089] A "Fab' fragment" is a Fab fragment that has one or more cysteine ​​residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0090] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by an inter-heavy chain disulfide bridge at the hinge region.

[0091] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of an Fv fragment containing only three antigen-specific CDRs) retains the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site comprising both VH and VL.

[0092] A "single-chain variable antibody fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0093] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks and nurse sharks, and Camelidae, including camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region; i.e., these functional antibodies are heavy chain homodimers with only the H2L2 structure (referred to as "heavy chain antibodies" or "HCAbs"). Camelized VHH reportedly contain hinge, CH2, and CH3 domains and are recombined with IgG2 and IgG3 constant regions lacking the CH1 domain. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold and may provide a framework for long transmembrane loop structures.

[0094] As used herein, the term "antigen-binding protein" refers to a protein that specifically binds to one or more target antigens. An antigen-binding protein is generally a protein that comprises an antigen-binding fragment that specifically binds to an antigen, and optionally comprises a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. In some embodiments, the antigen-binding protein is an antibody or antibody fragment. In some embodiments, the antigen-binding protein may include a protein comprising one or more antigen-binding fragments incorporated into a single polypeptide chain or into multiple polypeptide chains.For example, antigen-binding proteins include diabodies (see, e.g., EP 404,097, WO 93 / 11161, and Houinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90:6444-6448, 1993); intrabodies; domain antibodies (a single VL or VH domain, or two or more VH domains linked by a peptide linker; see Ward et al., Nature, Vol. 341:544-546, 1989); maxibodies (two scFvs fused to an Fc region; Fredericks et al., Protein Engineering, Design & Selection, Vol. 17:95-106, 2004 and Powers et al., Journal of Immunological Methods, Vol. 251:123-135, 2001); triabodies; tetrabodies; minibodies (scFv fused to a CH3 domain; see Olafsen et al., Protein Eng Des Sel., Vol. 17:315-23, 2004); peptibodies (one or more peptides attached to an Fc region; see WO 00 / 24782); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with complementary light chain polypeptides; see Zzjpate et al., Protein Eng., Vol. 8:1057-1062, 1995); small modular immunopharmaceuticals (see U.S. Patent Publication No. 20030133939); and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc; see, e.g., Spiess et al., Mol. Immunol., Vol. 67(2 Pt A):95-106, 2015).

[0095] As used herein, a "mesothelin-binding protein" is an antigen-binding protein that specifically binds to mesothelin. In some embodiments, a mesothelin-binding protein may also bind to one or more target antigens other than mesothelin.

[0096] The antibodies and antibody fragments (e.g., heavy-chain-only antibodies and three-chain antibody-like molecules) of the present disclosure include multispecific antibodies and antibody fragments, which are antibodies and antibody fragments with two or more binding specificities. As used herein, the term "multispecific" includes "bispecific" (i.e., two binding specificities) and "trispecific" (i.e., three binding specificities), as well as higher orders of independent specific binding affinities, e.g., higher orders of polyepitopic specificity.

[0097] As used herein, an "isolated" molecule (e.g., an antibody, antibody fragment, single-domain antibody, mesothelin-binding protein, etc.) is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the molecule, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an isolated molecule is purified (1) to greater than 95% by weight, e.g., greater than 99% by weight, of the molecule isolated as determined by the Lowry method; (2) to the extent that at least 15 residues of N-terminal or internal amino acid sequence are sufficient to be obtained by use of a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, for example, silver staining. In some embodiments, an isolated molecule is prepared by a process that includes one purification step.

[0098] As used herein, an "antibody-drug conjugate" refers to an antibody or antibody fragment that is attached to another moiety, such as a payload, such as a radionuclide.

[0099] As used herein, an "epitope" is a site on the surface of an antigen molecule to which a single antibody or antibody fragment binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies and antibody fragments. This term specifically includes linear epitopes and conformational epitopes. Conformational epitopes and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. An epitope may include amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide).

[0100] As used herein, "polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets.

[0101] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or treated. The subject can be a human, but also includes other mammals, such as mammals useful as laboratory models for human disease, e.g., mice, rats, etc. In some embodiments, the mammal is a human.

[0102] As used herein, the term "treatment" encompasses any improvement of a disease in a subject, including slowing or halting the progression of the disease in a patient, reducing the number or severity of the subject's symptoms, or increasing the frequency or length of periods in which the subject is free of symptoms of the disease.

[0103] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors (FcRs) to perform specific immune functions. In some embodiments, effector cells can induce ADCC, such as natural killer cells. For example, FcR-expressing monocytes and macrophages are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to cells that present antigens.

[0104] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector, a "plasmid," refers to a circular double-stranded DNA loop to which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors." In some embodiments, expression vectors for use in recombinant DNA techniques are in the form of plasmids.

[0105] As used herein, "host cell" refers to a cell into which an expression vector has been introduced. "Host cell" refers not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not be, in fact, identical to the parent cell but are still included within the scope of the term "host cell" as used herein. Exemplary recombinant host cells include, but are not limited to, transfectomas such as CHO cells, HEK293 cells, NS / 0 cells, and lymphocytic cells.

[0106] "K D The term "(M)" as used herein refers to the dissociation equilibrium constant of a specific antigen-binding interaction as determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, an anti-mouse Fc sensor is loaded with a mouse Fc fusion antigen and then immersed in an antibody-containing well to measure the concentration-dependent binding rate (k on ) is measured. off ) is measured in the final step, where the sensor is immersed in a well containing buffer only. D is k off / k on (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0107] As used herein, a molecule (e.g., a protein, antibody, or antibody fragment, etc.) "specifically binds" to a target antigen if it has a significantly higher binding affinity for the target antigen compared to its affinity for other unrelated proteins under similar binding assay conditions, such that it is able to distinguish between the target antigens. A molecule that specifically binds to an antigen has a binding affinity of ≦1×10 -6 The equilibrium dissociation constant (K D ) can bind to its antigen. Dis ≦1×10 -8 In some embodiments, the molecules described herein specifically bind to an antigen with "high affinity" when the binding affinity is ≦5×10 for human MSLN and / or human CD3. -7 K of M D In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 at a density of ≦1×10 -7 K of M D In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 at a density of ≦5×10 -8 K of M D In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 at a density of ≦2×10 -8 K of M D In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 at a density of ≦1×10 -8 K of M D In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 at a density of ≦1×10 -9 K of M D Combine with.

[0108] Affinity may be determined using a variety of techniques, non-limiting examples of which include affinity ELISA assays. In some embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the binding rate constant (k a Unit: M -1 s -1 ) and dissociation rate constant (k d Unit: s -1 ) can be measured. Then, the equilibrium dissociation constant (K D Unit: M) is the ratio of reaction rate constants (k d / k aIn some embodiments, affinity is determined by a kinetic method, such as the equilibrium exclusion binding assay (KExA) as described in Rathanaswami et al., Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (K D , M) and the binding rate constant (k a , M -1 s -1 From these values, the dissociation rate constant (k d , s -1 ) can be calculated (K D ×k a In another embodiment, affinity is determined by biolayer interferometry, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015, and used in the Octet® system (Pall ForteBio). The rate constant (k a and k d ) and affinity constant (K D ) can be calculated in real time using biolayer interferometry.

[0109] As used herein, "a [Y]-binding VH CDR" refers to a CDR of a VH region, wherein the VH region specifically binds to a target [Y].

[0110] As used herein, the term "amino acid" or "amino acid residue" refers to an amino acid having an art-recognized definition, such as an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0111] As used herein, "amino acid modification" includes, but is not limited to, deletion from, and / or insertion into, and / or substitution of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution can be made to arrive at a final construct, provided that the final construct has the desired properties. Amino acid changes may also alter post-translational processes of the antibody construct, such as changing the number or location of glycosylation sites. Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions) is contemplated as long as the final construct retains the ability to bind to the target antigen.

[0112] Those skilled in the art will recognize that conservative variants of the antibodies and antibody fragments described herein can be produced. Such conservative variants employed in antibody fragments, such as dsFv or scFv fragments, include those having a V H and V L Retain key amino acid residues necessary for correct folding and stabilization between domains, and maintain the charge characteristics of the residues to preserve the molecule's low pI and low toxicity. In some embodiments, amino acid substitutions (e.g., up to 1, up to 2, up to 3, up to 4, or up to 5 amino acid substitutions) are made to the V domain to increase yield. H and / or V L Conservative amino acid substitution tables providing functionally similar amino acids, such as those set forth in Table A1, are well known to those of skill in the art.

[0113] [Table 1]

[0114] As used herein, a "synthetic immune receptor" is an artificial cell receptor (e.g., an artificial T cell receptor, an artificial NK cell receptor, etc.) that is expressed on an immune effector cell and engineered to specifically bind to a target antigen.

[0115] As used herein, "percent (%) amino acid sequence identity" or "percent (%) sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0116] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such compositions are sterile. "Pharmaceutically acceptable" excipients (e.g., vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0117] As used herein, a "sterile" composition is sterile or free or essentially free of all living microorganisms and their spores.

[0118] As used herein, a "frozen" composition is one that is at a temperature below 0°C.

[0119] As used herein, a "stable" composition is one in which the protein therein essentially retains its physical stability, and / or chemical stability, and / or biological activity upon storage. In some embodiments, the composition essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the composition. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessment of antibody biological activity or antigen-binding function, etc. Instability can include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differential glycosylation, etc.

[0120] Some embodiments of the present disclosure relate to single-domain antibodies that specifically bind to mesothelin (MSLN). Specifically, the present disclosure provides a family of closely related single-domain antibodies that specifically bind to human mesothelin (MSLN). This family of single-domain antibodies comprises the set of CDR sequences defined herein and shown in Tables S1 and S2, and is exemplified by the heavy chain variable region (VH) sequences provided in SEQ ID NOS: 15-35 in Table S3. This family of single-domain antibodies offers many benefits that contribute to their usefulness as clinical therapeutics. Illustratively, single-domain antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities.

[0121] [Table 2]

[0122] [Table 3]

[0123] [Table 4]

[0124] [Table 5]

[0125] In some embodiments, the single domain antibody comprises: (i) a VH complementarity-determining region 1 (CDR1) comprising the sequence GFX1FX2SSYX3 (SEQ ID NO: 80), a VH complementarity determining region 1 (CDR1) in which X1 is T or S, X2 is S or T, and X3 is D, P, or A; (ii) a VH CDR2 comprising the sequence IX4X5X6GX7X8X9 (SEQ ID NO: 81), X4 is S or T, X5 is Y, G, or S, X6 is D or S, X7 is S, G, or D, X8 is K, G, or S, and X9 is K or T; and (iii) sequence AX 10 PPSYYDFLSDPDY (SEQ ID NO: 82), In the formula, X 10 is R or K; and a VH CDR3,

[0126] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35.

[0127] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO:20.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 23. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 26.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 27. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 28. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 32.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 34. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 35.

[0128] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and (ii) a VH CDR2 comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; (iii) a VH CDR3 comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 13 or SEQ ID NO: 14.

[0129] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 13.

[0130] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0131] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0132] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 8; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0133] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0134] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0135] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:9; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0136] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:10; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:13.

[0137] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:12; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0138] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:3; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0139] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0140] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:4; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0141] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:5; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:11; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0142] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution. In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution listed in Table A1.

[0143] In some embodiments, the VH CDR1 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the VH CDR3 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:13 or SEQ ID NO:14. In some embodiments, the at most one amino acid modification is an amino acid substitution. In some embodiments, the at most one amino acid modification is a conservative amino acid substitution. In some embodiments, the at most one amino acid modification is an amino acid deletion. In some embodiments, the at most one amino acid modification is an amino acid addition.

[0144] In some embodiments, VH CDR1 comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, VH CDR2 comprises a sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, VH CDR3 comprises a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0145] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (ii) a VH CDR2 comprising a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; and (iii) a heavy chain variable (VH) region comprising: a VH CDR3 comprising a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0146] In some embodiments, the single domain antibody comprises: (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively; (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively; (k) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively; (l) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively; or (m) a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively.

[0147] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively.In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively.

[0148] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 20. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 23. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 26. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 28.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 32. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 34. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 35.

[0149] In some embodiments, the VH CDR1, VH CDR2 and VH CDR3 sequences are present in a human VH framework.

[0150] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 95% sequence identity to any one of SEQ ID NOs: 15-35.

[0151] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 20. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 23.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 26. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 27. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 28. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 32.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 34. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 35.

[0152] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region selected from SEQ ID NOs: 15-35.

[0153] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 15. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 16. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 17. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 18. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 19. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 20. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 21. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 22. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 23. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 24. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 25. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 26. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 28. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 30. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 31. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 32. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 33. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 34. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 35.

[0154] In some embodiments, the single domain antibody specifically binds to human MSLN.

[0155] In some embodiments, the single domain antibody has a molecular weight of about 10 -9 M ~ about 10 -6 K of M D In some embodiments, the single domain antibody binds to human MSLN at a binding affinity of ≦5×10 -7 K of M D In some embodiments, the single domain antibody binds to human MSLN at a concentration of ≦1×10 -7 K of M D In some embodiments, the single domain antibody binds to human MSLN at a binding affinity of ≦5×10 -8 K of M D In some embodiments, the single domain antibody binds to human MSLN at a binding affinity of ≦2×10 -8 K of M D In some embodiments, the single domain antibody binds to human MSLN at a concentration of ≦1×10 -8 K of M D In some embodiments, the single domain antibody binds to human MSLN at a concentration of ≦1×10 -9 K of M D It binds to human MSLN.

[0156] In some embodiments, the single domain antibody is a human single domain antibody.

[0157] In some embodiments, the single domain antibody is an isolated single domain antibody. In some embodiments, the single domain antibody is an isolated human single domain antibody.

[0158] Some embodiments of the present disclosure relate to mesothelin binding proteins, including single domain antibodies that specifically bind to mesothelin, as described herein.

[0159] In some embodiments, the single domain antibody comprises: (i) a VH complementarity-determining region 1 (CDR1) comprising the sequence GFX1FX2SSYX3 (SEQ ID NO: 80), a VH complementarity determining region 1 (CDR1) in which X1 is T or S, X2 is S or T, and X3 is D, P, or A; (ii) a VH CDR2 comprising the sequence IX4X5X6GX7X8X9 (SEQ ID NO: 81), X4 is S or T, X5 is Y, G, or S, X6 is D or S, X7 is S, G, or D, X8 is K, G, or S, and X9 is K or T; and (iii) sequence AX 10 PPSYYDFLSDPDY (SEQ ID NO: 82), In the formula, X 10 is R or K; and a VH CDR3,

[0160] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15-35.

[0161] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO:20.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 23. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 26.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 27. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 28. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 32.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 have at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 34. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 35.

[0162] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and (ii) a VH CDR2 comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; (iii) a VH CDR3 comprising a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 13 or SEQ ID NO: 14.

[0163] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 13.

[0164] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0165] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0166] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 8; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0167] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0168] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0169] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:9; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0170] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:10; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:13.

[0171] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:2; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:12; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0172] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:3; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0173] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 14.

[0174] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:4; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:7; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0175] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising: (i) a VH CDR1 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:5; (ii) a VH CDR2 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:11; and (iii) a VH CDR3 comprising a sequence with up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO:14.

[0176] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution. In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution listed in Table A1.

[0177] In some embodiments, the VH CDR1 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. In some embodiments, the VH CDR3 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:13 or SEQ ID NO:14. In some embodiments, the at most one amino acid modification is an amino acid substitution. In some embodiments, the at most one amino acid modification is a conservative amino acid substitution. In some embodiments, the at most one amino acid modification is an amino acid deletion. In some embodiments, the at most one amino acid modification is an amino acid addition.

[0178] In some embodiments, VH CDR1 comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, VH CDR2 comprises a sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, VH CDR3 comprises a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0179] In some embodiments, the single domain antibody comprises: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (ii) a VH CDR2 comprising a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; and (iii) a heavy chain variable (VH) region comprising: a VH CDR3 comprising a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14.

[0180] In some embodiments, the single domain antibody comprises: (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively; (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively; (k) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively; (l) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively; or (m) a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively.

[0181] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively.In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively.

[0182] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 20. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 23. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 26. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 28.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 32. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 34. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 35.

[0183] In some embodiments, the VH CDR1, VH CDR2 and VH CDR3 sequences are present in a human VH framework.

[0184] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 15-35. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region having at least 95% sequence identity to any one of SEQ ID NOs: 15-35.

[0185] In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 15. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 16. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) affinity to SEQ ID NO: 17. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 18. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 19. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 20. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 21. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 22. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 23.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 24. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 25. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 26. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 27. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 28. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 29. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 30. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 31. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 32.In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 33. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 34. In some embodiments, a single domain antibody comprises a heavy chain variable (VH) region that has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) matching to SEQ ID NO: 35.

[0186] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region selected from SEQ ID NOs: 15-35.

[0187] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 15. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 16. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 17. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 18. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 19. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 20. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 21. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 22. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 23. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 24. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 25. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 26. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 28. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 29. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 30. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 31. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 32. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 33. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 34. In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region of SEQ ID NO: 35.

[0188] In some embodiments, the mesothelin binding protein specifically binds to human MSLN.

[0189] In some embodiments, the mesothelin binding protein is about 10 -9 M ~ about 10 -6 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at ≦5×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at a concentration of ≦1×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at ≦5×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at a concentration of ≦2×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at a concentration of ≦1×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN at a concentration of ≦1×10 -9 K of M D It binds to human MSLN.

[0190] In some embodiments, the mesothelin-binding protein further binds to one or more target antigens other than mesothelin. In some embodiments, the mesothelin-binding protein is multispecific. In some embodiments, the mesothelin-binding protein is bispecific.

[0191] In some embodiments, the mesothelin binding protein further specifically binds to CD3. In some embodiments, the mesothelin binding protein further specifically binds to human CD3. In some embodiments, the mesothelin binding protein is about 10 -9 M ~ about 10 -6 K of M DIn some embodiments, the mesothelin-binding protein binds to human CD3 at ≦5×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human CD3 at a concentration of ≦1×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human CD3 at ≦5×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human CD3 at a concentration of ≦2×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human CD3 at a concentration of ≦1×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human CD3 at a concentration of ≦1×10 -9 K of M D It binds to human CD3.

[0192] In some embodiments, the mesothelin binding protein is about 10 -9 M ~ about 10 -6 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦5×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦1×10 -7 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦5×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦2×10 -8 K of M D In some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦1×10 -8 K of M DIn some embodiments, the mesothelin-binding protein binds to human MSLN and / or CD3 at a concentration of ≦1×10 -9 K of M D It binds to human MSLN and / or CD3.

[0193] In some embodiments, the mesothelin-binding protein further specifically binds to human CD3 epsilon. In some embodiments, the mesothelin-binding protein binds to an epitope on CD3 comprising at least one residue selected from CD3 epsilon (SEQ ID NO: 86): K73 and S83; and CD3 delta (SEQ ID NO: 87) K82 and C93. In some embodiments, the epitope on CD3 comprises a region of CD3 epsilon defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. In some embodiments, the epitope on CD3 comprises a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, S83. In some embodiments, the epitope comprises a conformational epitope comprising residues of both CD3 delta and CD3 epsilon, hi some embodiments, the conformational epitope comprises residues CD3ε K73 and S83; CD3δ K82 and C93, respectively.

[0194] In some embodiments, the mesothelin-binding protein further comprises a CD3-binding VH region. In some embodiments, the mesothelin-binding protein further comprises a CD3-binding VH region paired with a light chain (VL) region.

[0195] In some embodiments, the CD3-binding VH region can belong to a family of closely related single-domain antibodies that specifically bind to human CD3. This family of single-domain antibodies is defined herein and comprises the set of CDR sequences shown in Tables S4 and S5, and is exemplified by the provided heavy chain variable region (VH) sequences of SEQ ID NOS: 47-64 in Table S6. Multispecific molecules comprising these CD3-binding VH domains and their associated light chain variable domains (as described in Tables S7 and S8) have advantageous properties, for example, as described in published PCT application WO 2018 / 052503, the disclosure of which is incorporated herein by reference in its entirety. Any of the single-domain antibodies described herein that specifically bind to MSLN can be combined with a CD3-binding domain and a fixed light chain domain described herein to generate multispecific mesothelin-binding proteins.

[0196] [Table 6]

[0197] [Table 7]

[0198] [Table 8]

[0199] [Table 9]

[0200] [Table 10]

[0201] [Table 11]

[0202] In some embodiments, the CD3-binding VH region is (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having up to two (e.g., 0, 1, or 2) amino acid modifications compared to any one of SEQ ID NOs: 36 to 41; and (ii) a VH CDR2 comprising a sequence having up to two (e.g., zero, one, or two) amino acid modifications relative to SEQ ID NO: 42; and (iii) a VH CDR3 comprising a sequence having up to two (e.g., 0, 1, or 2) amino acid modifications compared to any one of SEQ ID NOs: 43 to 46.

[0203] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 36, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 43. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0204] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 36, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0205] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 36, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 45. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0206] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 36, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 46. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0207] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 37, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0208] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 38, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0209] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 39, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0210] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 40, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0211] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 40, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 45. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0212] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity determining region 1 (CDR1) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 41, (ii) a VH complementarity determining region 2 (CDR2) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 42, and (iii) a VH complementarity determining region 3 (CDR3) comprising a sequence with up to two (e.g., zero, one, or two) amino acid modifications compared to SEQ ID NO: 45. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42.

[0213] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution. In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution listed in Table A1.

[0214] In some embodiments, the CD3-binding VH CDR1 comprises a sequence with at most one amino acid modification compared to any one of SEQ ID NOs: 36-41. In some embodiments, the CD3-binding VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO: 42. In some embodiments, the CD3-binding VH CDR3 comprises a sequence with at most one amino acid modification compared to any one of SEQ ID NOs: 43-46. In some embodiments, the at most one amino acid modification is an amino acid substitution. In some embodiments, the at most one amino acid modification is a conservative amino acid substitution. In some embodiments, the at most one amino acid modification is an amino acid deletion. In some embodiments, the at most one amino acid modification is an amino acid addition.

[0215] In some embodiments, the CD3-binding VH CDR1 comprises a sequence selected from SEQ ID NOs: 36-41. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 36. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 37. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 38. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 39. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 40. In some embodiments, the CH3-binding VH CDR1 comprises the sequence of SEQ ID NO: 41.

[0216] In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO:42.

[0217] In some embodiments, the CD3-binding VH CDR3 comprises a sequence selected from SEQ ID NOs: 43-46. In some embodiments, the CH3-binding VH CDR3 comprises the sequence of SEQ ID NO: 43. In some embodiments, the CH3-binding VH CDR3 comprises the sequence of SEQ ID NO: 44. In some embodiments, the CH3-binding VH CDR3 comprises the sequence of SEQ ID NO: 45. In some embodiments, the CH3-binding VH CDR3 comprises the sequence of SEQ ID NO: 46.

[0218] In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64.

[0219] In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in a CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 47. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in a CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 48. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in a CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 49. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in a CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 50. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 51. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 52. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO:53.In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 54. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 55. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 56. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 57. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 58. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 59. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO:60.In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 61. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 62. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 63. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to CDR1, 2, and 3 of SEQ ID NO: 64.

[0220] In some embodiments, the CDR3-binding VH region comprises the CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 47. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 48. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 49. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 50. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 51. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 52. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 53. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 54. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 55. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 56. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 57. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 58. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 59. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 60. In some embodiments, the CD3-binding VH region comprises the CDR1, CDR2, and CDR3 of SEQ ID NO: 61. In some embodiments, the CD3-binding VH region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 62. In some embodiments, the CD3-binding VH region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 63. In some embodiments, the CD3-binding VH region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 64.

[0221] In some embodiments, the CD3-binding VH region is (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 43, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 44, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 45, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 46, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 37, 42, and 44, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 38, 42, and 44, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 39, 42, and 44, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 44, respectively; (i) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 45, respectively; or (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 41, 42, and 45, respectively.

[0222] In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 43, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 44, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 45, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 46, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 37, 42, and 44, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 38, 42, and 44, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 39, 42, and 44, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 44, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 45, respectively. In some embodiments, the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 41, 42, and 45, respectively.

[0223] In some embodiments, the CD3-binding VH region is (i) Array GFTFX 11 X 12 A VH complementarity determining region 1 (CDR1) comprising YA (SEQ ID NO: 83), In the formula, X 11 is D, A, or H, and X 12 is D or N; and (ii) a VH CDR2 comprising the sequence ISWNSGSI (SEQ ID NO: 42); and (iii) sequence AKDSRGYGX 13 YX 14 X 15 a VH CDR3 comprising GGAY (SEQ ID NO: 84), In the formula, X 13 is D or S, and X 14 is R or S, and X 15 is L or R.

[0224] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are present within a human VH framework.

[0225] In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to any one of SEQ ID NOs: 47-64. In some embodiments, the CD3-binding VH region has at least 95% sequence identity to any one of SEQ ID NOs: 47-64.

[0226] In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 47. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 48. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 49. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 50. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:51. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:52. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:53. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:54. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:55.In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 56. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 57. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 58. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 59. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 60. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 61. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 62. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 63. In some embodiments, the CD3-binding VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO:64.

[0227] In some embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 68. In some embodiments, the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3 comprising the sequences of SEQ ID NOs: 65, 66, and 67, respectively. In some embodiments, the VL CDR1, VL CDR2, and VL CDR3 sequences are present in a human VH framework.

[0228] In some embodiments, the light chain variable region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 90% (e.g., 90%, 95%, at least 95%, etc.) sequence identity to SEQ ID NO: 68. In some embodiments, the light chain variable region has at least 95% sequence identity to SEQ ID NO: 68.

[0229] In some embodiments, the mesothelin binding protein is an anti-mesothelin antibody or fragment thereof.

[0230] In some embodiments, the anti-mesothelin antibody or fragment thereof is a monoclonal antibody or fragment thereof. In some embodiments, the anti-mesothelin antibody or fragment thereof is an isolated monoclonal antibody or fragment thereof.

[0231] In some embodiments, the anti-mesothelin antibody is an intact IgG molecule. In some embodiments, the anti-mesothelin antibody is an intact IgG1 molecule. In some embodiments, the anti-mesothelin antibody is an intact IgG2 molecule. In some embodiments, the anti-mesothelin antibody is an intact IgG4 molecule.

[0232] In some embodiments, the mesothelin-binding protein is an antibody fragment. In some embodiments, the antibody fragment is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody fragment is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody fragment is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody fragment is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody fragment is a heavy chain-only antibody. In some embodiments, the antibody fragment is a TCA.

[0233] In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises an Fc region. In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0234] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366 in one chain). Y and F405A and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A and Y407V in the other polypeptide. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain, etc.).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain. 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E in the other chain. 356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain, etc.

[0235] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution of one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effector-less mutation (e.g., N297A, N297G, a DANA mutation (D265A+N297A), or a DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0236] In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the anti-mesothelin antibody or fragment thereof comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 61). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 62) comprising an S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 63). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising an F234A mutation, an L235A mutation, or both an F234A mutation and an L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 65). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0237] Table S9 provides sequences for human IgG1 and IgG4 Fc region sequences, as well as versions of these sequences that incorporate additional mutations (variants) that may confer additional desirable properties to the anti-mesothelin antibodies and fragments thereof described herein. Table S10 provides exemplary human CD3 epsilon and CD3 delta sequences.

[0238] [Table 12]

[0239] [Table 13]

[0240] [Table 14]

[0241] Some embodiments of the present disclosure relate to polynucleotides that encode single domain antibodies that specifically bind to mesothelin described herein.

[0242] Some embodiments of the present disclosure relate to compositions comprising one or more polynucleotides encoding mesothelin-binding proteins described herein. In some embodiments, the mesothelin-binding protein is an anti-mesothelin antibody or a fragment thereof.

[0243] Some embodiments of the present disclosure relate to recombinant expression vectors comprising the single domain antibodies that specifically bind to mesothelin described herein, as well as host cells comprising the recombinant expression vectors.

[0244] Some embodiments of the present disclosure relate to one or more recombinant expression vectors comprising one or more polynucleotides encoding the mesothelin-binding proteins described herein, as well as host cells comprising the one or more recombinant expression vectors.

[0245] Some embodiments of the present disclosure relate to synthetic immunoreceptors comprising single domain antibodies that specifically bind to mesothelin described herein, as well as cells comprising the synthetic immunoreceptors.

[0246] In some embodiments, the single domain antibody is connected to a transmembrane domain via a hinge region and further connected to a costimulatory domain, such as a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the synthetic immunoreceptor further comprises a sequence encoding an intracellular signaling domain, such as, for example, 4-1BB and / or CD3 zeta.

[0247] Some embodiments of the present disclosure relate to producing a mesothelin binding protein (e.g., an anti-mesothelin antibody or fragment thereof) described herein, comprising growing a cell described herein under conditions permissive for expression of the antibody and isolating the mesothelin binding protein (e.g., an anti-mesothelin antibody or fragment thereof) from the cell.

[0248] Some embodiments of the present disclosure relate to antibody-drug conjugates comprising a single domain antibody that specifically binds to mesothelin as described herein. In some embodiments, the drug in the antibody-drug conjugate is a chemotherapeutic agent. In some embodiments, the drug in the antibody-drug conjugate is a radionuclide. In some embodiments, the antibody-drug conjugate is for use in diagnostic applications, such as the detection or monitoring of a disease associated with expression of mesothelin, such as a proliferative disease or cancer.

[0249] Some embodiments of the present disclosure relate to pharmaceutical compositions comprising a mesothelin binding protein, an antibody-drug conjugate, or an anti-mesothelin antibody or fragment thereof, and a pharmaceutically acceptable excipient.

[0250] Non-limiting examples of pharmaceutically acceptable excipients include adjuvants, solid carriers, water, buffers, or other carriers used in the art to carry therapeutic ingredients, or combinations thereof.

[0251] Pharmaceutical compositions of the present disclosure are prepared by mixing a protein having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers, such as in the form of a lyophilized formulation or an aqueous solution for storage (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0252] In some embodiments, pharmaceutical compositions may include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides. salt-forming counterions (e.g., sodium, etc.); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide, etc.); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohols (e.g., mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapar, etc.); stability enhancers (e.g., sucrose or sorbitol); isotonicity enhancers (e.g., alkali metal halides, sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.Methods and suitable materials for formulating molecules for therapeutic use are known in the pharmaceutical art; see, eg, REMINGTON'S PHARMACEUTICAL SCIENCES, 18. th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company. Pharmaceutical compositions of the present disclosure include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0253] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (e.g., a dosage form for a single administration). The formulation will depend on the selected route of administration. The mesothelin-binding proteins (e.g., antimesothelin antibodies and fragments thereof) and antibody-drug conjugates described herein can be administered by intravenous injection or infusion, or subcutaneously. For administration by injection, the mesothelin-binding proteins (e.g., antimesothelin antibodies and fragments thereof) and antibody-drug conjugates described herein are formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain carriers, excipients, or stabilizers, as discussed above. Alternatively, the mesothelin-binding proteins (e.g., antimesothelin antibodies and fragments thereof) and antibody-drug conjugates described herein may be in lyophilized form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. This lyophilized material can be reconstituted in, for example, bacteriostatic water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was present prior to lyophilization.

[0254] For example, U.S. Patent No. 9,034,324 discloses antibody formulations. Similar formulations can be used for the anti-mesothelin antibodies and fragments thereof described herein. Subcutaneous antibody formulations are described, for example, in U.S. Patent Application Publication Nos. 20160355591 and 20160166689.

[0255] Some embodiments of the present disclosure relate to methods of treating a disease associated with expression of mesothelin in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein.

[0256] In some embodiments, administration results in a slowing or inhibition of tumor growth or metastasis of mesothelin-expressing cancers. Measuring the reduction in tumor cell proliferation can be determined by several different methodologies well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison to control subjects, imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, and the like. In some embodiments, administration of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment results in at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in in vivo tumor cell proliferation compared to a control antigen-binding agent, with a 100% reduction in tumor growth indicating a complete response and disappearance of the tumor. In some embodiments, administration of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment results in about a 50-100%, about 75-100%, or about 90-100% reduction in tumor cell proliferation in vivo compared to a control antigen-binding agent. In some embodiments, administration of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment results in about a 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% reduction in tumor cell proliferation in vivo compared to a control antigen-binding agent.

[0257] The effective dose for treating a disease will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or an animal, the administration of other agents, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, e.g., companion animals such as dogs, cats, and horses, and laboratory mammals such as rabbits, mice, and rats, can also be treated. Treatment dosages can be titrated to optimize safety and efficacy.

[0258] Dosage levels can be readily determined by a clinician of ordinary skill and can be modified as needed, for example, to modify a subject's response to treatment. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0259] In some embodiments, the mesothelin binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment is administered to a patient parenterally. Parenteral administration refers to the administration of a molecule by a route other than through the gastrointestinal tract and can include intraperitoneal, intramuscular, intravenous, intraarterial, intradermal, subcutaneous, intracerebral, intraventricular, and intrathecal administration.

[0260] In some embodiments, the mesothelin binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment is administered intravenously to a subject.

[0261] Parenteral or intravenous administration can be by injection (e.g., using a needle and syringe) or infusion (e.g., via a catheter and pump system). Administration according to the present disclosure is contemplated to be by intravenous injection or infusion. Typically, intravenous (IV) infusion is administered through a line, port, or catheter (a small, flexible tube), such as a central venous access, or central venous catheter (CVC), which is a catheter placed in a large vein, or a peripheral venous catheter (PVC), which is a catheter placed in a peripheral vein. Generally, the catheter or line can be placed through a vein in the neck (internal jugular vein), a vein in the chest (subclavian vein or axillary vein), a vein in the groin (femoral vein), or a vein in the arm (also known as a PICC line or peripherally inserted central catheter). A central IV line is advanced through a vein and has a catheter that drains into a large central vein (usually the superior vena cava, inferior vena cava), or even into the right atrium of the heart. A peripheral intravenous (PIV) line is used in peripheral veins (veins in the arms, hands, legs, and feet). A port is a central venous line that has no external connector; instead, it is coated with silicone rubber and has a small reservoir implanted under the skin. Medication is administered intermittently by inserting a small needle into the skin, piercing the silicone, and entering the reservoir. When the needle is withdrawn, the reservoir cover naturally reseals. The cover can tolerate hundreds of needle sticks over its lifespan.

[0262] In some embodiments, the disease associated with expression of mesothelin is selected from a proliferative disease and a cancer.

[0263] In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0264] The mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein can also be accompanied by the administration of other anti-cancer drugs or therapeutic treatments (such as surgical removal of tumors). Any suitable anti-cancer drug can be administered in combination with the mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments disclosed herein. Examples of anti-cancer drugs include, but are not limited to, chemotherapeutic agents, such as antimitotic agents, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibodies specific for targeting cancer cells.

[0265] In some embodiments, the mesothelin binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment is administered before, during, or after surgery.

[0266] Some embodiments of the present disclosure relate to a mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein for use in treating a disease associated with mesothelin expression. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease and a cancer. In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0267] Some embodiments of the present disclosure relate to the use of a mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein in the manufacture of a medicament for the treatment of a disease associated with mesothelin expression. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease and a cancer. In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0268] Some embodiments of the present disclosure relate to methods for confirming a diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with a single domain antibody or mesothelin-binding protein described herein and detecting binding of the single domain antibody or mesothelin-binding protein to the sample. An increase in binding of the single domain antibody or mesothelin-binding protein to the sample compared to binding of the single domain antibody or mesothelin-binding protein to a control sample confirms a diagnosis of cancer. In some embodiments, the method further comprises contacting the sample with a detection antibody that specifically recognizes the single domain antibody or mesothelin-binding protein and detecting binding of the detection antibody.

[0269] Some embodiments of the present disclosure relate to methods for detecting cancer associated with mesothelin expression in a subject. The method includes contacting a sample from the subject with a single domain antibody or mesothelin-binding protein described herein and detecting binding of the single domain antibody or mesothelin-binding protein to the sample. Increased binding of the single domain antibody or mesothelin-binding protein to the sample compared to a control sample detects cancer in the subject. In some embodiments, the method further includes contacting the sample with a detection antibody that specifically recognizes the single domain antibody or mesothelin-binding protein and detecting binding of the detection antibody.

[0270] Some embodiments of the present disclosure relate to kits comprising a mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described herein, or a pharmaceutical composition comprising the same, and instructions for use. The kit may further contain at least one additional agent, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the contents of the kit. The term "label," as used herein, includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit.

[0271] In some embodiments, the kit is a diagnostic kit. In some embodiments, a kit is provided for detecting mesothelin in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of soluble mesothelin protein or fragments. Kits for detecting polypeptides typically include a single domain antibody or mesothelin-binding protein according to the present disclosure. In some embodiments, the single domain antibody or mesothelin-binding protein is labeled (e.g., fluorescently, radioactively, or enzymatically labeled).

[0272] In some embodiments, the kit may additionally include means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, or the like). The kit may further include buffers and other reagents routinely used for the practice of a particular method. Such kits and suitable contents are well known to those of skill in the art.

[0273] Non-limiting exemplary embodiments: Some exemplary embodiments of the present disclosure include, but are not limited to, the following. 1. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15 to 35. 2. A single domain antibody according to embodiment 1, wherein the full set (combination) of VH CDR1, 2 and 3 has at least 85% sequence identity to CDR1, 2 and 3 of any one of SEQ ID NOs: 15 to 35. 3. A single domain antibody according to embodiment 1 or 2, in which the full set (combination) of VH CDR1, 2 and 3 has at least 90% sequence identity to CDR1, 2 and 3 of any one of SEQ ID NOs: 15 to 35. 4. A single domain antibody according to any one of embodiments 1 to 3, wherein the full set (combination) of VH CDR1, 2, and 3 has at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 15 to 35. 5. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody is (i) a VH complementarity determining region 1 (CDR1) comprising a sequence having up to two amino acid modifications compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and (ii) a VH CDR2 comprising a sequence having up to two amino acid modifications compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; and (iii) a VH CDR3 comprising a sequence having up to two amino acid modifications compared to SEQ ID NO: 13 or SEQ ID NO: 14; and a heavy chain variable (VH) region comprising: 6. The single domain antibody of embodiment 5, wherein each amino acid modification, if any, is a conservative amino acid substitution. 7. The single domain antibody of embodiment 5 or 6, wherein VH CDR1 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. 8. A single domain antibody according to any one of embodiments 5 to 7, wherein VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. 9. A single domain antibody according to any one of embodiments 5 to 8, wherein the VH CDR3 comprises a sequence with at most one amino acid modification compared to SEQ ID NO: 13 or SEQ ID NO: 14. 10. The single domain antibody of any one of embodiments 7 to 9, wherein at most one amino acid modification is an amino acid substitution. 11. The single domain antibody of any one of embodiments 7 to 9, wherein at most one amino acid modification is a conservative amino acid substitution. 12. The single domain antibody of any one of embodiments 7 to 9, wherein at most one amino acid modification is an amino acid deletion. 13. The single domain antibody of any one of embodiments 7 to 9, wherein at most one amino acid modification is an amino acid addition. 14. A single domain antibody according to any one of embodiments 1 to 13, wherein VH CDR1 comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. 15. A single domain antibody according to any one of embodiments 1 to 14, wherein VH CDR2 comprises a sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. 16. A single domain antibody according to any one of embodiments 1 to 15, wherein the VH CDR3 comprises a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14. 17. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody is: (i) a VH complementarity-determining region 1 (CDR1) comprising the sequence GFX1FX2SSYX3 (SEQ ID NO: 80), a VH complementarity determining region 1 (CDR1) in which X1 is T or S, X2 is S or T, and X3 is D, P, or A; (ii) a VH CDR2 comprising the sequence IX4X5X6GX7X8X9 (SEQ ID NO: 81), X4 is S or T, X5 is Y, G, or S, X6 is D or S, X7 is S, G, or D, X8 is K, G, or S, and X9 is K or T; and (iii) sequence AX 10 PPSYYDFLSDPDY (SEQ ID NO: 82), In the formula, X10 is R or K; and a VH CDR3, 18. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody is: (i) a VH complementarity determining region 1 (CDR1) comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; (ii) a VH CDR2 comprising a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; and (iii) a VH CDR3 comprising a sequence selected from SEQ ID NO: 13 and SEQ ID NO: 14; and a heavy chain variable (VH) region comprising: 19. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody is: (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 13, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 6, and 14, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 7, and 14, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 8, and 14, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 6, and 14, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 7, and 14, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 9, and 14, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 10, and 13, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 12, and 14, respectively; (j) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 14, respectively; (k) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 6, and 14, respectively; (l) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 4, 7, and 14, respectively; or (m) A single domain antibody comprising a heavy chain variable (VH) region comprising VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 5, 11, and 14, respectively. 20. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody comprises a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 15 to 35. 21. The single domain antibody according to any one of embodiments 1 to 20, wherein the VH CDR1, VH CDR2 and VH CDR3 sequences are present in a human VH framework. 22. A single domain antibody that specifically binds to mesothelin (MSLN), wherein the single domain antibody comprises a heavy chain variable (VH) region having at least 80% sequence identity to any one of SEQ ID NOs: 15 to 35. 23. A single domain antibody according to any one of embodiments 1 to 22, wherein the VH region has at least 85% sequence identity to any one of SEQ ID NOs: 15 to 35. 24. A single domain antibody according to any one of embodiments 1 to 23, wherein the VH region has at least 90% sequence identity to any one of SEQ ID NOs: 15 to 35. 25. A single domain antibody according to any one of embodiments 1 to 24, wherein the VH region has at least 95% sequence identity to any one of SEQ ID NOs: 15 to 35. 26. A single domain antibody that specifically binds to mesothelin (MSLN), the single domain antibody comprising a heavy chain variable (VH) region selected from SEQ ID NOs: 15 to 35. 27. The single domain antibody of any one of embodiments 1 to 26, wherein the single domain antibody specifically binds to human MSLN. 28. Single domain antibodies are approximately 10 -9 M ~ about 10 -6 K of M D 28. The single domain antibody of any one of embodiments 1 to 27, which binds to human MSLN at 29. The single domain antibody of any one of embodiments 1 to 28, wherein the single domain antibody is an isolated single domain antibody. 30. A mesothelin binding protein comprising a single domain antibody according to any one of embodiments 1 to 28. 31. The mesothelin-binding protein of embodiment 30, wherein the mesothelin-binding protein specifically binds to human MSLN. 32. Mesothelin-binding protein is approximately 10 -9 M ~ about 10 -6 K of M D 32. The mesothelin-binding protein of embodiment 30 or 31, which binds to human MSLN at 33. The mesothelin-binding protein of any one of embodiments 30-32, wherein the mesothelin-binding protein further specifically binds to CD3. 34. The mesothelin-binding protein of any one of embodiments 30-33, wherein the mesothelin-binding protein further specifically binds to human CD3. 35. The mesothelin-binding protein of any one of embodiments 30-34, wherein the mesothelin-binding protein further specifically binds to human CD3 epsilon. 36. The mesothelin-binding protein of any one of embodiments 30 to 34, wherein the mesothelin-binding protein binds to an epitope on CD3 comprising at least one residue selected from CD3 epsilon (SEQ ID NO: 86): K73 and S83; and CD3 delta (SEQ ID NO: 87) K82 and C93. 37. The mesothelin-binding protein of embodiment 36, wherein the epitope on CD3 comprises a region of CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. 38. The mesothelin-binding protein of embodiment 36 or 37, wherein the epitope on CD3 comprises a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, S83. 39. The mesothelin-binding protein of any one of embodiments 36 to 38, wherein the epitope comprises a conformational epitope having residues of both CD3 delta and CD3 epsilon. 40. The mesothelin-binding protein of any one of embodiments 36 to 39, wherein the conformational epitope comprises residues CD3ε K73 and S83; CD3δ K82 and C93, respectively. 41. The mesothelin-binding protein of any one of embodiments 30-40, wherein the mesothelin-binding protein is a monoclonal antibody. 42. The mesothelin-binding protein of any one of embodiments 30-41, wherein the mesothelin-binding protein is an isolated monoclonal antibody. 43. An antibody-drug conjugate comprising a single domain antibody according to any one of embodiments 1 to 28. 44. An anti-mesothelin antibody, comprising a single domain antibody according to any one of embodiments 1 to 28. 45. The anti-mesothelin antibody of embodiment 44, wherein the anti-mesothelin antibody binds to effector cells. 46. ​​The anti-mesothelin antibody of embodiment 44 or 45, wherein the anti-mesothelin antibody is multispecific. 47. An anti-mesothelin antibody according to any one of embodiments 44 to 46, wherein the anti-mesothelin antibody further specifically binds to a tumor-specific antigen other than MSLN. 48. The anti-mesothelin antibody of any one of embodiments 44-47, wherein the anti-mesothelin antibody is bispecific. 49. The anti-mesothelin antibody of any one of embodiments 44-48, wherein the anti-mesothelin antibody further specifically binds to CD3. 50. The anti-mesothelin antibody of any one of embodiments 44-49, wherein the anti-mesothelin antibody further specifically binds to human CD3. 51. The anti-mesothelin antibody of any one of embodiments 44 to 50, wherein the anti-mesothelin antibody further specifically binds to human CD3 epsilon. 52. The anti-mesothelin antibody of any one of embodiments 44 to 50, wherein the anti-mesothelin antibody binds to epsilon on CD3 comprising at least one residue selected from CD3 epsilon (SEQ ID NO: 86): K73 and S83; and CD3 delta (SEQ ID NO: 87) K82 and C93. 53. The anti-mesothelin antibody of embodiment 52, wherein the epitope on CD3 comprises a region of CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, C93. 54. The anti-mesothelin antibody of embodiment 52 or 53, wherein the epitope on CD3 comprises a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, S83. 55. The anti-mesothelin antibody of any one of embodiments 52 to 54, wherein the epitope comprises a conformational epitope having residues of both CD3 delta and CD3 epsilon. 56. The anti-mesothelin antibody of embodiment 55, wherein the conformational epitope comprises residues CD3ε K73 and S83; CD3δ K82 and C93, respectively. 57. The anti-mesothelin antibody of any one of embodiments 30-56, wherein the anti-mesothelin antibody further comprises a CD3-binding VH region. 58. The anti-mesothelin antibody of any one of embodiments 30 to 57, wherein the anti-mesothelin antibody is an IgG4 antibody. 59. The anti-mesothelin antibody of any one of embodiments 30 to 57, wherein the anti-mesothelin antibody is an IgG1 antibody. 60. The anti-mesothelin antibody of any one of embodiments 30-58, wherein the anti-mesothelin antibody further comprises a CD3-binding VH region paired with a light chain variable (VL) region. 61. The CD3-binding VH region is (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having up to two amino acid modifications compared to any one of SEQ ID NOs: 36 to 41; and (ii) a VH CDR2 comprising a sequence with up to two amino acid modifications compared to SEQ ID NO: 42; and (iii) a VH CDR3 comprising a sequence having up to two amino acid modifications compared to any one of SEQ ID NOs: 43 to 46. 62. The anti-mesothelin antibody of embodiment 61, wherein the CD3-binding VH CDR1 comprises a sequence having at most one amino acid modification compared to any one of SEQ ID NOs: 36-41. 63. The anti-mesothelin antibody of embodiment 61 or 62, wherein the CD3-binding VH CDR2 comprises a sequence with at most one amino acid modification compared to SEQ ID NO: 42. 64. The anti-mesothelin antibody of any one of embodiments 61-63, wherein the CD3-binding VH CDR3 comprises a sequence having at most one amino acid modification compared to any one of SEQ ID NOs: 43-46. 65. The anti-mesothelin antibody of any one of embodiments 62-64, wherein at most one amino acid modification is an amino acid substitution. 66. The anti-mesothelin antibody of any one of embodiments 62-65, wherein at most one amino acid modification is a conservative amino acid substitution. 67. The anti-mesothelin antibody of any one of embodiments 62-64, wherein at most one amino acid modification is an amino acid deletion. 68. The anti-mesothelin antibody of any one of embodiments 62-64, wherein at most one amino acid modification is an amino acid addition. 69. The anti-mesothelin antibody of any one of embodiments 62-68, wherein the CD3-binding VH CDR1 comprises a sequence selected from SEQ ID NOs: 36-41. 70. The anti-mesothelin antibody of any one of embodiments 62-69, wherein the CD3-binding VH CDR2 comprises the sequence of SEQ ID NO: 42. 71. The anti-mesothelin antibody of any one of embodiments 62-70, wherein the CD3-binding VH CDR3 comprises a sequence selected from SEQ ID NOs: 43-46. 72. The anti-mesothelin antibody of any one of embodiments 57 or 60-71, wherein the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 80% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. 73. The anti-mesothelin antibody of any one of embodiments 57 or 60-72, wherein the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 85% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. 74. An anti-mesothelin antibody according to any one of embodiments 57 or 60-73, wherein the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 90% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. 75. An anti-mesothelin antibody according to any one of embodiments 57 or 60-74, wherein the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 47-64. 76. The CD3-binding VH region is (i) Array GFTFX 11 X 12 A VH complementarity determining region 1 (CDR1) comprising YA (SEQ ID NO: 83), In the formula, X 11 is D, A, or H, and X12 is D or N; and (ii) a VH CDR2 comprising the sequence ISWNSGSI (SEQ ID NO: 42); and (iii) sequence AKDSRGYGX 13 YX 14 X 15 a VH CDR3 comprising GGAY (SEQ ID NO: 84), In the formula, X 13 is D or S, and X 14 is R or S, and X 15 and a VH CDR3 wherein: 77. The anti-mesothelin antibody of embodiment 57 or 60, wherein the CD3-binding VH region comprises CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 47-64. 78. The anti-mesothelin antibody of any one of embodiments 57-77, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are in a human VH framework. 79. The anti-mesothelin antibody of any one of embodiments 57-78, wherein the CD3-binding VH region has at least 80% sequence identity to any one of SEQ ID NOs: 47-64. 80. The anti-mesothelin antibody of any one of embodiments 57-79, wherein the CD3-binding VH region has at least 85% sequence identity to any one of SEQ ID NOs: 47-64. 81. The anti-mesothelin antibody of any one of embodiments 57-80, wherein the CD3-binding VH region has at least 90% sequence identity to any one of SEQ ID NOs: 47-64. 82. The anti-mesothelin antibody of any one of embodiments 57-81, wherein the CD3-binding VH region has at least 95% sequence identity to any one of SEQ ID NOs: 47-64. 83. The CD3-binding VH region is (a) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 43, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 44, respectively; (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 45, respectively; (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 46, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 37, 42, and 44, respectively; (f) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 38, 42, and 44, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 39, 42, and 44, respectively; (h) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 44, respectively; (i) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 40, 42, and 45, respectively; or (j) An anti-mesothelin antibody described in any one of embodiments 57 to 82, comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 41, 42, and 45, respectively. 84. The anti-mesothelin antibody of embodiment 83, wherein the CD3-binding VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 36, 42, and 43. 85. The anti-mesothelin antibody of any one of embodiments 60 to 84, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 68. 86. The anti-mesothelin antibody of any one of embodiments 60 to 85, wherein the light chain variable region comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising the sequences of SEQ ID NOs: 65, 66, and 67, respectively. 87. The anti-mesothelin antibody of any one of embodiments 60-86, wherein the VL CDR1, VL CDR2, and VL CDR3 sequences are in a human VH framework. 88. The anti-mesothelin antibody of any one of embodiments 60-87, wherein the light chain variable region has at least 80% sequence identity to SEQ ID NO: 68. 89. The anti-mesothelin antibody of any one of embodiments 60 to 88, wherein the light chain variable region has at least 85% sequence identity to SEQ ID NO: 68. 90. The anti-mesothelin antibody of any one of embodiments 60-89, wherein the light chain variable region has at least 90% sequence identity to SEQ ID NO: 68. 91. The anti-mesothelin antibody of any one of embodiments 60 to 90, wherein the light chain variable region has at least 95% sequence identity to SEQ ID NO: 68. 92. The anti-mesothelin antibody of any one of embodiments 44 to 91, wherein the anti-mesothelin antibody further comprises an Fc region. 93. The anti-mesothelin antibody of any one of embodiments 44-92, wherein the anti-mesothelin antibody further comprises a variant Fc region. 94. The anti-mesothelin antibody of embodiment 93, wherein the variant Fc region comprises a heterodimerization modification. 95. An anti-mesothelin antibody according to any one of embodiments 44 to 94, wherein the Fc region is a silenced Fc region. 96. The anti-mesothelin antibody of any one of embodiments 44-95, wherein the anti-mesothelin antibody specifically binds to human MSLN. 97. Anti-mesothelin antibodies are present in approximately 10 -9 M ~ about 10 -6 K of M D The anti-mesothelin antibody of any one of embodiments 44 to 96, which binds to human MSLN at 98. The anti-mesothelin antibody of any one of embodiments 44-97, wherein the anti-mesothelin antibody is an isolated antibody. 99. An antibody fragment that specifically binds to mesothelin, wherein the antibody fragment comprises a fragment of an anti-mesothelin antibody described in any one of embodiments 44 to 98. 100. The antibody fragment of embodiment 99, wherein the antibody fragment specifically binds to human MSLN. 101. The antibody fragment of embodiment 99 or 100, wherein the antibody fragment is an isolated antibody fragment. 102. A polynucleotide encoding a single domain antibody according to any one of embodiments 1 to 29. 103. A composition comprising one or more polynucleotides encoding a mesothelin-binding protein according to any one of embodiments 30 to 42. 104. A composition comprising one or more polynucleotides encoding an anti-mesothelin antibody according to any one of embodiments 44 to 98. 105. A recombinant expression vector comprising a polynucleotide or composition according to any one of embodiments 102 to 104. 106. A host cell comprising the recombinant expression vector of embodiment 105. 107. A synthetic immunoreceptor comprising a single domain antibody according to any one of embodiments 1 to 28. 108. A cell comprising a synthetic immune receptor according to embodiment 107. 109. A method of treating a disease associated with expression of mesothelin in a subject in need thereof, comprising administering to the subject at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described in any one of embodiments 30 to 101. 110. A method of treating a disease associated with expression of mesothelin in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment described in any one of embodiments 30 to 101. 111. The method of embodiment 110, wherein the disease associated with the expression of mesothelin is selected from proliferative diseases and cancer. 112. The method of embodiment 111, wherein the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. 113. A mesothelin binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment according to any one of embodiments 30 to 101 for use in treating a disease associated with the expression of mesothelin. 114. The mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment for use according to embodiment 113, wherein the disease associated with the expression of mesothelin is selected from proliferative diseases and cancer. 115. The mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment for use according to embodiment 114, wherein the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. 116. Use of a mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment according to any one of embodiments 30 to 101 in the manufacture of a medicament for the treatment of a disease associated with the expression of mesothelin. 117. The use according to embodiment 116, wherein the disease associated with the expression of mesothelin is selected from proliferative diseases and cancer. 118. The use according to embodiment 117, wherein the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. 119. A pharmaceutical composition comprising at least one mesothelin binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment according to any one of embodiments 30 to 101, and a pharmaceutically acceptable excipient. [Example]

[0274] In order that the present disclosure may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the disclosure in any manner.

[0275] Example 1. Binding to MSLN-expressing cell lines and off-target cell lines Cell binding for exemplary anti-MSLN single domain antibodies of the present disclosure was evaluated using CHO cells expressing human MSLN protein (CHO_huMSLN) and CHO cells not expressing MSLN protein (CHO-OFFtgt). Pelleted CHO cells were diluted to 1.25 x 10 in flow buffer (1x phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% NaN). 6 The cells were resuspended at 1.25 μg / mL. The antibody-containing supernatant was diluted 1:5 in flow buffer, and 40 μL of cells plus 10 μL of diluted antibody supernatant were incubated at 4°C for 30 minutes. The cells were washed with flow buffer and resuspended in 50 μL of phycoerythrin (PE)-conjugated secondary antibody (Southern Biotech #2042-09) diluted in flow buffer to 1.25 μg / mL and incubated at 4°C for 20 minutes. After two washing steps, the cells were resuspended in flow buffer and analyzed using a Guava easyCyte 8HT system. Table E1 summarizes the target binding activity of anti-MSLN single domain antibodies as fold over background mean fluorescence intensity (MFI) signals.

[0276] [Table 15]

[0277] Example 2. Binding to MSLN-expressing cell lines and off-target cell lines Cell binding dose curves for exemplary anti-MSLN single domain antibodies described herein were generated using CHO cells (FIG. 1A), HeLa cells (FIG. 1B), and CHO-OFFtgt cells (FIG. 1C) expressing human MSLN. Single domain antibodies were tested in an 8-point dose curve, starting at 150 nM followed by 3-fold serial dilutions. All washes and dilutions of cells, antibodies, and reagents were performed using flow buffer (1× phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% NaN). Cells were pelleted and diluted to 1×10 in flow buffer. 6Cells were resuspended at 1000 cells / mL. 50 μL of cells were then combined with 50 μL of test antibody and incubated at 4°C for 30 minutes, followed by two washing steps with flow buffer. Cells were then resuspended in 50 μL of PE-conjugated secondary antibody (Southern Biotech #2042-09) diluted to 1.25 μg / mL and incubated at 4°C for 20 minutes. After two washing steps, cells were resuspended in flow buffer and analyzed using a BD Celesta system. PE mean fluorescence intensity was plotted as fold of background (cells incubated with secondary detection antibody only).

[0278] Example 3. Cell binding EC50 values ​​in MSLN-expressing cell lines To determine cell binding EC50 values ​​for exemplary anti-MSLN single domain antibodies of the present disclosure, cell binding dose curves were generated using HeLa cells and CHO cells expressing human MSLN. Single domain antibodies were tested in an 8-point dose curve, starting at a 150 nM starting dose followed by 3-fold serial dilutions, as described in Example 2. The transformed data were plotted as an xy graph using a nonlinear regression curve fit (available in GraphPad Prism 8.4.3) to obtain the EC50 (nM), which is summarized in Table E2.

[0279] [Table 16]

[0280] Example 4. Binding affinity and epitope binning Table E3 summarizes affinity and epitope bin information for exemplary anti-MSLN single domain antibodies of the present disclosure. The affinity of each single domain antibody to recombinant human MSLN (R&D Systems, #3265-MS) was measured by biolayer interferometry (BLI) using an Octet HTX. Test antibodies were immobilized at 5 μg / mL using an AHC (Anti-hIgG Fc Capture) sensor. After a baseline reading, the sensor was immersed in antigen solution (starting at 500 nM, followed by a 7-point, 2-fold dilution series). Association and dissociation were measured for 180 and 240 seconds, respectively. Data were analyzed using Octet Data Analysis v11.0 HT (ForteBio) using a standard 1:1 binding model. Epitope bins for the single domain antibodies were determined by in-tandem competitive BLI binding experiments using an Octet HTX. Antigen (5 μg / mL) was loaded onto the sensor, a Ni-NTA sensor, followed by a baseline measurement in kinetic buffer. The antigen-coated center was then immersed in a saturating concentration of antibody 1. Here, a second baseline was set for 300 seconds. The antigen-antigen 1 complex was then immersed in antibody 2 solution for 180 seconds and then dissociated.

[0281] [Table 17]

[0282] Example 5. CAR-T-mediated T cell activation with human tumor cell lines CAR-T cell activity was measured by transfecting Jurkat T lymphocytes with an anti-MSLN CAR and a 6xNFAT TK nanoluciferase reporter according to the manufacturer's protocol (Lonza 4D-Nucleofector X; SE Cell Line 4D-Nucleofector X Kit L, #V4XC-1012; program CL-120). Figure 2A is a schematic diagram of an exemplary CAR-T construct containing an anti-MSLN extracellular binding domain comprising an antibody sequence of the present disclosure. Transfected Jurkat cells were co-cultured with MSLN+ CHO cells, HeLa, or MSLN-negative K562 cells stably transfected to express human MSLN in RPMI-1640 medium (Fisher Scientific, Waltham, Massachusetts, USA) containing 10% FBS (ThermoFisher) in a humidified incubator at 37°C and 8% CO for 24 hours. Luciferase activity was measured using the Promega Nano-Glo Luciferase Assay System on a SpectraMax i3x multimode microplate reader (Molecular Devices) using the manufacturer's protocol (catalog #N1110). Data were normalized to cocultures containing CAR-transfected Jurkat and MSLN-negative K562 cell lines. Statistical significance was determined using an unpaired, two-tailed t-test. Results are shown in Figure 2B.

[0283] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and journal articles, are expressly incorporated herein by reference. Anything described in an embodiment of the present disclosure can be combined with one or more other embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0284] The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, but instead as non-limiting exemplifications of particular aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. Indeed, various modifications of the disclosed subject matter in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and accompanying figures. Such modifications are intended to be within the scope of the disclosed subject matter.

[0285] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for purposes of illustration and are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has been selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the marketplace, and / or to enable those skilled in the art to understand the disclosed subject matter.

Claims

1. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody comprises a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 95% sequence identity with any one of CDR1, 2, and 3 of SEQ ID NOs. 15 to 35.

2. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) A VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications compared to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, (ii) VH CDR2 having a sequence having up to two amino acid modifications compared to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, (iii) VH CDR3 containing a sequence having up to two amino acid modifications compared to SEQ ID NO: 13 or SEQ ID NO: 14 A single-domain antibody containing a heavy chain variable (VH) region.

3. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) Array GFX １ FX ２ SSYX ３ VH complementarity determination region 1 (CDR1) including (Sequence ID 80), In the formula, X １ is T or S, X ２ is S or T, and X ３ VH complementarity determination region 1 (CDR1) is D, P, or A, (ii) Array IX ４ X ５ X ６ GX ７ X ８ X ９ a VH CDR2 containing (sequence number 81), In the formula, X ４ is S or T, X ５ is Y, G, or S, and X ６ is D or S, X ７ is S, G, or D, X ８ is K, G, or S, and X ９ VH CDR2 is K or T, (iii) Array AX １０ VH CDR3 containing PPSYYDFLSPDY (Sequence ID 82), In the formula, X １０ VH CDR3 and A single-domain antibody containing a heavy chain variable (VH) region.

4. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) A VH complementarity determination region 1 (CDR1) including sequences selected from sequence number 1, sequence number 2, sequence number 3, sequence number 4, and sequence number 5, (ii) VH CDR2 containing an array selected from SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 9, SEQ ID NOs. 10, SEQ ID NOs. 11, and SEQ ID NOs. 12, (iii) VH CDR3 containing sequences selected from sequence numbers 13 and 14 A single-domain antibody containing a heavy chain variable (VH) region.

5. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 6, and 13, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 6, and 14, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 14, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 8, and 14, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 2, 6, and 14, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 2, 7, and 14, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 2, 9, and 14, respectively; (h) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 2, 10, and 13, respectively; (i) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 2, 12, and 14, respectively; (j) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 3, 6, and 14, respectively; (k) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 4, 6, and 14, respectively; (l) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 4, 7, and 14, respectively; or (m) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 5, 11, and 14, respectively. A single-domain antibody containing a heavy chain variable (VH) region.

6. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody includes a heavy chain variable (VH) region comprising one of the CDR1, CDR2, and CDR3 of SEQ ID NOs.

7. The single-domain antibody according to any one of claims 1 to 6, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences are present in the human VH framework.

8. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody includes a heavy chain variable (VH) region having at least 95% sequence identity with any one of SEQ ID NOs. 15 to 35.

9. The single-domain antibody according to claim 8, wherein the single-domain antibody comprises a heavy chain variable (VH) region selected from SEQ ID NOs. 15 to 35.

10. The single-domain antibody according to any one of claims 1 to 6 or 8, wherein the single-domain antibody specifically binds to human MSLN.

11. The aforementioned single-domain antibody is approximately 10 －９ M-10 －６ M's K Ｄ A single-domain antibody according to any one of claims 1 to 6 or 8, which binds to human MSLN.

12. The single-domain antibody according to any one of claims 1 to 6 or 8, wherein the single-domain antibody is an isolated single-domain antibody.

13. A mesothelin-binding protein comprising a single-domain antibody according to any one of claims 1 to 6 or 8.

14. The mesothelin-binding protein according to claim 13, wherein the mesothelin-binding protein specifically binds to human MSLN.

15. The mesothelin-binding protein according to claim 13, wherein the mesothelin-binding protein further specifically binds to CD3.

16. The mesothelin-binding protein according to claim 13, wherein the mesothelin-binding protein further specifically binds to human CD3.

17. The mesothelin-binding protein according to claim 13, wherein the mesothelin-binding protein is an isolated monoclonal antibody.

18. An antibody-drug conjugate comprising a single-domain antibody according to any one of claims 1 to 6 or 8.

19. An anti-mesothelin antibody comprising a single-domain antibody according to any one of claims 1 to 6 or 8.

20. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody is multispecific.

21. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody further specifically binds to tumor-specific antigens other than MSLN.

22. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody is bispecific.

23. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody further specifically binds to CD3.

24. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody further specifically binds to human CD3.

25. The anti-mesothelin antibody according to claim 19, further comprising a CD3-binding VH region.

26. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody is of the IgG1 or IgG4 subtype.

27. The anti-mesothelin antibody according to claim 19, further comprising a CD3-binding VH region that pairs with a light chain variable (VL) region.

28. The CD3-bound VH region is (i) A VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications compared to any one of sequence numbers 36 to 41, (ii) VH CDR2 containing a sequence having up to two amino acid modifications compared to SEQ ID NO: 42, (iii) VH CDR3 containing a sequence having up to two amino acid modifications compared to any one of sequence numbers 43-46 The anti-mesothelin antibody according to claim 25, comprising:

29. The anti-mesothelin antibody according to claim 28, wherein the CD3-conjugated VH CDR1 comprises a sequence selected from sequence numbers 36 to 41.

30. The anti-mesothelin antibody according to claim 28, wherein the CD3-bound VH CDR2 comprises the sequence of SEQ ID NO:

42.

31. The anti-mesothelin antibody according to claim 28, wherein the CD3-conjugated VH CDR3 comprises a sequence selected from sequence numbers 43 to 46.

32. The anti-mesothelin antibody according to claim 25, wherein the full set (combination) of VH CDR1, 2, and 3 in the CD3-binding VH region has at least 95% sequence identity with respect to any one of the CDR1, 2, and 3 in SEQ ID NOs: 47 to 64.

33. The CD3-bound VH region is (i) Sequence GFTFX １１ X １２ VH complementarity determination region 1 (CDR1) including YA (SEQ ID NO: 83), In the formula, X １１ is D, A, or H, and X １２ VH complementarity determination region 1 (CDR1) is D or N, (ii) VH CDR2 containing sequence ISWNSGSI (sequence number 42), (iii) Sequence AKDSRGYGX １３ YX １４ X １５ A VH CDR3 containing GGAY (SEQ ID NO: 84), In the formula, X １３ is D or S, X １４ is R or S, and X １５ VH CDR3 where L or R The anti-mesothelin antibody according to claim 25, comprising:

34. The anti-mesothelin antibody according to claim 25, wherein the CD3-binding VH region comprises one of the CDR1, CDR2, and CDR3 of SEQ ID NOs: 47 to 64.

35. The anti-mesothelin antibody according to claim 25, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are present in the human VH framework.

36. The anti-mesothelin antibody according to claim 25, wherein the CD3-binding VH region has at least 95% sequence identity with any one of SEQ ID NOs: 47 to 64.

37. The CD3-bound VH region is (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 36, 42, and 43, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 36, 42, and 44, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 36, 42, and 45, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 36, 42, and 46, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 37, 42, and 44, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 38, 42, and 44, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 39, 42, and 44, respectively; (h) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 40, 42, and 44, respectively; (i) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 40, 42, and 45, respectively; or (j) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 41, 42, and 45, respectively. The anti-mesothelin antibody according to claim 25, comprising:

38. The anti-mesothelin antibody according to claim 37, wherein the CD3-binding VH region comprises VH CDR1, VH CDR2, and VH CDR3, which include the sequences of SEQ ID NOs: 36, 42, and 43.

39. The anti-mesothelin antibody according to claim 27, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO:

68.

40. The anti-mesothelin antibody according to claim 27, wherein the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3, each containing the sequence of SEQ ID NOs. 65, 66, and 67, respectively.

41. The anti-mesothelin antibody according to claim 27, wherein the VLCDR1, VLCDR2, and VLCDR3 sequences are present in the human VH framework.

42. The anti-mesothelin antibody according to claim 27, wherein the light chain variable region has at least 95% sequence identity with respect to SEQ ID NO:

68.

43. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody specifically binds to human MSLN.

44. The anti-mesothelin antibody according to claim 19, wherein the anti-mesothelin antibody is an isolated antibody.

45. An antibody fragment that specifically binds to mesothelin, wherein the antibody fragment comprises a fragment of the anti-mesothelin antibody described in claim 19.

46. The antibody fragment according to claim 45, wherein the antibody fragment is a tri-chain antibody-like molecule.

47. A pharmaceutical composition comprising at least one of the mesothelin-binding protein described in claim 13, the antibody-drug conjugate described in claim 18, the anti-mesothelin antibody described in claim 19, or the antibody fragment described in claim 45, and a pharmaceutically acceptable excipient.

48. A polynucleotide encoding a single-domain antibody according to any one of claims 1 to 6 or 8.

49. A composition comprising one or more polynucleotides encoding a mesothelin-binding protein according to claim 13, an anti-mesothelin antibody according to claim 19, or an antibody fragment according to claim 45.

50. A recombinant expression vector comprising the polynucleotide described in claim 48 or the composition described in claim 49.

51. A host cell comprising the recombinant expression vector described in claim 50.

52. A pharmaceutical composition for treating a mesothelin expression-related disease in a subject requiring treatment for a disease associated with mesothelin expression, comprising at least one of the following: the mesothelin-binding protein described in claim 13, the antibody-drug conjugate described in claim 18, the anti-mesothelin antibody described in claim 19, or the antibody fragment described in claim 45.

53. The pharmaceutical composition according to claim 52, wherein the disease associated with the expression of the mesothelin is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

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