Mesothelin-binding proteins and their use

Abstract

To provide additional and effective therapeutic and diagnostic agents for diseases associated with mesothelin expression. [Solution] Disclosed are single-domain antibodies and mesothelin-binding proteins that specifically bind to mesothelin (MSLN), anti-mesothelin antibodies and their antibody fragments, antibody-drug conjugates, synthetic immune receptors, and diagnostic agents comprising the same. Also disclosed are pharmaceutical compositions comprising any of the foregoing, and any of the foregoing uses in the treatment and / or diagnosis and / or monitoring of diseases associated with MSLN expression.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 255,887, filed on 14 October 2021, U.S. Provisional Patent Application No. 63 / 255,891, filed on 14 October 2021, U.S. Provisional Patent Application No. 63 / 303,422, filed on 26 January 2022, and U.S. Provisional Patent Application No. 63 / 392,569, filed on 27 July 2022, the contents of each of these, in whole, are incorporated herein by reference.

[0002] The computer-readable amino acid sequence listing submitted concurrently with this specification is incorporated in its entirety by reference and identified as follows: a 70,499-byte XML file named "10184-WO02_SEC_Sequence_Listing" created on 12 October 2022.

[0003] This specification discloses single-domain antibodies and mesothelin-binding proteins that specifically bind to mesothelin (MSLN), anti-mesothelin antibodies and their antibody fragments, antibody-drug conjugates, synthetic immune receptors, and diagnostic agents comprising the same. Pharmaceutical compositions comprising any of the foregoing, and any of the foregoing uses in the treatment and / or diagnosis and / or monitoring of diseases associated with MSLN expression are also disclosed. [Background technology]

[0004] Mesothelin (MSLN; CAK1; UniProt Q13421; also known as 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 monoclonal antibody (mAb) K1 produced by immunization of mice with human ovarian cancer (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 ability. The MSLN gene encodes a 71 kDa precursor protein, which is cleaved by Furin into two products: an amino-terminus shed fragment called megakaryocyte-enhancing factor (MPF; 31 kDa) and a glycosyl phosphatidylinositol (GPI)-anchored glycoprotein MSLN (40 kDa), which remains ligated to the cell membrane via GPI binding. The MSLN protein is organized into a superhelical structure with ARM-type repeats.

[0005] Both MPF and MDLN exhibit physiological activity, but their precise biological functions remain unknown. Knockout mice were prepared in which the mesothelin gene was disrupted by homologous recombination (Bera, TK 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, previously identified as an ovarian cancer antigen. MSLN is associated with tumor cell proliferation and migration (Rump A et al., J Biol Chem. 2004 Mar 5;279(10):9190-8), and the mesothelin-MUC16 interaction has been proposed to function in cell adhesion, invasion, and metastasis. As evidence, mesothelin expression within the peritoneum is correlated with the preferred sites for metastatic formation in ovarian cancer, 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, the expression of MSLN in normal tissues 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 therapies (Hassan R et al., J Clin Oncol. 2016 Dec; 34(34): 4171-4179). Furthermore, MSLN conjugates 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 cancer (Cristaudo et al., Clin. Cancer Res. 13:5076-5081, 2007). Overexpression of MSLN has been associated with poor prognosis in lung adenocarcinoma and triple-negative breast cancer, for example.

[0007] In addition to its expression on the cell surface, mesothelin is also desorbed into the serum through the action of ADAM17 / TACE. Serum levels of desorbed mesothelin are elevated in patients with ovarian cancer and other cancers. MESOMARK®, an ELISA test for desorbed serum mesothelin, is approved by the FDA for humanitarian use and may be helpful in the diagnosis or monitoring of mesothelioma. Desorbed mesothelin is also used alone or in combination with other markers to aid in the diagnosis or prognosis of other cancer types. The correlation between serum levels of desorbed mesothelin and disease suggests a potential role of the mesothelin protein in cancer progression.

[0008] Therefore, additional and effective therapeutic and diagnostic agents are needed 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 [Overview of the project] [Means for solving the problem]

[0010] This specification discloses single-domain antibodies that specifically bind to mesothelin (MSLN), for example, human MSLN.

[0011] In some embodiments, a single-domain antibody is used. (i) array VH complementarity determination region 1 (CDR1) including GGSISX1SYY (sequence number 53), In the formula, VH CDR1 is N or S, (ii) Array A VH CDR2 containing IYX2SGX3X4 (sequence number 68), In the formula, VH CDR2 is such that X2 is H or Y, X3 is N or S, and X4 is T or I. (iii) array A VH CDR3 containing X5X6QX7GVGATTTEEY (Sequence ID 54), The formula includes a heavy-chain variable (VH) region comprising VH CDR3, where X5 is T, V, or A, X6 is S or T, and X7 is D or N.

[0012] In some embodiments, a single-domain antibody contains 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%) sequence identity with any one of the CDR1, 2, and 3 of SEQ ID NOs. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to any one of the CDR1, 2, and 3 of SEQ ID NOs. 11-19.

[0013] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 1 or SEQ ID NO: 9, (ii) VH CDR2 having a sequence with up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 having a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

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

[0015] In some embodiments, VH CDR1 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 1 or SEQ ID NO: 9. In some embodiments, VH CDR2 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10. In some embodiments, VH CDR3 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the up to one amino acid modification is an amino acid substitution. In some embodiments, the up to one amino acid modification is a conservative amino acid substitution. In some embodiments, the up to one amino acid modification is an amino acid deletion. In some embodiments, the up to one amino acid modification is an amino acid addition.

[0016] In some embodiments, VH CDR1 includes sequences selected from SEQ ID NO: 1 and SEQ ID NO: 9. In some embodiments, VH CDR2 includes sequences selected from SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 10. In some embodiments, VH CDR3 includes sequences selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8.

[0017] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing sequences selected from sequence number 1 and sequence number 9, (ii) A VH CDR2 containing sequences selected from sequence number 2, sequence number 7, and sequence number 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 containing a sequence selected from sequence numbers 3, 4, 5, 6, and 8.

[0018] In some embodiments, a single-domain antibody is used. (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 3, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 4, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 5, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 6, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 8, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 6, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 9, 2, and 4, respectively; or (h) Includes heavy chain variable (VH) regions containing VH CDR1, VH CDR2, and VH CDR3, respectively, which contain sequences of sequence numbers 1, 10, and 4.

[0019] In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising one of the CDR1, CDR2, and CDR3 sequences from SEQ ID NOs. 11-19.

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

[0021] In some embodiments, the single-domain antibody includes 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%) sequence identity to any one of SEQ ID NOs. 11-19.

[0022] In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region selected from SEQ ID NOs: 11-19.

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

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

[0025] Furthermore, this specification discloses a mesothelin-binding protein comprising a single-domain antibody that specifically binds 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 binds to approximately 10 -9 M~10-6 M's K D It then 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 binds more specifically to CD3. In some embodiments, the mesothelin-binding protein binds more specifically to human CD3. In some embodiments, the mesothelin-binding protein binds more specifically to human CD3 epsilon. In some embodiments, the mesothelin-binding protein binds to an epitope on CD3 containing at least one residue selected from CD3 epsilon (SEQ ID NO: 69): K73 and S83; and CD3 delta (SEQ ID NO: 70): K82 and C93. In some embodiments, the epitope on CD3 contains a region of CD3 epsilon defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. In some embodiments, the epitope on CD3 includes the CD3 epsilon region defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, and S83. In some embodiments, the epitope includes a structural epitope having both CD3 delta and CD3 epsilon residues. In some embodiments, the structural epitope includes 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 comprises a CD3-binding VH region that pairs with a light chain (LV) region.

[0030] In some embodiments, the CD3-bound VH region is (i) A VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., 0, 1, or 2) compared to any one of sequence numbers 20-25, (ii) VH CDR2 containing a sequence having up to two amino acid modifications (e.g., 0, 1, or 2) compared to SEQ ID NO: 26, (iii) The VH CDR3 comprises a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to any one of sequence numbers 27-30.

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

[0032] In some embodiments, CD3-bound VH CDR1 includes a sequence having up to one amino acid modification compared to any one of SEQ ID NOs. 20-25. In some embodiments, CD3-bound VH CDR2 includes a sequence having up to one amino acid modification compared to SEQ ID NOs. 26. In some embodiments, CD3-bound VH CDR3 includes a sequence having up to one amino acid modification compared to any one of SEQ ID NOs. 27-30. In some embodiments, the up to one amino acid modification is an amino acid substitution. In some embodiments, the up to one amino acid modification is a conservative amino acid substitution. In some embodiments, the up to one amino acid modification is an amino acid deletion. In some embodiments, the up to one amino acid modification is an amino acid addition.

[0033] In some embodiments, CD3-bound VH CDR1 includes a sequence selected from sequence numbers 20-25. In some embodiments, CH3-bound VH CDR2 includes the sequence of sequence number 26. In some embodiments, CD3-bound VH CDR3 includes a sequence selected from sequence numbers 27-30.

[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 any one of CDR1, 2, and 3 of SEQ ID NOs: 31-48. 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 any one of CDR1, 2, and 3 of SEQ ID NOs: 31-48. 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%, etc.) sequence identity to any one of CDR1, 2, and 3 of SEQ ID NOs: 31-48. 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 any one of CDR1, 2, and 3 of SEQ ID NOs: 31-48.

[0035] In some embodiments, the CDR3-binding VH region comprises any one of CDR1, CDR2, and CDR3 of SEQ ID NOs: 31-48.

[0036] In some embodiments, the CD3-binding VH region (i) a VH complementarity determining region 1 (CDR1) comprising the sequence GFTFX8X9YA (SEQ ID NO: 55), wherein X8 is D, A, or H, and X9 is D or N, the VH CDR1, (ii) a VH CDR2 comprising the sequence ISWNSGSI (SEQ ID NO: 26), and (iii) a VH CDR3 comprising the sequence AKDSRGYGX 10 Y X 11 X 12 GGAY (SEQ ID NO: 56), wherein X 10is D or S, X 11 is R or S, and X 12 Includes VH CDR3, where L is or R.

[0037] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are located within the 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%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 95% sequence identity with any one of sequence numbers 31-48.

[0039] In some embodiments, the CD3-bound VH region is (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 27, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 20, 26, and 28, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 29, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 21, 26, and 28, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 22, 26, and 28, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 23, 26, and 28, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 24, 26, and 28, respectively; (h) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 30, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 25, 26, and 29, respectively; or (j) Includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 24, 26, and 29, respectively.

[0040] In some embodiments, the light chain variable region includes CDR1, CDR2, and CDR3 of sequence number 52. In some embodiments, the light chain variable region includes VL CDR1, VL CDR2, and VL CDR3, each containing the sequences of sequence numbers 49, 50, and 51, respectively. In some embodiments, the VL CDR1, VL CDR2, and VL CDR3 sequences are present in the 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%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 95% sequence identity with respect to sequence number 52.

[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 (TCA).

[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 includes a heterodimerization modification. In some embodiments, the Fc region is a silenced Fc region.

[0046] Furthermore, this specification discloses polynucleotides encoding single-domain antibodies that specifically bind to the mesothelin described herein.

[0047] Furthermore, this specification discloses compositions comprising one or more polynucleotides encoding the mesothelin-binding protein described herein. In some embodiments, the mesothelin-binding protein is an anti-mesothelin antibody or a fragment thereof.

[0048] Furthermore, this specification discloses a recombinant expression vector comprising a single-domain antibody that specifically binds to the mesothelin described herein, as well as a host cell comprising the recombinant expression vector.

[0049] Furthermore, this specification discloses one or more recombinant expression vectors comprising one or more polynucleotides encoding the mesothelin-binding protein described herein, as well as host cells comprising one or more recombinant expression vectors.

[0050] Furthermore, this specification discloses a synthetic immune receptor comprising a single-domain antibody that specifically binds to the mesothelin described herein, as well as cells comprising the synthetic immune receptor.

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

[0052] Furthermore, this specification discloses pharmaceutical compositions comprising a mesothelin-binding protein, an antibody-drug conjugate, or an anti-mesothelin antibody or a fragment thereof, and a pharmaceutically acceptable excipient.

[0053] Furthermore, this specification discloses a method for treating a subject in need of treatment for a disease associated with mesothelin expression, 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 diseases associated with mesothelin expression are selected from proliferative disorders and cancers. In some embodiments, the cancers are selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0054] Furthermore, the Specified Mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein are disclosed for use in the treatment of diseases associated with mesothelin expression. In some embodiments, the diseases associated with mesothelin expression are selected from proliferative disorders and cancers. In some embodiments, the cancers are selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0055] Furthermore, this specification discloses the use of mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein in the manufacture of pharmaceuticals for the treatment of diseases associated with mesothelin expression. In some embodiments, the diseases associated with mesothelin expression are selected from proliferative disorders and cancers. In some embodiments, the cancers are selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer. [Brief explanation of the drawing]

[0056] [Figure 1A] Representative CHO cell-binding dose curves for the exemplary single-domain antibodies of this disclosure are shown, where CHO cells express human MSLN. [Figure 1B] Representative HeLa cell binding dose curves for the exemplary single-domain antibodies of this disclosure are shown. [Figure 1C] Representative CHO cell binding dose curves for the exemplary single-domain antibodies of this disclosure are shown, where CHO cells do not express the MSLN protein. In Figures 1A–1C, the mean fluorescence intensity of PE is plotted as a multiple of the background (i.e., cells incubated with secondary detection antibody only). [Figure 2A] This is a schematic diagram of a CAR-T structure containing an anti-MSLN extracellular binding domain with the antibody sequence described herein. [Figure 2B]This shows the T cell activity of Jurkat cells transfected with anti-MSLN 394556 CAR containing CHO-huMSLN (**p=0.0075) and HeLa (**p=0.0015). [Modes for carrying out the invention]

[0057] Definition: In some embodiments, "approximately" when used in relation to a measurable numerical variable refers to all values ​​of the variable that are greater than the indicated value of the variable and the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or ±10% of the indicated value. In some embodiments, the numerical range includes a number (i.e., endpoints) that defines the range.

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

[0059] As used herein, the terms “a” and “an” mean “one or more” unless otherwise indicated. Furthermore, “one or more” and “at least one” are used interchangeably herein. In addition, unless the context requires otherwise, singular terms include plural forms, and plural terms include singular forms.

[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 term “light chain” or “immunoglobulin light chain” as used herein refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) may 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 a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4), arranged from the amino terminus to the carboxyl terminus. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG 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), and the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). The constant domains of the immunoglobulin heavy chain may be derived from any immunoglobulin isotype, including subtypes. The antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain) and between the hinge regions of the two antibody heavy chains. In some embodiments, the antibodies of this disclosure are human antibodies or humanized antibodies and may be of type IgG1, IgG2, IgG3, or IgG4.

[0061] The variable regions of immunoglobulin chains generally exhibit an identical overall structure, including a relatively conserved framework region (FR) joined by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs, derived from the two chains of each heavy / light chain pair, are typically aligned by the framework region to form a structure that specifically binds to a particular epitope of a target protein (e.g., MSLN or CD3). From the N-terminus to the C-terminus, both the naturally occurring light and heavy chain variable regions typically follow this order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering schemes have been devised to assign numbers to the amino acids occupying positions within each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or in Chothia & Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDR and FR of a given antibody can be identified using this system. Other numbering systems for amino acids in 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 this disclosure, “CDR” means 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] A "framework region" or "FR" residue is a variable domain residue other than a hypervariable region / CDR residue as defined herein.

[0063] The antibody residues described herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1 to 113 of the heavy chain) (e.g., 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 to refer to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al., cited above). "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers of constant domains such as antibodies, single-domain antibodies, and antibody fragments refer to residue numbering according to the EU numbering system.

[0064] As used herein, "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 substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any naturally occurring variations that may be present. Typically, in contrast to polyclonal antibody preparations, which contain different antibodies targeting different determinants (epitopes), monoclonal antibodies generally target a single determinant on an antigen. As a non-limiting example, monoclonal antibodies according to this disclosure may be prepared by the hybridoma method first described by Kohler et al (1975) Nature 256:495, and may also be prepared by recombinant protein preparation methods (see, for example, U.S. Patent No. 4,816,567).

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

[0067] As used herein, “antibody fragment” generally refers to fragments of full-length antibodies such as 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 chain and a light chain), or modified fragments of full-length antibodies, such as tri-chain antibody-like molecules, heavy chain-only antibodies, single-chain variable fragments (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, diabody, single-chain diabody, tandem diabody (Tandabs), tandem di-scFv, tandem tri-scFv, and the following: (VH-V This refers to "mini-bodies" exemplified by structures such as L-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multi-bodies (e.g., tria-bodies or tetra-bodies), and single-domain antibodies (e.g., nano-bodies or single-variable-domain antibodies containing only one variable region, which may be VHH, VH, or VL, that specifically binds to an antigen or target independently of other variable regions or domains).

[0068] As used herein, the term “heavy chain only antibody” refers to a dimeric immunoglobulin protein consisting of two heavy chain polypeptides (e.g., heavy chain polypeptides each approximately 50–70 kDa). A “heavy chain only antibody” is an antibody fragment lacking the two light chain polypeptides found in conventional antibodies. In some embodiments, a “heavy chain only antibody” is a homodimeric antibody containing 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 consists of a variable region antigen-binding domain comprising Framework 1, CDR1, Framework 2, CDR2, Framework 3, CDR3, and Framework 4. In some embodiments, a heavy chain only antibody consists 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 consists 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 consists of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. While the heavy chain-only antibodies described herein may belong to the IgG subclass, heavy chain-only antibodies belonging to other subclasses such as the IgM, IgA, IgD, and IgE subclasses are also included herein. In some embodiments, the heavy chain-only antibody may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, for example, the IgG1 or IgG4 subtype. In some embodiments, the heavy chain-only antibody is of the IgG1 or IgG4 subtype, and one or more 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 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 CH domains are modified to alter the effector function of the antibody. Modifications of CH domains that alter effector function are further described herein.Non-limiting examples of heavy-chain-only antibodies are described, for example, in International Publication No. 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0069] As used herein, “single-domain antibody” refers to a single-chain polypeptide containing all or part of the heavy-chain variable domains or all or part of the light-chain variable domains of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody.

[0070] As used herein, the terms “tri-chain antibody-like molecule” or “TCA” refer to an antibody-like molecule comprising, essentially comprising, or comprising three polypeptide subunits, two of which comprising, essentially comprising, or comprising 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 to a first antigen. The third polypeptide subunit comprises an Fc portion including CH2, and / or CH3, and / or CH4 domains 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 a first antigen, comprising, essentially comprising, or comprising a heavy chain antibody in which such binding domains originate from or have sequence identity with a variable region of the antibody heavy chain or light chain. A portion of such a variable region is V H and / or V L Gene segments, D and J H Gene segment or J L It can be encoded by gene segments. The variable region is a rearranged V H DJ H , V L DJ H , V H J L or V L J L It can be encoded by a gene segment.

[0071] As used herein, “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 chain, 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 camel heavy-chain antibodies; 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 originate from any mammalian source such as humans, mice, rats, rabbits, or camels.

[0072] When an antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments (each possessing a single antigen-binding site) and the remaining "Fc" fragment (containing all but the first domain of the constant region of the immunoglobulin heavy chain) are produced. The Fab fragment contains the variable domains derived from the light chain and heavy chain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the "Fab fragment" consists of one immunoglobulin 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 the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH domain and CH1 domain derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0073] An immunoglobulin “Fc fragment” or “Fc region” generally comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain. In some embodiments of this disclosure, a mesothelin-binding protein (e.g., an anti-mesothelin antibody fragment (e.g., a TCA or heavy-chain-only antibody)) comprises an Fc region derived from an immunoglobulin. The Fc region may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cell-mediated 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 functions.

[0074] "Functional Fc regions" possess "effector functions" of naturally occurring Fc regions. Non-limited examples of effector functions include C1q binding, CDC;Fc receptor binding, ADCC, ADCP, and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions generally require Fc regions that interact with receptors, such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and low-affinity FcRn receptors, and can be evaluated using various assays known in the art.

[0075] "Dead" or "silenced" Fc cells are those that have been mutated to retain activity, for example, with respect to the extension of their serum half-life, but do not activate high-affinity Fc receptors or have reduced affinity for Fc receptors.

[0076] The "native sequence Fc region" contains an amino acid sequence identical to that of a naturally occurring Fc region. Examples of native human Fc regions include the natural sequence human IgG1 Fc region (non-A and A allotypes), the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region, as well as their natural variants.

[0077] The "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, for example, one or more (e.g., two or more, three or more, four or more) amino acid substitutions. Exemplarily, in some embodiments, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, for example, about 1 to about 5 amino acid substitutions in the native sequence Fc region or the parent polypeptide Fc region, compared to the native sequence Fc region or the parent polypeptide Fc region. In some embodiments, the variant Fc region as described herein has at least about 80% homology with the native sequence Fc region and / or the parent polypeptide Fc region, for example, at least about 85% homology, for example, at least about 90% homology, for example, at least about 95% homology, for example, at least about 99% homology.

[0078] As used herein, “heterodimerization change” refers to a change in the A and B chains of an Fc region (i.e., two chains containing an Fc region, where one chain is referred to as the “A” chain and the other as the “B” chain) that promotes the formation of a heterodimer Fc region, i.e., an Fc region in which the A and B chains do not have the same amino acid sequence. In some embodiments, the heterodimerization change may be asymmetric, i.e., an A chain with a particular change may pair with a B chain with a different change. These changes promote heterodimerization and discourage homodimerization. Whether hetero- or homodimerization has been formed can be determined, for example, by polyacrylamide gel electrophoresis in a situation where one polyethylene chain is a dummy Fc and the other is scFv-Fc, and evaluated by the size difference. One non-limiting example of such paired heterodimerization changes is the so-called “knob and hole” substitution. See, for example, U.S. Patent No. 7,695,936 and U.S. Patent Application Publication No. 2003 / 0078385. As used herein, an Fc region containing a pair of knob-and-hole substitutions contains 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 heterodimerization compared to those found in unmodified A and B chains and may be employed in non-limiting embodiments of this 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 T3 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.Substitutions that create new disulfide crosslinks instead of, or in addition to, such changes can promote heterodimerization. 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 Fc polypeptide chain; Y349C in one Fc polypeptide chain and E356C in the other Fc polypeptide chain; Y349C in one Fc polypeptide chain and E357C in the other Fc polypeptide chain; L351C in one Fc polypeptide chain and S354C in the other Fc polypeptide chain; T394C in one Fc polypeptide chain and E397C in the other Fc polypeptide chain; or D399C in one Fc polypeptide chain and K392C in the other Fc polypeptide chain. Additionally or alternatively, substitutions that alter the charge of one or more residues at the CH3-CH3 interface can enhance heterodimer formation, as described, for example, in International Publication No. 2009 / 089004, incorporated herein by reference. Such substitutions are referred 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-restrictive 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. Furthermore, 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 combination with other charge pair substitutions.Specific examples of single pairs and combinations of charge pair substitutions 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 K and E356K; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain. Any of these heterodimerization changes can be used in polypeptides containing the variant Fc region 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 at 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 of human IgG1 or IgG2 residues, and substitutions at positions 327, 330, and 331 of IgG4 residues, significantly reduce ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613–24; and Shields R. Let al., 2001. J Biol Chem. 276(9):6591–604). The Fc amino acid sequence of human IgG4 (UniProtKB number P01861) is shown herein as Sequence ID No. 76. Silenced IgG1 is described, for example, Boesch, AW, et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014. 6(4):p.915–27, and these disclosures are incorporated herein by reference in their entirety.

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

[0081] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to result in complement binding or Fc receptor binding. For example, deletions may occur at complement binding sites, such as C1q binding sites, but are not limited to these. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Publications 97 / 34631 and 96 / 32478. In addition, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

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

[0083] In some embodiments, effector function is reduced by mutations in the constant region that eliminate glycosylation, such as “effectorless mutations.” 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, for example, the variant Fc region is a variant Fc region lacking glycosylation at N297, as described in International Publication No. 2014 / 153063, incorporated herein by reference. Alternatively, 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 non-glycosylating host cells (e.g., Escherichia coli (E. coli)) or expression in host cells that result in altered glycosylation patterns that are 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 replaced 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 Fc and the tryptophan residues W87 and W110 of FcgRIII (Sondermann et al., Nature 406, 267-273 (20 Jul. 2000)). In some further embodiments, at least one further amino acid substitution in the Fc variant region is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, at least one further amino acid substitution is L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, all according to EU numbering (see, for example, U.S. Patent No. 8,969,526, which is 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 includes at least two further amino acid substitutions in L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, where the residues are numbered according to EU numbering (see, for example, U.S. Patent No. 8,969,526). In some embodiments, the variant Fc region including the P329G, L234A and L235A (EU numbering) substitutions exhibits reduced affinity for human FcγRIIIA and FcγRIIA.

[0086] In some embodiments, the variant Fc region includes a triple mutation: amino acid substitutions (P329 / LALA) at positions P329, L234A and L235A according to EU numbering (see, for example, U.S. Patent No. 8,969,526). In some embodiments, the variant Fc region includes 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 containing the 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 containing the T366S mutation, the L368A mutation, and the Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein may be used in any preferred manner 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 the proper pairing (heterodimization) of desired pairs of heavy chain polypeptide subunits in the antibody.

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

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

[0090] The "F(ab')2 fragment" is a divalent fragment containing two Fab' fragments linked by disulfide bridges between heavy chains in the hinge region.

[0091] The "Fv" fragment is the smallest fragment containing a complete antigen recognition and binding site derived from the antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in a tightly covalently bound state. In this configuration, the three CDRs of each variable region interact to define the 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 the three antigen-specific CDRs) has lower affinity than the entire binding site containing both VH and VL, but still possesses the ability to recognize and bind to the antigen.

[0092] A "single-chain variable antibody fragment" or "scFv fragment" comprises a VH region and a VL region of the antibody, which are present on a single polypeptide chain and optionally include a peptide linker between the VH and VL regions that allows Fv to form a desired structure for antigen binding (see, for example, 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 a light chain occur naturally in certain animal species, e.g., nurse sharks, pygmy sharks, and camelids (Camelidae), e.g., camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is reduced to a single-domain VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding domain; that is, these functional antibodies are homodimers of the heavy chain (referred to as "heavy chain antibodies" or "HCAbs") that have only the structure H2L2. Camelized VHH domains, according to reports, contain hinge domains, CH2 domains, and CH3 domains, and recombine with the IgG2 and IgG3 constant regions, which lack a CH1 domain. Camelized VHH domains have been shown to bind to 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 No. 2005 / 0136049 and U.S. Patent Application Publication No. 2005 / 0037421. Another scaffold can be fabricated from human variable-like domains that are more closely compatible with the shark V-NAR scaffold and may provide a framework of long, penetrating loop structures.

[0094] As used herein, the term “antigen-binding protein” refers to a protein that specifically binds to one or more target antigens. Generally, an antigen-binding protein is a protein comprising an antigen-binding fragment that specifically binds to an antigen, and optionally comprising a backbone or framework portion that allows the antigen-binding fragment to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen. In some embodiments, the antigen-binding protein is an antibody or an antibody fragment. In some embodiments, the antigen-binding protein may comprise a protein comprising one or more antigen-binding fragments incorporated into a single-chain polypeptide or into multiple polypeptide chains.For example, antigen-binding proteins include diabodies (see, e.g., European Patent No. 404,097, International Publication No. 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); and maxibodies (two scFv fused to an Fc region; see Fredericks et al, Protein Engineering, Design & Selection, Vol. 17:95-106, 2004 and Powers et al, Journal of Immunological Sciences). See Methods, Vol.251:123-135, 2001); triabody; tetrabody; minibody (scFv fused to the CH3 domain; see Olafsen et al, Protein Eng Des Sel., Vol.17:315-23, 2004); peptidebody (one or more peptides attached to the Fc region; see International Publication No. 00 / 24782); linear antibody (a pair of tandem Fd segments (VH-CHl-VH-CHl) forming a pair of antigen-binding regions with a complementary light chain polypeptide; see Zzjpate et al, Protein Eng., Vol.8: See 1057-1062, 1995; small modular immunotherapies (see U.S. Patent Publication No. 20030133939 brochure); and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc; see, for example, Spiess et al, Mol.Immunol., Vol. 67(2 Pt A):95-106, 2015), but are not limited to these.

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

[0096] The antibodies and antibody fragments of this disclosure (e.g., heavy-chain-only antibodies and triple-chain antibody-like molecules) include multispecific antibodies and antibody fragments which are antibodies and antibody fragments having two or more binding specificities. As used herein, the term “multispecific” includes “double specificity” (i.e., two binding specificities) and “triple specificity” (i.e., three binding specificities), as well as higher-order independent specific binding affinities, such as higher-order polyepitope specificity.

[0097] As used herein, “isolated” molecules (e.g., antibodies, antibody fragments, single-domain antibodies, mesothelin-binding proteins, etc.) are molecules identified and separated and / or recovered from components of their natural environment. Contaminants from the natural environment are substances that may interfere with the diagnostic or therapeutic use of the molecule and may include, for example, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the isolated molecule is purified by (1) determining the isolated molecule by Lowry method until it exceeds 95% by weight, e.g., 99% by weight; (2) obtaining at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) becoming homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, for example, silver staining. In some embodiments, the isolated molecule is prepared by a process that includes one purification step.

[0098] As used herein, “antibody-drug conjugate” refers to an antibody or antibody fragment that is bound to another part, such as a payload, such as a radionuclide.

[0099] As used herein, “epitope” refers to 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. Specifically, this term includes linear epitopes and structural epitopes. Structural and non-structural epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. An epitope may include an amino acid residue 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, “polyepitope 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 the mammal being evaluated and / or treated for a treatment. The subject may be human, but may also include other mammals, such as mice and rats, which are useful as laboratory models for human diseases. In some embodiments, the mammal is human.

[0102] As used herein, the term “treatment” encompasses any improvement of the disease in the subject, including delaying or cessation of disease progression in the patient, a reduction in the number or severity of the symptoms of the subject, or an increase in the frequency or length of periods in which the subject is free from symptoms of the disease.

[0103] As used herein, the term “effector cell” refers to an immune cell 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 bone marrow or lymphoid origin, e.g., lymphocytes (e.g., T cells including B cells and cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, e.g., 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 ADCCs, such as natural killer cells. For example, monocytes and macrophages expressing FcRs are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system or binding to antigen-presenting cells.

[0104] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which this nucleic acid molecule is ligated. A 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, to which additional DNA segments may be ligated to a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the genome of the host cell upon introduction, thereby replicating together with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors.” In some embodiments, expression vectors for use in recombinant DNA technology 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 a specific target cell but also to the offspring of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such offspring may not be, in effect, identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Examples of recombinant host cells include, but are not limited to, CHO cells, HEK293 cells, NS / 0 cells, and transfectomas such as lymphocytes.

[0106] "K D The term (M), as used herein, refers to the dissociation equilibrium constant of a specific antigen-binding interaction determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, a mouse Fc fusion antigen is placed on an anti-mouse Fc sensor and then immersed in an antibody-containing well to determine the concentration-dependent binding rate (k on ) is measured. Antibody dissociation rate (k off ) is measured in the final stage, where the sensor is immersed in a well containing only the buffer solution. D is, k off / k on This is the ratio. (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) "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity to the target antigen compared to its affinity to other unrelated proteins, and as a result can identify the target antigen. A molecule that specifically binds to an antigen is defined as having an affinity of ≤1 × 10⁻⁶. -6 The equilibrium dissociation constant (K) of M D ) can bind to that antigen. The molecule is K D≤ 1 × 10 -8 When M is present, it binds specifically to the antigen with "high affinity". In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 ≤ 5 × 10⁻¹⁴ -7 M's K D They bind together. In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 by ≤1 × 10⁻¹⁰ -7 M's K D They bind together. In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 by ≤5 × 10 -8 M's K D They bind together. In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 by ≤2 × 10⁻¹⁰ -8 M's K D They bind together. In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 by ≤1 × 10⁻¹⁰ -8 M's K D They bind together. In some embodiments, the molecules described herein bind to human MSLN and / or human CD3 by ≤1 × 10⁻¹⁰ -9 M's K D They are joined together.

[0108] Affinity may be determined using various techniques, a non-limiting example being affinity ELISA assays. In some embodiments, affinity is determined by surface plasmon resonance assays (e.g., BIAcore®-based assays). 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. Next, the equilibrium dissociation constant (K D The unit (M) is the ratio of the velocity constants (k d / k aThe affinity can be calculated from the equilibrium dissociation constant (K). In some embodiments, affinity is determined by kinetic methods such as the binding equilibrium exclusion method (KinExA) as described in Rathanaswami et al., Analytical Biochemistry, Vol.373:52-60, 2008. Using the KinExA assay, the equilibrium dissociation constant (K) can be calculated. D , M) and coupling rate constant (k a M -1 s -1 ) can be measured. From these values, the dissociation rate constant (k d , s -1 (K) can be calculated. D ×k a In other embodiments, affinity is determined by biolayer interferometry, as described in Kumaraswamy et al., Methods Mol. Biol., Vol.1278:165-82, 2015, and used in the Octet® system (Pall ForteBio). Rate constant (k a and k d ) and affinity constant (K D This can be calculated in real time using biolayer interferometry.

[0109] As used herein, "[Y]-bound VH CDR" refers to a CDR in the VH region, where the VH region specifically binds to the target [Y].

[0110] As used herein, the terms “amino acid” or “amino acid residue” refer to amino acids having definitions accepted in the art, such as amino acids 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, synthesized, or rare amino acids may be used as needed. Generally, amino acids can be classified by the presence of 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, deletions from and / or insertions of residues in an amino acid sequence, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions may be performed to arrive at the 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 changes in the number or location of glycosylation sites. Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions) is conceivable, as long as the final construct retains its ability to bind to the target antigen.

[0112] Those skilled in the art will recognize that conserved variants of the antibodies and antibody fragments described herein can be produced. Such conserved variants employed in antibody fragments such as dsFv fragments or scFv fragments are V H and V L The key amino acid residues necessary for accurate folding and stabilization between regions are retained, and the charge properties of the residues are maintained to keep the molecule 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 increase yield. H and / or V L This can be done within the domain. For example, tables of conservative amino acid substitutions that provide functionally similar amino acids, such as those listed in Table A1, are well known to those skilled in the art.

[0113] [Table 1]

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

[0115] As used herein, “amino acid sequence identity percentage (%)” or “sequence identity percentage (%)” relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum possible sequence identity percentage, without considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the scope 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 algorithm necessary to achieve the maximum possible alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity percentage value is generated using the sequence comparison computer program ALIGN-2.

[0116] The term “pharmaceutical composition” refers to a formulation that is in a form that enables the biological activity of the active ingredient to be effective and does not contain any additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered. Such a composition is sterile. “Pharmacologically acceptable” excipients (e.g., vehicles, additives) are those that can be reasonably administered to the target mammal to provide an effective dose of the active ingredient used.

[0117] As used herein, a “sterile” composition is sterile or free from or essentially free from all living microorganisms and their spores. As used herein, a “frozen” composition is at a temperature below 0°C.

[0118] As used herein, a “stable” composition is one in which the proteins within it essentially retain their physical and / or chemical stability and / or biological activity during storage. In some embodiments, the composition essentially retains its physical and chemical stability and its biological activity during 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 outlined, 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 may be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively assessed by a variety of different methods, including evaluation of aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or visual inspection); assessment of charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; analysis of amino-terminus or carboxy-terminus sequences; mass spectrometry; SDS-PAGE analysis for comparison of reduced intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; and evaluation of the biological activity or antigen-binding function of the antibody. Instability may include 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 cysteine, N-terminal elongation, C-terminal processing, and differences in glycosylation.

[0119] Some embodiments of this disclosure relate to single-domain antibodies that specifically bind to mesothelin (MSLN). Specifically, this disclosure provides a family closely related to single-domain antibodies that specifically bind to human mesothelin (MSLN). The single-domain antibodies of this family include a set of CDR sequences defined herein and shown in Tables S1 and S2, and are exemplified by the provided heavy chain variable region (VH) sequences, SEQ ID NOs. 11-19, listed in Table S3. This family of single-domain antibodies offers many benefits that contribute to their usefulness as clinical therapeutic agents. As an example, the single-domain antibodies include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities.

[0120] [Table 2]

[0121] [Table 3]

[0122] [Table 4]

[0123] In some embodiments, a single-domain antibody is used. (i) array VH complementarity determination region 1 (CDR1) including GGSISX1SYY (sequence number 53), In the formula, VH CDR1 is N or S, (ii) Array A VH CDR2 containing IYX2SGX3X4 (sequence number 68), In the formula, VH CDR2 is such that X2 is H or Y, X3 is N or S, and X4 is T or I. (iii) array A VH CDR3 containing X5X6QX7GVGATTTEEY (Sequence ID 54), The formula includes a heavy-chain variable (VH) region comprising VH CDR3, where X5 is T, V, or A, X6 is S or T, and X7 is D or N.

[0124] In some embodiments, a single-domain antibody contains 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%) sequence identity with any one of the CDR1, 2, and 3 of SEQ ID NOs. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to any one of the CDR1, 2, and 3 of SEQ ID NOs. 11-19.

[0125] In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 11. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 12. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 13. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 14. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 15. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 16.In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 17. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 18. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 19.

[0126] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 1 or SEQ ID NO: 9, (ii) VH CDR2 having a sequence with up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 having a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

[0127] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 3.

[0128] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0129] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 5.

[0130] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 6.

[0131] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 7; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 8.

[0132] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 9; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6.

[0133] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 9; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0134] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 10; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0135] In some embodiments, each amino acid modification is a conserved amino acid substitution, if any. In some embodiments, each amino acid modification is a conserved amino acid substitution, if any, listed in Table A1.

[0136] In some embodiments, VH CDR1 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 1 or SEQ ID NO: 9. In some embodiments, VH CDR2 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10. In some embodiments, VH CDR3 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the up to one amino acid modification is an amino acid substitution. In some embodiments, the up to one amino acid modification is a conservative amino acid substitution. In some embodiments, the up to one amino acid modification is an amino acid deletion. In some embodiments, the up to one amino acid modification is an amino acid addition.

[0137] In some embodiments, VH CDR1 includes sequences selected from SEQ ID NO: 1 and SEQ ID NO: 9. In some embodiments, VH CDR2 includes sequences selected from SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 10. In some embodiments, VH CDR3 includes sequences selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8.

[0138] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing sequences selected from sequence number 1 and sequence number 9, (ii) A VH CDR2 containing sequences selected from sequence number 2, sequence number 7, and sequence number 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 containing a sequence selected from sequence numbers 3, 4, 5, 6, and 8.

[0139] In some embodiments, a single-domain antibody is used. (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 3, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 4, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 5, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 6, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 8, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 6, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 9, 2, and 4, respectively; or (h) Includes heavy chain variable (VH) regions containing VH CDR1, VH CDR2, and VH CDR3, respectively, which contain sequences of sequence numbers 1, 10, and 4.

[0140] In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 4, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 5, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 6, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 7, and 8, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 9, 2, and 6, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 9, 2, and 4, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 1, 10, and 4, respectively.

[0141] In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region containing one of the CDR1, CDR2, and CDR3 of SEQ ID NOs. 11-19. 2. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 11. 3. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 12. 4. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 13. 5. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 14. 6. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 15. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 16. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 17. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 18. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 19.

[0142] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences are present in the human VH framework.

[0143] In some embodiments, the single-domain antibody includes 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%) sequence identity to any one of SEQ ID NOs. 11-19.

[0144] In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 11. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 12. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 13. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 14. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 15. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 16. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 17. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 18. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 19.

[0145] In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region selected from SEQ ID NOs: 11-19.

[0146] In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 11. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 12. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 13. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 14. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 15. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 16. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 17. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 18. In some embodiments, the single-domain antibody includes the heavy chain variable (VH) region of SEQ ID NO: 19.

[0147] In some embodiments, the single-domain antibody specifically binds to human MSLN.

[0148] In some embodiments, the single-domain antibody is approximately 10 -9 M ~ about 10 -6 M's K D It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤5 × 10 -7 M's K D It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤1 × 10⁻⁶ -7 M's K D It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤5 × 10 -8 M's K D It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤2 × 10⁻⁶ -8 M's KD It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤1 × 10⁻⁶ -8 M's K D It binds to human MSLN. In some embodiments, the single-domain antibody has a domain of ≤1 × 10⁻⁶ -9 M's K D It then binds to human MSLN.

[0149] In some embodiments, the single-domain antibody is a human single-domain antibody.

[0150] 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.

[0151] Some embodiments of this disclosure relate to mesothelin-binding proteins, including single-domain antibodies that specifically bind to mesothelin as described herein.

[0152] In some embodiments, a single-domain antibody is used. (i) array VH complementarity determination region 1 (CDR1) including GGSISX1SYY (sequence number 53), In the formula, VH CDR1 is N or S, (ii) Array A VH CDR2 containing IYX2SGX3X4 (sequence number 68), In the formula, VH CDR2 is such that X2 is H or Y, X3 is N or S, and X4 is T or I. (iii) array A VH CDR3 containing X5X6QX7GVGATTTEEY (Sequence ID 54), The formula includes a heavy-chain variable (VH) region comprising VH CDR3, where X5 is T, V, or A, X6 is S or T, and X7 is D or N.

[0153] In some embodiments, a single-domain antibody contains 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%) sequence identity with any one of the CDR1, 2, and 3 of SEQ ID NOs. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity to any one of the CDR1, 2, and 3 of SEQ ID NOs. 11-19.

[0154] In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 11. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 12. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 13. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 14. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 15. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 16.In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 17. In some embodiments, the single-domain antibody includes 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 18. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 19.

[0155] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 1 or SEQ ID NO: 9, (ii) VH CDR2 having a sequence with up to two amino acid modifications (e.g., one, two, or zero) compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 having a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8.

[0156] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 3.

[0157] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0158] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 5.

[0159] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3 comprising a sequence having up to two (e.g., one, two, zero) amino acid modifications compared to SEQ ID NO: 6.

[0160] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 7; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 8.

[0161] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 9; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 6.

[0162] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 9; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 2; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0163] In some embodiments, the single-domain antibody includes a heavy-chain variable (VH) region comprising: (i) VH CDR1, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 1; (ii) VH CDR2, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 10; and (iii) VH CDR3, which comprises a sequence having up to two (e.g., one, two, or zero) amino acid modifications compared to SEQ ID NO: 4.

[0164] In some embodiments, each amino acid modification is a conserved amino acid substitution, if any. In some embodiments, each amino acid modification is a conserved amino acid substitution, if any, listed in Table A1.

[0165] In some embodiments, VH CDR1 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 1 or SEQ ID NO: 9. In some embodiments, VH CDR2 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10. In some embodiments, VH CDR3 includes a sequence having up to one amino acid modification compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the up to one amino acid modification is an amino acid substitution. In some embodiments, the up to one amino acid modification is a conservative amino acid substitution. In some embodiments, the up to one amino acid modification is an amino acid deletion. In some embodiments, the up to one amino acid modification is an amino acid addition.

[0166] In some embodiments, VH CDR1 includes sequences selected from SEQ ID NO: 1 and SEQ ID NO: 9. In some embodiments, VH CDR2 includes sequences selected from SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 10. In some embodiments, VH CDR3 includes sequences selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8.

[0167] In some embodiments, a single-domain antibody is used. (i) VH complementarity determination region 1 (CDR1) containing sequences selected from sequence number 1 and sequence number 9, (ii) A VH CDR2 containing sequences selected from sequence number 2, sequence number 7, and sequence number 10, (iii) A heavy chain variable (VH) region comprising a VH CDR3 containing a sequence selected from sequence numbers 3, 4, 5, 6, and 8.

[0168] In some embodiments, a single-domain antibody is used. (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 3, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 4, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 5, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 6, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 8, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 6, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 9, 2, and 4, respectively; or (h) Includes heavy chain variable (VH) regions containing VH CDR1, VH CDR2, and VH CDR3, respectively, which contain sequences of sequence numbers 1, 10, and 4.

[0169] In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 4, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 5, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 2, and 6, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs: 1, 7, and 8, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 9, 2, and 6, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 9, 2, and 4, respectively. In some embodiments, the single-domain antibody includes heavy chain variable (VH) regions comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 1, 10, and 4, respectively.

[0170] In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region containing one of the CDR1, CDR2, and CDR3 of SEQ ID NOs. 11-19. 2. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 11. 3. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 12. 4. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 13. 5. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 14. 6. The single-domain antibody includes a heavy chain variable (VH) region containing CDR1, CDR2, and CDR3 of SEQ ID NO. 15. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 16. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 17. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 18. In some embodiments, the single-domain antibody includes a heavy chain variable (VH) region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 19.

[0171] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences are present in the human VH framework.

[0172] In some embodiments, the single-domain antibody includes 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%) sequence identity to any one of SEQ ID NOs. 11-19.

[0173] In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 11. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 12. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 13. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 14. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 15. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 16. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 17. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 18. In some embodiments, the single-domain antibody includes 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%) relative to SEQ ID NO: 19.

[0174] In some embodiments, the single domain antibody comprises a heavy chain variable (VH) region selected from SEQ ID NOs: 11-19.

[0175] In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 11. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 12. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 13. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 14. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 15. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 16. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 17. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 18. In some embodiments, the single domain antibody comprises the heavy chain variable (VH) region of SEQ ID NO: 19.

[0176] In some embodiments, the mesothelin binding protein specifically binds to human MSLN.

[0177] In some embodiments, the mesothelin binding protein binds to human MSLN with a K -9 of about 10 -6 M to about 10 D M. In some embodiments, the mesothelin binding protein binds to human MSLN with a K -7 of ≦ 5×10 s D M. In some embodiments, the mesothelin binding protein binds to human MSLN with a K -7 of ≦ 1×10 D M. In some embodiments, the mesothelin binding protein binds to human MSLN with a K -8 of ≦ 5×10 D M. In some embodiments, the mesothelin binding protein binds to human MSLN with a K of ≦ 2×10-8 K of M D It binds to human MSLN. In some embodiments, the mesothelin-binding protein is ≤ 1 × 10 -8 K of M D It binds to human MSLN. In some embodiments, the mesothelin-binding protein is ≤ 1 × 10 -9 K of M D It binds to human MSLN.

[0178] 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.

[0179] 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 binds to human CD3 with a K of about 10 -9 M to about 10 -6 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 5 × 10 -7 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 1 × 10 -7 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 5 × 10 -8 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 2 × 10 -8 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 1 × 10 -8 K of M D It binds to human CD3. In some embodiments, the mesothelin-binding protein binds to human CD3 with a K of ≤ 1 × 10 -9 K of M D It binds to human CD3.

[0180] In some embodiments, the mesothelin-binding protein is approximately 10 -9 M ~ about 10 -6 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤5 × 10⁻⁶ -7 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤1 × 10⁻⁶ -7 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤5 × 10⁻⁶ -8 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤2 × 10⁻⁶ -8 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤1 × 10⁻⁶ -8 M's K D It binds to human MSLN and / or CD3. In some embodiments, the mesothelin-binding protein has a capacity of ≤1 × 10⁻⁶ -9 M's K D It binds to human MSLN and / or CD3.

[0181] In some embodiments, the mesothelin-binding protein binds more specifically to human CD3 epsilon. In some embodiments, the mesothelin-binding protein binds to an epitope on CD3 containing at least one residue selected from CD3 epsilon (SEQ ID NO: 69): K73 and S83; and CD3 delta (SEQ ID NO: 70): K82 and C93. In some embodiments, the epitope on CD3 contains a region of CD3 epsilon defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. In some embodiments, the epitope on CD3 contains a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, and S83. In some embodiments, the epitope includes a structural epitope having both CD3 delta and CD3 epsilon residues. In some embodiments, the structural epitope includes residues CD3ε K73 and S83; and CD3δ K82 and C93, respectively.

[0182] In some embodiments, the mesothelin-binding protein further comprises a CD3-binding VH region. In some embodiments, the mesothelin-binding protein comprises a CD3-binding VH region that pairs with a light chain (LV) region.

[0183] In some embodiments, the CD3-binding VH domain may belong to a family of single-domain antibodies closely associated with specific binding to human CD3. This family of single-domain antibodies comprises the set of CDR sequences defined herein and shown in Tables S4 and S5, and is exemplified by the heavy-chain variable region (VH) sequences provided as Sequence IDs 31-48, listed in Table S6. These multispecific molecules, comprising these CD3-binding VH domains and their associated light-chain variable domains (as shown in Tables S7 and S8), possess advantageous properties, such as those described in the published PCT application brochure WO2018 / 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 MSLNs can be combined with the CD3-binding domain and the immobilized light-chain domain described herein to generate a multispecific mesothelin-binding protein.

[0184] [Table 5]

[0185] [Table 6]

[0186] [Table 7]

[0187] [Table 8]

[0188] [Table 9]

[0189] [Table 10]

[0190] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to any one of SEQ ID NOs: 20-25, (ii) a VH CDR2 comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 26, (iii) a VH CDR3 comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to any one of SEQ ID NOs: 27-30.

[0191] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 20, (ii) a VH complementarity-determining region 2 (CDR2) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 26, and (iii) a VH complementarity-determining region 3 (CDR3) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 27. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 26.

[0192] In some embodiments, the CD3-binding VH region comprises (i) a VH complementarity-determining region 1 (CDR1) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 20, (ii) a VH complementarity-determining region 2 (CDR2) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 26, and (iii) a VH complementarity-determining region 3 (CDR3) comprising a sequence having a maximum of 2 (e.g., 0, 1, or 2, etc.) amino acid modifications compared to SEQ ID NO: 28. In some embodiments, the CH3-binding VH CDR2 comprises the sequence of SEQ ID NO: 26.

[0193] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 20; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 29. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0194] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 21; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 28. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0195] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 22; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 28. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0196] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 23; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 28. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0197] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 24; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 28. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0198] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 20; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 30. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0199] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 25; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 29. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0200] In some embodiments, the CD3-binding VH region includes (i) a VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 24; (ii) a VH complementarity determination region 2 (CDR2) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 26; and (iii) a VH complementarity determination region 3 (CDR3) containing a sequence having up to two amino acid modifications (e.g., zero, one, or two) compared to SEQ ID NO: 29. In some embodiments, the CH3-binding VH CDR2 contains the sequence of SEQ ID NO: 26.

[0201] In some embodiments, each amino acid modification is a conserved amino acid substitution, if any. In some embodiments, each amino acid modification is a conserved amino acid substitution, if any, listed in Table A1.

[0202] In some embodiments, CD3-bound VH CDR1 includes a sequence having up to one amino acid modification compared to any one of SEQ ID NOs. 20-25. In some embodiments, CD3-bound VH CDR2 includes a sequence having up to one amino acid modification compared to SEQ ID NOs. 26. In some embodiments, CD3-bound VH CDR3 includes a sequence having up to one amino acid modification compared to any one of SEQ ID NOs. 27-30. In some embodiments, the up to one amino acid modification is an amino acid substitution. In some embodiments, the up to one amino acid modification is a conservative amino acid substitution. In some embodiments, the up to one amino acid modification is an amino acid deletion. In some embodiments, the up to one amino acid modification is an amino acid addition.

[0203] In some embodiments, the CD3-linked VH CDR1 includes a sequence selected from sequence numbers 20 to 25. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 20. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 21. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 22. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 23. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 24. In some embodiments, the CH3-linked VH CDR1 includes the sequence of sequence number 25.

[0204] In some embodiments, the CH3-linked VH CDR2 includes the sequence of SEQ ID NO: 26.

[0205] In some embodiments, the CD3-linked VH CDR3 includes a sequence selected from sequence numbers 27-30. In some embodiments, the CH3-linked VH CDR3 includes the sequence of sequence number 27. In some embodiments, the CH3-linked VH CDR3 includes the sequence of sequence number 28. In some embodiments, the CH3-linked VH CDR3 includes the sequence of sequence number 29. In some embodiments, the CH3-linked VH CDR3 includes the sequence of sequence number 30.

[0206] 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%) sequence identity with any one of the CDR1, 2, and 3 in sequence numbers 31 to 48. 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 with any one of the CDR1, 2, and 3 in sequence numbers 31-48.

[0207] 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 31. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 32. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 33. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 34. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 35. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 36. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-bound VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 37.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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 38. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 39. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 40. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 41. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 42. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 43. In some embodiments, the full set (combination) of VH CDR1, 2, and 3 in the CD3-bound VH region has at least 80% (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%) sequence identity with respect to CDR1, 2, and 3 of sequence number 44.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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 45. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 46. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 47. 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%) sequence identity with respect to CDR1, 2, and 3 of SEQ ID NO: 48.

[0208] In some embodiments, the CDR3-bound VH region includes one of the CDR1, CDR2, and CDR3 from sequence numbers 31 to 48. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 31. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 32. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 33. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 34. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 35. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 36. In some embodiments, the CD3-bound VH region includes the CDR1, CDR2, and CDR3 of sequence number 37. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 38. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 39. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 40. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 41. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 42. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 43. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 44. In some embodiments, the CD3-binding VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 45. In some embodiments, the CD3-bound VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 46. In some embodiments, the CD3-bound VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 47. In some embodiments, the CD3-bound VH region includes CDR1, CDR2, and CDR3 of SEQ ID NO: 48.

[0209] In some embodiments, the CD3-binding VH region is (a) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 20, 26, and 27; (b) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 20, 26, and 28; (c) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 20, 26, and 29; (d) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 21, 26, and 28; (e) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 22, 26, and 28; (f) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 23, 26, and 28; (g) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 24, 26, and 28; (h) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 20, 26, and 30; (i) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 25, 26, and 29; or (j) VH CDR1, VH CDR2, and VH CDR3 each comprising the sequences of SEQ ID NO: 24, 26, and 29.

[0210] In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 27, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 28, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 29, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 21, 26, and 28, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 22, 26, and 28, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 23, 26, and 28, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 24, 26, and 28, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 20, 26, and 30, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 25, 26, and 29, respectively. In some embodiments, the CD3-bound VH region includes VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 24, 26, and 29, respectively.

[0211] In some embodiments, the CD3-bound VH region is (i) array VH complementarity determination region 1 (CDR1) including GFTFX8X9YA (SEQ ID NO: 55), In the formula, VH CDR1 is such that X8 is D, A, or H, and X9 is D or N. (ii) VH CDR2 containing sequence ISWNSGSI (sequence number 26), (iii) array AKDSRGYGX 10 YX 11 X 12 A VH CDR3 containing GGAY (SEQ ID NO: 56), In the formula, X 10 is D or S, X 11 is R or S, and X 12 Includes VH CDR3, where L is or R.

[0212] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are located within the human VH framework.

[0213] 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%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity with any one of sequence numbers 31-48. In some embodiments, the CD3-binding VH region has at least 95% sequence identity with any one of sequence numbers 31-48.

[0214] 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%) sequence identity with SEQ ID NO: 31. 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%) sequence identity with SEQ ID NO: 32. 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%) sequence identity with SEQ ID NO: 33. 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%) sequence identity with SEQ ID NO: 34. 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%) sequence identity with SEQ ID NO: 35. 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%) sequence identity with SEQ ID NO: 36. 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%) sequence identity with SEQ ID NO: 37. 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%) sequence identity with SEQ ID NO: 38. In some embodiments, the CD3-binding VH region has at least 80% sequence identity with respect to sequence number 39 (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.).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%) sequence identity with SEQ ID NO: 40. 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%) sequence identity with SEQ ID NO: 41. 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%) sequence identity with SEQ ID NO: 42. 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%) sequence identity with SEQ ID NO: 43. 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%) sequence identity with SEQ ID NO: 44. 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%) sequence identity with SEQ ID NO: 45. 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%) sequence identity with SEQ ID NO: 46. 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%) sequence identity with SEQ ID NO: 47. In some embodiments, the CD3-binding VH region has at least 80% sequence identity with respect to sequence number 48 (e.g., 80%, 85%, 90%, 95%, at least 85%, at least 90%, at least 95%, etc.).

[0215] In some embodiments, the light chain variable region includes CDR1, CDR2, and CDR3 of sequence number 52. In some embodiments, the light chain variable region includes VL CDR1, VL CDR2, and VL CDR3, each containing the sequences of sequence numbers 49, 50, and 51, respectively. In some embodiments, the VL CDR1, VL CDR2, and VL CDR3 sequences are present in the human VH framework.

[0216] 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%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 85% (e.g., 85%, 90%, 95%, at least 90%, at least 95%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 90% (e.g., 90%, 95%, at least 95%) sequence identity with respect to sequence number 52. In some embodiments, the light chain variable region has at least 95% sequence identity with respect to sequence number 52.

[0217] In some embodiments, the mesothelin-binding protein is an anti-mesothelin antibody or a fragment thereof.

[0218] 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.

[0219] 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.

[0220] In some embodiments, the mesothelin-binding protein is an antibody fragment. In some embodiments, the antibody fragment is the immunoactive portion of an intact IgG molecule. In some embodiments, the antibody fragment is the immunoactive portion of an intact IgG1 molecule. In some embodiments, the antibody fragment is the immunoactive portion of an intact IgG2 molecule. In some embodiments, the antibody fragment is the immunoactive 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.

[0221] 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 with the native sequence Fc region.

[0222] In some embodiments, the variant Fc region includes heterodimerization modifications. In some embodiments, the heterodimerization modifications include knob-and-hole substitutions (for example, in the variant IgG1 Fc region, 1) Y407T in one strand and T366Y in the other strand; 2) Y407A in one strand and T366W in the other strand; 3) F405A in one strand and T394W in the other strand; 4) F405W in one strand and T394S in the other strand; 5) Y407T in one strand and T366Y in the other strand; 6) T366 7) Y and F405A and T394W and Y407T in the other chain; 8) T366W and F405W in one chain and T394S and Y407A in the other chain; or 9) T366W in one polypeptide of Fc and T366S in the other polypeptide, and L368A and Y407V). In some embodiments, heterodimerization modifications include substitutions that create new disulfide crosslinks (e.g., in the 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 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.

[0223] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region includes one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region includes substitutions that alter glycosylation. In some embodiments, the silenced Fc region includes effectorless mutations (e.g., N297A, N297G, DANA mutations (D265A+N297A), or DANG mutations (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region includes K322A and L234A / L235A mutations.

[0224] In some embodiments, the anti-mesothelin antibody or fragment thereof further comprises a heavy chain constant region sequence in the absence of the 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 containing the S228P mutation (SEQ ID NO: 62). 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 containing the F234A mutation, the L235A mutation, or both the F234A and L235A mutations. 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 containing the T366W mutation. In some embodiments, the CH3 domain includes variant human IgG4 CH3 domain sequences containing T366S, L368A mutations and Y407V mutations.

[0225] Table S9 provides sequences of human IgG1 and IgG4 Fc region sequences, as well as versions of these sequences incorporating additional mutations (variants) that may confer additional desired properties to the anti-mesothelin antibodies and fragments described herein. Table S10 provides exemplary human CD3 epsilon and CD3 delta sequences.

[0226] [Table 11]

[0227] [Table 12]

[0228] [Table 13]

[0229] Some embodiments of this disclosure relate to polynucleotides encoding single-domain antibodies that specifically bind to mesothelin as described herein.

[0230] Some embodiments of this disclosure relate to compositions comprising one or more polynucleotides encoding a mesothelin-binding protein described herein. In some embodiments, the mesothelin-binding protein is an anti-mesothelin antibody or a fragment thereof.

[0231] Some embodiments of this disclosure relate to recombinant expression vectors comprising single-domain antibodies that specifically bind to mesothelin as described herein, as well as host cells comprising the recombinant expression vectors.

[0232] Some embodiments of this disclosure relate to one or more recombinant expression vectors comprising one or more polynucleotides encoding the mesothelin-binding proteins described herein, and to host cells comprising one or more recombinant expression vectors.

[0233] Some embodiments of this disclosure relate to synthetic immune receptors comprising single-domain antibodies that specifically bind to mesothelin as described herein, as well as cells comprising synthetic immune receptors.

[0234] In some embodiments, a single-domain antibody is connected to a transmembrane domain via a hinge region, and further connected to a co-stimulatory domain such as a functional signaling domain derived from, for example, OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the synthetic immune receptor further includes a sequence encoding an intracellular signaling domain such as, for example, 4-1BB and / or CD3 zeta.

[0235] Some embodiments of the present disclosure relate to the production of a mesothelin-binding protein described herein (e.g., an anti-mesothelin antibody or a fragment thereof), comprising growing cells described herein under conditions that allow antibody expression, and isolating the mesothelin-binding protein (e.g., an anti-mesothelin antibody or a fragment thereof) from the cells.

[0236] Some embodiments of this 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 intended for use in diagnostic applications such as the detection or monitoring of diseases associated with mesothelin expression, such as proliferative disorders or cancer.

[0237] 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.

[0238] Non-limiting examples of pharmaceutically acceptable excipients include adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0239] The pharmaceutical compositions of this disclosure are prepared for storage by mixing proteins of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers, such as in the form of lyophilized formulations or aqueous solutions (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include, but are not limited to, buffers such as phosphates, citrates, 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; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin Examples include proteins such as methyl esters, gelatin, or immunoglobulins; 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 dextrin; 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 nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0240] In some embodiments, the pharmaceutical composition may include, for example, formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis. In such embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); and low molecular weight polypeptides. Examples of substances that may be used include, but are not limited to, salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, etc., triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (e.g., sucrose or sorbitol); isotonic enhancers (e.g., alkali metal halides, sodium chloride, or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.Methods for formulating molecules for therapeutic use and suitable materials are known in the pharmaceutical field, for example, as described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company. Pharmaceutical compositions of this disclosure include, but are not limited to, liquid compositions, freeze-dried compositions, and lyophilized compositions.

[0241] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage forms (e.g., single-dose dosage forms). The formulation depends on the chosen route of administration. The mesothelin-binding proteins (e.g., anti-mesothelin antibodies and their fragments) and antibody-drug conjugates described herein may be administered by intravenous injection or infusion, or subcutaneously. For administration by injection, the mesothelin-binding proteins (e.g., anti-mesothelin antibodies and their fragments) and antibody-drug conjugates described herein may be formulated in 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., anti-mesothelin antibodies and their fragments) 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, for example, in bacteriostatic water for injection (BWFI), physiological saline, phosphate-buffered saline (PBS), or in the same formulation in which the protein was present before lyophilization.

[0242] For example, an antibody formulation is disclosed in U.S. Patent No. 9,034,324. Similar formulations may be used with the anti-mesothelin antibodies and fragments thereof described herein. Subcutaneous antibody formulations are described, for example, in U.S. Patent Publication No. 20160355591 and U.S. Patent Publication No. 20160166689.

[0243] Some embodiments of this disclosure relate to methods for treating subjects in need of treatment for diseases associated with mesothelin expression, 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 as described herein.

[0244] In some embodiments, administration results in slowing or inhibiting tumor growth or metastasis of mesothelin-expressing cancers. The measurement of the reduction in tumor cell growth can be determined by several different methodologies known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison with control subjects, imaging techniques (e.g., CT or MRI) with or without the use of 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in tumor growth compared to a control antigen-binding agent, where a 100% reduction in tumor growth indicates 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 a reduction of in vivo tumor cell proliferation by approximately 50–100%, approximately 75–100%, or approximately 90–100% compared to a control antigen conjugate. In some embodiments, administration of at least one mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment results in a reduction of in vivo tumor cell proliferation by approximately 50–60%, approximately 60–70%, approximately 70–80%, approximately 80–90%, or approximately 90–100% compared to a control antigen conjugate.

[0245] The effective dose for treating a disease varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, and whether the administration and treatment of other drugs are prophylactic or therapeutic. While the patient is usually human, non-human mammals, such as companion animals like dogs, cats, and horses, and laboratory mammals like rabbits, mice, and rats, may also be treated. Treatment doses may be dose-set to optimize safety and efficacy.

[0246] Dosage levels can be easily determined by a clinician with standard skills and may be modified as needed, for example, to alter the subject's response to treatment. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the host being treated and the specific administration method.

[0247] In some embodiments, the mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment is administered to the patient parenterally. Parenteral administration refers to the administration of molecules via routes other than the gastrointestinal tract, and may include intraperitoneal, intramuscular, intravenous, intraarterial, intradermal, subcutaneous, intracerebral, intraventricular, and intrathecal administration.

[0248] In some embodiments, a mesothelin-binding protein, an antibody-drug conjugate, an anti-mesothelin antibody, or an antibody fragment is administered intravenously to the subject.

[0249] Parenteral or intravenous administration may be performed by injection (e.g., using a needle and syringe) or infusion (e.g., via a catheter and pump system). Administration according to this disclosure is assumed to be by intravenous injection or intravenous infusion. Typically, intravenous (IV) infusion is administered via a line, port, or catheter (a small flexible tube), such as a central venous access, or a central venous catheter (CVC), which is a catheter placed in a vena cava, or a peripheral venous catheter (PVC), which is a catheter placed in a peripheral vein. Generally, catheters or lines may be placed through veins in the neck (internal jugular vein), thoracic veins (subclavian or axillary vein), inguinal veins (femoral vein), or arm veins (also known as PICC lines or peripherally inserted central venous catheters). Central IV lines are advanced through veins and have catheters that drain into large central veins (usually the superior or inferior vena cava) or further into the right atrium of the heart. Peripheral venous (PIV) lines are used in peripheral veins (veins in the arms, hands, legs, and feet). A PIV is a central venous line without an external connector; instead, it has a small reservoir covered in silicone rubber and implanted under the skin. Medications are administered intermittently by inserting a small needle into the skin, piercing the silicone, and transferring the medication into the reservoir. The reservoir cover reseals spontaneously when the needle is withdrawn. The cover can withstand hundreds of needle sticks during its service life.

[0250] In some embodiments, the diseases associated with mesothelin expression are selected from proliferative disorders and cancers.

[0251] In some embodiments, the cancer is selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0252] The mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein may be administered in conjunction with other anticancer agents or therapeutic procedures (such as surgical resection of tumors). Any suitable anticancer agent may be administered in combination with the mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments disclosed herein. Examples of anticancer agents include, but are not limited to, chemotherapeutic agents, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalation antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvivators, bioresponse modifiers, antihormonal agents (e.g., antiandrogens), and anti-angiogenic agents. Other anticancer therapies include radiotherapy and other antibodies specific to target cancer cells.

[0253] In some embodiments, the mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment is administered before, during, or after surgery.

[0254] Some embodiments of this disclosure relate to mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein for use in the treatment of diseases associated with mesothelin expression. In some embodiments, the diseases associated with mesothelin expression are selected from proliferative disorders and cancers. In some embodiments, the cancers are selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0255] Some embodiments of this disclosure relate to the use of mesothelin-binding proteins, antibody-drug conjugates, anti-mesothelin antibodies, or antibody fragments described herein in the manufacture of pharmaceuticals for the treatment of diseases associated with mesothelin expression. In some embodiments, the diseases associated with mesothelin expression are selected from proliferative disorders and cancers. In some embodiments, the cancers are selected from mesothelioma, pancreatic cancer, gastric cancer, ovarian cancer, lung cancer, and triple-negative breast cancer.

[0256] Some embodiments of this disclosure relate to a method for confirming a cancer diagnosis 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 the binding of the single-domain antibody or mesothelin-binding protein to the sample. An increase in the binding of the single-domain antibody or mesothelin-binding protein to the sample compared to the binding of the single-domain antibody or mesothelin-binding protein to a control sample confirms the cancer diagnosis. 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 the binding of the detection antibody.

[0257] Some embodiments of this disclosure relate to a method for detecting cancer associated with mesothelin expression in a subject. The method comprises contacting a sample from the subject with a single-domain antibody or mesothelin-binding protein as described herein, and detecting the binding of the single-domain antibody or mesothelin-binding protein to the sample. An increase in the binding of the single-domain antibody or mesothelin-binding protein to the sample compared to a control sample indicates the detection of cancer in the subject. 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 the binding of the detection antibody.

[0258] Some embodiments of this disclosure relate to a kit comprising a mesothelin-binding protein, an antibody-drug conjugate, an anti-mesothelin antibody, or an antibody fragment or a pharmaceutical composition containing the same, as described herein, and instructions for use. The kit may further contain at least one additional drug, such as a chemotherapeutic agent. The kit typically includes a label indicating the intended use of the kit's contents. The term “label,” as used herein, includes any written or documentary material that is on or supplied with the kit, or otherwise attached to the kit.

[0259] In some embodiments, the kit is a diagnostic kit. In some embodiments, the kit is provided for detecting mesothelin in a biological sample, such as a blood sample or a tissue sample. For example, in a subject, a biopsy can be performed to confirm a cancer diagnosis 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. A kit for detecting polypeptides typically comprises a single-domain antibody or mesothelin-binding protein according to the disclosure. In some embodiments, the single-domain antibody or mesothelin-binding protein is labeled (e.g., fluorescently labeled, radioactively labeled, or enzymatically labeled).

[0260] In some embodiments, the kit may additionally include means for detecting the label (e.g., an enzyme substrate for enzyme labeling, a filter set for detecting a fluorescent label, a suitable secondary label such as a secondary antibody, or the like). The kit may further include buffers and other reagents routinely used for practicing a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0261] Non-exclusive exemplary embodiments: Some exemplary embodiments of this 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 contains a heavy chain variable (VH) region in which the full set (combination) of VH CDR1, 2, and 3 has at least 80% sequence identity with any one of the CDR1, 2, and 3 sequences from sequence numbers 11 to 19. 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 with any one of CDR1, 2, and 3 of SEQ ID NOs. 11-19. 3. A single-domain antibody according to Embodiment 1 or 2, wherein the full set (combination) of VH CDR1, 2, and 3 has at least 90% sequence identity with any one of CDR1, 2, and 3 of SEQ ID NOs. 11 to 19. 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 with any one of CDR1, 2, and 3 of SEQ ID NOs. 11 to 19. 5. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications compared to SEQ ID NO: 1 or SEQ ID NO: 9, (ii) VH CDR2 having a sequence with up to two amino acid modifications compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10, (iii) A single-domain antibody comprising a heavy chain variable (VH) region comprising a VH CDR3 having a sequence having up to two amino acid modifications compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. 6. The single-domain antibody according to Embodiment 5, wherein each amino acid modification, if any, is a conservative amino acid substitution. 7. A single-domain antibody according to Embodiment 5 or 6, wherein VH CDR1 comprises a sequence having up to one amino acid modification compared to SEQ ID NO: 1 or SEQ ID NO: 9. 8. A single-domain antibody according to any one of Embodiments 5 to 7, wherein VH CDR2 comprises a sequence having up to one amino acid modification compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10. 9. A single-domain antibody according to any one of Embodiments 5 to 8, wherein VH CDR3 contains a sequence having up to one amino acid modification compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. 10. A single-domain antibody according to any one of embodiments 7 to 9, wherein the maximum of one amino acid modification is an amino acid substitution. 11. A single-domain antibody according to any one of embodiments 7 to 9, wherein the maximum of one amino acid modification is a conservative amino acid substitution. 12. A single-domain antibody according to any one of embodiments 7 to 9, wherein the maximum of one amino acid modification is an amino acid deletion. 13. A single-domain antibody according to any one of embodiments 7 to 9, wherein the maximum of one amino acid modification is 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 and SEQ ID NO: 9. 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: 2, SEQ ID NO: 7, and SEQ ID NO: 10. 16. A single-domain antibody according to any one of Embodiments 1 to 15, wherein VH CDR3 comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8. 17. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) array VH complementarity determination region 1 (CDR1) including GGSISX1SYY (sequence number 53), In the formula, VH CDR1 is N or S, (ii) Array A VH CDR2 containing IYX2SGX3X4 (sequence number 68), In the formula, VH CDR2 is such that X2 is H or Y, X3 is N or S, and X4 is T or I. (iii) array A VH CDR3 containing X5X6QX7GVGATTTEEY (Sequence ID 54), A single-domain antibody comprising a heavy chain variable (VH) region containing VH CDR3, wherein X5 is T, V, or A, X6 is S or T, and X7 is D or N. 18. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) VH complementarity determination region 1 (CDR1) containing sequences selected from sequence number 1 and sequence number 9, (ii) A VH CDR2 containing sequences selected from sequence number 2, sequence number 7, and sequence number 10, (iii) A single-domain antibody comprising a heavy chain variable (VH) region comprising a VH CDR3 comprising a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8. 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, each containing the sequences of sequence numbers 1, 2, and 3, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 4, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 5, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 6, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 8, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 6, respectively; (g) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 9, 2, and 4, respectively; or (h) Single-domain antibodies containing heavy chain variable (VH) regions including VH CDR1, VH CDR2, and VH CDR3, respectively, which contain sequences of SEQ ID NOs. 1, 10, and 4. 20. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody includes a heavy chain variable (VH) region containing one of the CDR1, CDR2, and CDR3 sequences from sequence numbers 11 to 19. 21. A 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 the human VH framework. 22. 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 80% sequence identity with any one of sequence numbers 11 to 19. 23. A single-domain antibody according to any one of Embodiments 1 to 22, wherein the VH region has at least 85% sequence identity with any one of Sequence IDs 11 to 19. 24. A single-domain antibody according to any one of Embodiments 1 to 23, wherein the VH region has at least 90% sequence identity with any one of Sequence IDs 11 to 19. 25. A single-domain antibody according to any one of Embodiments 1 to 24, wherein the VH region has at least 95% sequence identity with any one of Sequence IDs 11 to 19. 26. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody includes a heavy chain variable (VH) region selected from SEQ ID NOs. 11-19. 27. A single-domain antibody according to 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 M's K D A single-domain antibody according to any one of embodiments 1 to 27, which binds to human MSLN. 29. A single-domain antibody according to 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 as described in any one of Embodiments 1 to 28. 31. The mesothelin-binding protein according to Embodiment 30, wherein the mesothelin-binding protein specifically binds to human MSLN. 32. Mesothelin-binding protein, approximately 10 -9 M ~ about 10 -6 M's K D A mesothelin-binding protein according to embodiment 30 or 31, which binds to human MSLN. 33. The mesothelin-binding protein according to any one of embodiments 30 to 32, wherein the mesothelin-binding protein binds more specifically to CD3. 34. The mesothelin-binding protein according to any one of embodiments 30 to 33, wherein the mesothelin-binding protein binds more specifically to human CD3. 35. The mesothelin-binding protein according to any one of embodiments 30 to 34, wherein the mesothelin-binding protein further specifically binds to human CD3 epsilon. 36. A mesothelin-binding protein according to 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: 69): K73 and S83; and CD3 delta (SEQ ID NO: 70): K82 and C93. 37. The mesothelin-binding protein according to Embodiment 36, wherein the epitope on CD3 includes a region of the CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. 38. The mesothelin-binding protein according to Embodiment 36 or 37, wherein the epitope on CD3 includes a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, and S83. 39. A mesothelin-binding protein according to any one of embodiments 36 to 38, wherein the epitope comprises a structural epitope having both CD3 delta and CD3 epsilon residues. 40. A mesothelin-binding protein according to any one of embodiments 36 to 39, wherein the three-dimensional epitope comprises residues CD3εK73 and S83; and CD3δK82 and C93, respectively. 41. The mesothelin-binding protein according to any one of Embodiments 30 to 40, wherein the mesothelin-binding protein is a monoclonal antibody. 42. The mesothelin-binding protein according to any one of embodiments 30 to 41, wherein the mesothelin-binding protein is an isolated monoclonal antibody. 43. An antibody-drug conjugate comprising a single-domain antibody as described in any one of Embodiments 1 to 28. 44. An anti-mesothelin antibody comprising a single-domain antibody as described in any one of Embodiments 1 to 28. 45. The anti-mesothelin antibody according to Embodiment 44, wherein the anti-mesothelin antibody binds to effector cells. 46. ​​The anti-mesothelin antibody according to 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 tumor-specific antigens other than MSLN. 48. An anti-mesothelin antibody according to any one of embodiments 44 to 47, wherein the anti-mesothelin antibody is bispecific. 49. An anti-mesothelin antibody according to any one of embodiments 44 to 48, wherein the anti-mesothelin antibody binds more specifically to CD3. 50. An anti-mesothelin antibody according to any one of embodiments 44 to 49, wherein the anti-mesothelin antibody binds more specifically to human CD3. 51. An anti-mesothelin antibody according to any one of embodiments 44 to 50, wherein the anti-mesothelin antibody further specifically binds to human CD3 epsilon. 52. An anti-mesothelin antibody according to any one of embodiments 44 to 50, wherein the anti-mesothelin antibody binds to epsilon on CD3 containing at least one residue selected from CD3 epsilon (SEQ ID NO: 69): K73 and S83; and CD3 delta (SEQ ID NO: 70): K82 and C93. 53. An anti-mesothelin antibody according to Embodiment 52, wherein the epitope on CD3 includes a region of the CD3 delta defined by K82, E83, S84, T85, V86, Q87, V88, H89, Y90, R91, M92, and C93. 54. An anti-mesothelin antibody according to Embodiment 52 or 53, wherein the epitope on CD3 includes a region of CD3 epsilon defined by K73, N74, 175, G76, S77, D78, E79, D80, H81, L82, S83. 55. An anti-mesothelin antibody according to any one of embodiments 52 to 54, wherein the epitope comprises a structural epitope having both CD3 delta and CD3 epsilon residues. 56. The anti-mesothelin antibody according to Embodiment 55, wherein the three-dimensional epitope comprises residues CD3εK73 and S83; and CD3δK82 and C93, respectively. 57. An anti-mesothelin antibody according to any one of embodiments 30 to 56, wherein the anti-mesothelin antibody further comprises a CD3-binding VH region. 58. An anti-mesothelin antibody according to any one of embodiments 30 to 57, wherein the anti-mesothelin antibody is an IgG4 antibody. 59. An anti-mesothelin antibody according to any one of embodiments 30 to 57, wherein the anti-mesothelin antibody is an IgG1 antibody. 60. An anti-mesothelin antibody according to any one of embodiments 30 to 58, further comprising a CD3-binding VH region paired with a light chain variable (LV) region. 61. The CD3-bound VH region, (i) VH complementarity determination region 1 (CDR1) containing a sequence having up to two amino acid modifications compared to any one of sequence numbers 20-25, (ii) VH CDR2 containing a sequence with up to two amino acid modifications compared to SEQ ID NO: 26, (iii) An anti-mesothelin antibody according to Embodiment 57 or 60, comprising a VH CDR3 having a sequence having up to two amino acid modifications compared to any one of SEQ ID NOs. 27 to 30. 62. The anti-mesothelin antibody according to Embodiment 61, wherein the CD3-bound VH CDR1 contains a sequence having up to one amino acid modification compared to any one of SEQ ID NOs. 20-25. 63. An anti-mesothelin antibody according to Embodiment 61 or 62, wherein the CD3-bound VH CDR2 contains a sequence having up to one amino acid modification compared to SEQ ID NO: 26. 64. An anti-mesothelin antibody according to any one of embodiments 61 to 63, wherein the CD3-bound VH CDR3 comprises a sequence having up to one amino acid modification compared to any one of sequence numbers 27 to 30. 65. An anti-mesothelin antibody according to any one of embodiments 62 to 64, wherein the maximum of one amino acid modification is an amino acid substitution. 66. An anti-mesothelin antibody according to any one of embodiments 62 to 65, wherein the maximum of one amino acid modification is a conservative amino acid substitution. 67. An anti-mesothelin antibody according to any one of embodiments 62 to 64, wherein the maximum number of amino acid modifications is an amino acid deletion. 68. An anti-mesothelin antibody according to any one of embodiments 62 to 64, wherein the maximum of one amino acid modification is amino acid addition. 69. An anti-mesothelin antibody according to any one of embodiments 62 to 68, wherein the CD3-conjugated VH CDR1 comprises a sequence selected from SEQ ID NOs. 20 to 25. 70. An anti-mesothelin antibody according to any one of embodiments 62 to 69, wherein the CD3-conjugated VH CDR2 comprises the sequence of SEQ ID NO: 26. 71. An anti-mesothelin antibody according to any one of embodiments 62 to 70, wherein the CD3-conjugated VH CDR3 comprises a sequence selected from SEQ ID NOs. 27 to 30. 72. An anti-mesothelin antibody according to Embodiment 57 or any one of 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 with respect to any one of the CDR1, 2, and 3 of SEQ ID NOs. 31-48. 73. An anti-mesothelin antibody according to Embodiment 57 or any one of 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 with respect to any one of the CDR1, 2, and 3 of SEQ ID NOs. 31-48. 74. An anti-mesothelin antibody according to Embodiment 57 or any one of 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 with respect to any one of the CDR1, 2, and 3 of SEQ ID NOs. 31-48. 75. An anti-mesothelin antibody according to Embodiment 57 or any one of 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 with respect to any one of the CDR1, 2, and 3 of SEQ ID NOs. 31-48. 76. The CD3-bound VH region, (i) array VH complementarity determination region 1 (CDR1) including GFTFX8X9YA (SEQ ID NO: 55), In the formula, VH CDR1 is such that X8 is D, A, or H, and X9 is D or N. (ii) VH CDR2 containing sequence ISWNSGSI (sequence number 26), (iii) array AKDSRGYGX 10 YX 11 X 12 A VH CDR3 containing GGAY (SEQ ID NO: 56), In the formula, X 10 is D or S, X 11 is R or S, and X 12 An anti-mesothelin antibody according to Embodiment 57 or 60, comprising VH CDR3, wherein is L or R. 77. An anti-mesothelin antibody according to Embodiment 57 or 60, wherein the CD3-binding VH region comprises one of the CDR1, CDR2, and CDR3 of SEQ ID NOs. 31 to 48. 78. An anti-mesothelin antibody according to any one of embodiments 57 to 77, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences in the CD3-binding VH region are present in the human VH framework. 79. An anti-mesothelin antibody according to any one of embodiments 57 to 78, wherein the CD3-binding VH region has at least 80% sequence identity with any one of sequence numbers 31 to 48. 80. An anti-mesothelin antibody according to any one of embodiments 57 to 79, wherein the CD3-binding VH region has at least 85% sequence identity with any one of sequence numbers 31 to 48. 81. An anti-mesothelin antibody according to any one of embodiments 57 to 80, wherein the CD3-binding VH region has at least 90% sequence identity with any one of sequence numbers 31 to 48. 82. An anti-mesothelin antibody according to any one of embodiments 57 to 81, wherein the CD3-binding VH region has at least 95% sequence identity with any one of sequence numbers 31 to 48. 83. The CD3-bound VH region, (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 27, respectively; (b) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 20, 26, and 28, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 29, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 21, 26, and 28, respectively; (e) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 22, 26, and 28, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 23, 26, and 28, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 24, 26, and 28, respectively; (h) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 30, respectively; (i) VH CDR1, VH CDR2, and VH CDR3 containing sequences of sequence numbers 25, 26, and 29, respectively; or (j) An anti-mesothelin antibody according to any one of embodiments 57 to 82, comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of SEQ ID NOs. 24, 26, and 29, respectively. 84. An anti-mesothelin antibody according to Embodiment 83, wherein the CD3-binding VH region comprises VH CDR1, VH CDR2, and VH CDR3, which include the sequences of SEQ ID NOs. 20, 26, and 27. 85. An anti-mesothelin antibody according to any one of embodiments 60 to 84, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 52. 86. An anti-mesothelin antibody according to any one of embodiments 60 to 85, wherein the light chain variable regions comprise VL CDR1, VL CDR2, and VL CDR3, each containing the sequences of SEQ ID NOs. 49, 50, and 51, respectively. 87. An anti-mesothelin antibody according to any one of embodiments 60 to 86, wherein the VL CDR1, VL CDR2, and VL CDR3 sequences are present in the human VH framework. 88. An anti-mesothelin antibody according to any one of embodiments 60 to 87, wherein the light chain variable region has at least 80% sequence identity with respect to SEQ ID NO: 52. 89. An anti-mesothelin antibody according to any one of embodiments 60 to 88, wherein the light chain variable region has at least 85% sequence identity with respect to SEQ ID NO: 52. 90. An anti-mesothelin antibody according to any one of embodiments 60 to 89, wherein the light chain variable region has at least 90% sequence identity with respect to SEQ ID NO: 52. 91. An anti-mesothelin antibody according to any one of embodiments 60 to 90, wherein the light chain variable region has at least 95% sequence identity with respect to SEQ ID NO: 52. 92. An anti-mesothelin antibody according to any one of embodiments 44 to 91, wherein the anti-mesothelin antibody further comprises an Fc region. 93. An anti-mesothelin antibody according to any one of embodiments 44 to 92, wherein the anti-mesothelin antibody further comprises a variant Fc region. 94. An anti-mesothelin antibody according to Embodiment 93, wherein the variant Fc region includes 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. An anti-mesothelin antibody according to any one of embodiments 44 to 95, wherein the anti-mesothelin antibody specifically binds to human MSLN. 97. Anti-mesothelin antibodies, approximately 10 -9 M ~ about 10 -6 M's K D An anti-mesothelin antibody according to any one of embodiments 44 to 96, which binds to human MSLN. 98. An anti-mesothelin antibody according to any one of Embodiments 44 to 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 according to Embodiment 99, wherein the antibody fragment specifically binds to human MSLN. 101. The antibody fragment according to Embodiment 99 or 100, wherein the antibody fragment is an isolated antibody fragment. 102. A polynucleotide encoding a single-domain antibody as described in any one of Embodiments 1 to 29. 103. A composition comprising one or more polynucleotides encoding a mesothelin-binding protein as described in any one of Embodiments 30 to 42. 104. A composition comprising one or more polynucleotides encoding an anti-mesothelin antibody as described in any one of Embodiments 44 to 98. 105. A recombinant expression vector comprising a polynucleotide or composition described in any one of Embodiments 102 to 104. 106. A host cell containing the recombinant expression vector described in Embodiment 105. 107. A synthetic immune receptor comprising a single-domain antibody as described in any one of Embodiments 1 to 28. 108. A cell comprising the synthetic immune receptor described in Embodiment 107. 109. A method for treating a subject in need of treatment for a disease associated with mesothelin expression, 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 for treating a subject in need of treatment for a disease associated with mesothelin expression, 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 in any one of embodiments 30 to 101. 111. The method according to Embodiment 110, wherein the disease associated with mesothelin expression is selected from proliferative disorders and cancer. 112. The method according to 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 the treatment of a disease associated with mesothelin expression. 114. A mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment for use according to Embodiment 113, wherein the disease associated with mesothelin expression is selected from proliferative disorders and cancer. 115. A 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 pharmaceutical product for the treatment of a disease associated with mesothelin expression. 117. The use according to Embodiment 116, wherein the disease associated with mesothelin expression is selected from proliferative disorders 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 described in any one of Embodiments 30 to 101, and a pharmaceutically acceptable excipient. [Examples]

[0262] Examples are provided below to help the disclosure be fully understood. These examples are for illustrative purposes only and should not be construed as limiting the disclosure in any way.

[0263] Example 1. Binding to MSLN-expressing cell lines and off-target cell lines The cell binding of the exemplary anti-MSLN single-domain antibody described herein was evaluated using CHO cells expressing human MSLN (CHO_huMSLN) and CHO cells not expressing MSLN protein (CHO-OFFtgt). Pellet-formed CHO cells were placed in a flow buffer (1× phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% NaN3) in a quantity of 1.25 × 10⁶ cells. 6 The cells were resuspended at a concentration of 1 / mL. The antibody-containing supernatant was diluted 1:5 in flow buffer, and 40 μL of cells plus 10 μL of the diluted antibody supernatant were incubated at 4°C for 30 minutes. The cells were washed in flow buffer and resuspended in 50 μL of phycoerythrin (PE) conjugate secondary antibody (Southern Biotech #2042-09) diluted to 1.25 μg / mL in flow buffer, and incubated at 4°C for 20 minutes. After two washing steps, the cells were resuspended in flow buffer and analyzed using the Guava easyCyte 8HT system. Table E1 summarizes the target binding activity of the anti-MSLN single-domain antibody as a multiple of the background MFI signal.

[0264] [Table 14]

[0265] Example 2. Binding to MSLN-expressing cell lines and off-target cell lines Cell-binding dose curves for the exemplary anti-MSLN single-domain antibodies described herein were performed using human MSLN-expressing CHO cells (Figure 1A), HeLa cells (Figure 1B), and CHO-OFFtgt cells (Figure 1C). Single-domain antibodies were tested using an 8-point dose curve, starting at a 150 nM concentration followed by 3-fold serial dilutions. All washing and dilution of cells, antibodies, and reagents was performed using flow buffer (1× phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% NaN3). Cells were pelleted and 1×10⁶ cells were placed in flow buffer. 6 The cells were resuspended at individual cells / mL. Next, 50 μL of cells were combined with 50 μL of test antibody and incubated at 4°C for 30 minutes, followed by two washes with flow buffer. The cells were then resuspended in 50 μL of PE conjugate secondary antibody (Southern Biotech #2042-09) diluted to 1.25 μg / mL and incubated at 4°C for 20 minutes. After two washes, the cells were resuspended in flow buffer and analyzed using the BD Celesta system. The mean PE fluorescence intensity was plotted as a multiple of the background (cells incubated with secondary detection antibody only).

[0266] Example 3. Cell-bound EC50 values ​​in MSLN-expressing cell lines To determine the cell-bound EC50 values ​​for the exemplary anti-MSLN single-domain antibodies of this disclosure, cell-bound dose curves were performed in HeLa cells and CHO cells expressing human MSLN. The single-domain antibodies were tested on an 8-point dose curve, starting at a 150 nM dose followed by 3-fold serial dilutions, as described in Example 2. The transformed data were plotted as xy graphs using a nonlinear regression curve fit (available in GraphPad Prism 8.4.3) to obtain EC50 (nM), which is summarized in Table E2.

[0267] [Table 15]

[0268] Example 4. Binding affinity and epitope binning Table E3 summarizes affinity and epitope bin information for the exemplary anti-MSLN single-domain antibodies of this disclosure. The affinity of each single-domain antibody to recombinant human MSLN (R&D Systems, #3265-MS) was measured by biolayer interferometry (BLI) using Octet HTX. The test antibody was immobilized at 5 μg / mL using an AHC (Anti-hIgG Fc Capture) sensor. After 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 seconds and 240 seconds, respectively. Data were analyzed using Octet Data Analysis v11.0 HT (ForteBio) with a standard 1:1 binding model. Epitope bins for single-domain antibodies were determined by in-tandem competitive BLI binding experiments using Octet HTX. The antigen (5 μg / mL) was loaded into the sensor and the Ni-NTA sensor, and then baseline measured in kinetic buffer. Next, the antigen-coated center was immersed in antibody 1 at a saturated concentration. A second baseline measurement was then performed for 300 seconds. The antigen-antibody 1 complex was then immersed in antibody 2 solution for 180 seconds and then dissociated.

[0269] [Table 16]

[0270] Example 5. CAR-T mediated T cell activation by human tumor cell lines CAR-T cell activity was measured by transfecting Jurkat T lymphocytes with anti-MSLN CAR and a 6×NFAT 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 structure containing the anti-MSLN extracellular binding domain, which includes the antibody sequence of this disclosure. Transfected Jurkat cells were co-cultured for 24 hours in RPMI-1640 (Fisher Scientific, Waltham, Massachusetts, USA) with 10% FBS (ThermoFisher) in a humidified, 37°C, 8% CO2 incubator with MSLN+CHO cells, HeLa, or MSLN-negative K562 cells stably transfected to express human MSLN. Luciferase activity was measured using the Promega Nano-Glo luciferase assay system with the manufacturer's protocol (catalog #N1110) on a SpectraMax i3x multimode microplate reader (Molecular Devices), and the data were normalized to co-cultures containing CAR-transfected Jurkat and MSLN-negative K562 cell lines. Statistical significance was determined using an unpaired two-sided t-test. The results are shown in Figure 2B.

[0271] All documents or parts of documents cited herein, including but not limited to patents, patent applications, papers, books, and academic articles, are expressly incorporated herein by reference. The embodiments described herein may be combined with one or more other embodiments of the herein unless the context expressly indicates otherwise.

[0272] The subject matter disclosed is not intended to be limited in scope by the specific embodiments described herein, but rather is intended as a non-limiting illustration of the individual aspects of this disclosure. Functionally equivalent methods and components are within the scope of this disclosure. In fact, in addition to those shown and described herein, various modifications of the disclosed subject matter will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be within the scope of the disclosed subject matter.

[0273] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for illustrative purposes only and are not intended to be exhaustive or restrictive in any way. Many variations and modifications will be apparent to those skilled in the art without departing from the scope and nature of the embodiments described. The terminology used herein has been chosen to best describe the principles of the embodiments, their practical applications, or technical improvements to the art found in the market, 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. 11 to 19.

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 or SEQ ID NO: 9, (ii) VH CDR2 having a sequence with up to two amino acid modifications compared to SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 10, (iii) A single-domain antibody comprising a heavy chain variable (VH) region comprising a VH CDR3 having up to two amino acid modifications compared to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

8.

3. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody is (i) Array GGSISX １ VH complementarity determination region 1 (CDR1) including SYY (Sequence ID 53), In the formula, X １ VH CDR1 is N or S, (ii) Array IYX ２ SGX ３ X ４ VH CDR2 containing (Sequence ID 68), In the formula, X ２ is H or Y, X ３ is N or S, and X ４ VH CDR2 is T or I, (iii) array X ５ X ６ QX ７ VH CDR3 containing GVGATTTEEY (SEQ ID NO: 54), In the formula, X ５ is T, V, or A, X ６ is S or T, and X ７ A single-domain antibody comprising a heavy chain variable (VH) region containing VH CDR3, wherein the region is D or N.

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) containing sequences selected from sequence number 1 and sequence number 9, (ii) VH CDR2 containing an array selected from sequence number 2, sequence number 7, and sequence number 10, (iii) A single-domain antibody comprising a heavy chain variable (VH) region comprising a VH CDR3 containing a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:

8.

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, 2, and 3, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 4, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 5, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 2, and 6, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 1, 7, and 8, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 6, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 9, 2, and 4, respectively; or (h) A single-domain antibody comprising heavy chain variable (VH) regions containing VH CDR1, VH CDR2, and VH CDR3, respectively, containing the sequences of SEQ ID NOs: 1, 10, and 4.

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. 11 to 19.

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. 11 to 19.

9. A single-domain antibody that specifically binds to mesothelin (MSLN), wherein the single-domain antibody includes a heavy chain variable (VH) region selected from SEQ ID NOs: 11 to 19.

10. The single-domain antibody according to any one of claims 1 to 9, 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 10, which binds to human MSLN.

12. The single-domain antibody according to any one of claims 1 to 11, 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 11.

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 or 14, wherein the mesothelin-binding protein further specifically binds to CD3.

16. The mesothelin-binding protein according to any one of claims 13 to 15, wherein the mesothelin-binding protein further specifically binds to human CD3.

17. The mesothelin-binding protein according to any one of claims 13 to 16, 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 11.

19. An anti-mesothelin antibody comprising a single-domain antibody according to any one of claims 1 to 11.

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 or 20, wherein the anti-mesothelin antibody further specifically binds to tumor-specific antigens other than MSLN.

22. The anti-mesothelin antibody according to any one of claims 19 to 21, wherein the anti-mesothelin antibody is bispecific.

23. The anti-mesothelin antibody according to any one of claims 19 to 22, wherein the anti-mesothelin antibody further specifically binds to CD3.

24. The anti-mesothelin antibody according to any one of claims 19 to 23, wherein the anti-mesothelin antibody further specifically binds to human CD3.

25. The anti-mesothelin antibody according to any one of claims 19 to 24, wherein the anti-mesothelin antibody further comprises a CD3-binding VH region.

26. The anti-mesothelin antibody according to any one of claims 19 to 25, wherein the anti-mesothelin antibody is of the IgG1 or IgG4 subtype.

27. The anti-mesothelin antibody according to any one of claims 19 to 26, further comprising a CD3-binding VH region that pairs with a light chain variable (LV) 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 20 to 25, (ii) VH CDR2 containing a sequence having up to two amino acid modifications compared to SEQ ID NO: 26, (iii) The anti-mesothelin antibody according to claim 25 or 27, comprising a VH CDR3 having a sequence having up to two amino acid modifications compared to SEQ ID NOs. 27-30.

29. The anti-mesothelin antibody according to claim 28, wherein the CD3-conjugated VH CDR1 comprises a sequence selected from SEQ ID NOs. 20 to 25.

30. The anti-mesothelin antibody according to claim 28 or 29, wherein the CD3-bound VH CDR2 comprises the sequence of SEQ ID NO:

26.

31. The anti-mesothelin antibody according to any one of claims 28 to 30, wherein the CD3-conjugated VH CDR3 comprises a sequence selected from sequence numbers 27 to 30.

32. The anti-mesothelin antibody according to claim 25 or any one of claims 27 to 31, 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: 31 to 48.

33. The CD3-bound VH region is (i) Array GFTFX ８ X ９ VH complementarity determination region 1 (CDR1) including YA (SEQ ID NO: 55), In the formula, X ８ is D, A, or H, and X ９ VH CDR1 is D or N, (ii) VH CDR2 containing sequence ISWNSGSI (Sequence ID 26), (iii) array AKDSRGYGX １０ YX １１ X １２ VH CDR3 containing GGAY (SEQ ID NO: 56), In the formula, X １０ is D or S, X １１ is R or S, and X １２ An anti-mesothelin antibody according to claim 25 or 27, comprising VH CDR3, wherein is L or R.

34. The anti-mesothelin antibody according to claim 25 or 27, wherein the CD3-binding VH region comprises one of the CDR1, CDR2, and CDR3 of SEQ ID NOs: 31 to 48.

35. The anti-mesothelin antibody according to any one of claims 25 to 34, 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 any one of claims 25 to 35, wherein the CD3-binding VH region has at least 95% sequence identity with any one of sequence numbers 31 to 48.

37. The CD3-bound VH region is (a) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 27, respectively; (b) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 28, respectively; (c) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 29, respectively; (d) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 21, 26, and 28, respectively; (e) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 22, 26, and 28, respectively; (f) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 23, 26, and 28, respectively; (g) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 24, 26, and 28, respectively; (h) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 20, 26, and 30, respectively; (i) VH CDR1, VH CDR2, and VH CDR3, each containing the sequences of sequence numbers 25, 26, and 29, respectively; or (j) An anti-mesothelin antibody according to any one of claims 25 to 36, comprising VH CDR1, VH CDR2, and VH CDR3, each containing the sequence of SEQ ID NOs. 24, 26, and 29, respectively.

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. 20, 26, and 27.

39. The anti-mesothelin antibody according to any one of claims 27 to 38, wherein the light chain variable region comprises CDR1, CDR2, and CDR3 of SEQ ID NO:

52.

40. The anti-mesothelin antibody according to any one of claims 27 to 39, wherein the light chain variable region comprises VL CDR1, VL CDR2, and VL CDR3, each containing the sequence of SEQ ID NOs: 49, 50, and 51, respectively.

41. The anti-mesothelin antibody according to any one of claims 27 to 40, wherein the VLCDR1, VLCDR2, and VLCDR3 sequences are present in the human VH framework.

42. The anti-mesothelin antibody according to any one of claims 27 to 41, wherein the light chain variable region has at least 95% sequence identity with respect to SEQ ID NO:

52.

43. The anti-mesothelin antibody according to any one of claims 19 to 42, wherein the anti-mesothelin antibody specifically binds to human MSLN.

44. The anti-mesothelin antibody according to any one of claims 19 to 43, 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 an anti-mesothelin antibody according to any one of claims 19 to 44.

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 mesothelin-binding protein, antibody-drug conjugate, anti-mesothelin antibody, or antibody fragment according to any one of claims 13 to 46, and a pharmaceutically acceptable excipient.

48. A polynucleotide encoding a single-domain antibody according to any one of claims 1 to 12.

49. A composition comprising one or more polynucleotides encoding a mesothelin-binding protein, an anti-mesothelin antibody, or an antibody fragment according to any one of claims 13 to 17 or 19 to 46.

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

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

52. A method for treating a subject in need of treatment for a disease associated with mesothelin expression, 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 claims 13 to 46.

53. The method 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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