Mesothelin constructs and their uses

Abstract

This application relates to anti-mesothelin constructs (such as anti-mesothelin antibodies, cytokine fusion proteins comprising said anti-mesothelin constructs), methods for preparing said anti-mesothelin constructs, and methods of using said constructs (such as methods of treating a disease or condition). This application also relates to a combination therapy for treating cancer, which comprises administering an anti-mesothelin agent and a cytokine (such as IL-21 or IL-15). The combination therapy may further comprise administering an anti-Her2 agent.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 686,481, filed Jun. 18, 2018, and U.S. Provisional Patent Application No. 62 / 809,496, filed Feb. 22, 2019. For all purposes, the entire contents of these applications are hereby incorporated herein by reference. FIELD OF THE INVENTION

[0003] This application relates to mesothelin constructs (such as mesothelin antibodies, cytokine fusion proteins comprising mesothelin constructs) and methods of using such constructs (such as methods of treating diseases). This application also relates to combination therapies for treating diseases (such as cancer). BACKGROUND OF THE INVENTION

[0004] Mesothelin is a glycoprotein present on the cell surface of the mesothelial lining of the peritoneal, pleural, and pericardial body cavities. It was initially purified from the human pancreatic cancer cell line HPC-Y5 and shown to have megakaryocyte potentiating ability, and thus was named megakaryocyte potentiating factor (MPF) (Yamaguchi et al., (1994) J. Biol. Chem. 269:805-808). The mesothelin cDNA could be cloned using a library prepared from the HPC-Y5 cell line (Kojima et al., (1995) J. Biol. Chem. 270:21984-21990). The mesothelin cDNA could also be cloned using the monoclonal antibody K1 that recognizes mesothelioma (Chang and Pastan (1996) Proc. Natl. Acad. Sci. USA 93:136-40). Structurally, mesothelin is expressed on the cell surface as a 60 kDa precursor polypeptide that is proteolytically processed into a 31 kDa shed component (corresponding to MPF) and a 40 kDa membrane-bound component (Hassan et al., (2004) Clin. Cancer Res. 10:3937-3942). In addition to being expressed on normal mesothelial cells, mesothelin is also overexpressed in several classes of human tumors, including all mesotheliomas, ovarian cancers, and pancreatic cancers, as well as some gastric, lung, and endometrial cancers. For example, mesothelin is expressed on approximately 70% of all ovarian cancers, approximately 82% of papillary serous adenocarcinomas, approximately 83% of all pancreatic cancers, and approximately 86% of all ductal pancreatic cancers.

[0005] Mesothelin specifically interacts with CA125 (also known as MUC16), a mucin-like glycoprotein present on the surface of tumor cells that has previously been identified as an ovarian cancer antigen. In addition, the binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion, and CA125 and mesothelin are co-expressed in advanced ovarian adenocarcinoma (Rump, A et al., (2004) J. Biol. Chem. 279:9190-9198). The expression of mesothelin in the peritoneal lining is associated with the preferred site of metastasis formation in ovarian cancer, and the mesothelin-CA125 binding is thought to facilitate peritoneal metastasis of ovarian tumors (Gubbels, J.A et al. (2006) Mol. Cancer 5:50).

[0006] In view of the foregoing, other agents for modulating mesothelin activity are of interest. Summary of the Invention

[0007] The present application provides an isolated anti-mesothelin (anti-MSLN) construct comprising an antibody portion, the antibody portion comprising an anti-MSLN heavy chain variable region (VH), the anti-MSLN heavy chain variable region comprising: a) heavy chain complementarity determining region (HCCDR) 1, the heavy chain complementarity determining region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, and SEQ ID NO:120;b) HC-CDR2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118 and SEQ ID NO:121; and c) HC-CDR3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:119 and SEQ ID NO:122.;

[0008] The present application also provides an isolated anti-mesothelin (anti-MSLN) construct comprising an antibody portion, the antibody portion comprising an anti-MSLN heavy chain variable region (VH), the anti-MSLN heavy chain variable region comprising: 1) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:14, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 2) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:15, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:16, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:17, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 3) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:20, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 4) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:23, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 5) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:25, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:26, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 6) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:27, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:29, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 7) an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:30, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:32, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions;8) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:33, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:35, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 9) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:36, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:38, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 10) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:39, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:40, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:41, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 11) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:42, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:44, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 12) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:45, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:46, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:47, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 13) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:48, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:49, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:50, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 14) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:51, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:52, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:53, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 15) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:54, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:55, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:56, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions;16) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:57, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:58, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:59, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 17) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:60, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:61, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:62, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 18) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:63, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:64, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:65, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 19) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:75, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:76, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:77, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 20) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:78, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:79, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:80, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 21) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:81, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:82, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 22) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:84, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:85, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:86, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 23) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:87, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:88, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:89, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions;24) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:90, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:91, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:92, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 25) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:93, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:94, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:95, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 26) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:96, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:97, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:98, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 27) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:99, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:100, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:101, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 28) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:102, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:103, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:104, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 29) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:105, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:106, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:107, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 30) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:108, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:109, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:110, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions; 31) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:111, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:112, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:113, or a variant thereof comprising at most about 5 (such as 4, 3, 2, 1) amino acid substitutions;32) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:114, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:115, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:116, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; 33) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:117, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:118, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:119, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions; or 34) An HC-CDR1 comprising the amino acid sequence of SEQ ID NO:120, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:121, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:122, or a variant thereof comprising up to about 5 (such as 4, 3, 2, 1) amino acid substitutions.;

[0009] In some embodiments of any of the constructs described above, HC-CDR1, HC-CDR2, and HC-CDR3 respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 of any one of anti-MSLN-1, anti-MSLN-2, anti-MSLN-3, anti-MSLN-4, anti-MSLN-5, anti-MSLN-6, anti-MSLN-7, anti-MSLN-8, anti-MSLN-9, anti-MSLN-10, anti-MSLN-11, anti-MSLN-12, anti-MSLN-13, anti-MSLN-14, anti-MSLN-15, anti-MSLN-16, anti-MSLN-17, anti-MSLN-18, anti-MSLN-19, anti-MSLN-20, anti-MSLN-21, anti-MSLN-22, anti-MSLN-23, anti-MSLN-24, anti-MSLN-25, anti-MSLN-26, anti-MSLN-27, anti-MSLN-28, anti-MSLN-29, anti-MSLN-30, anti-MSLN-31, anti-MSLN-32, anti-MSLN-33, and anti-MSLN-34, as shown in Table 10.

[0010] The present application provides an isolated anti-mesothelin (anti-MSLN) construct comprising an antibody portion, the antibody portion comprising an anti-MSLN heavy chain variable region (VH), the anti-MSLN heavy chain variable region comprising: HC-CDR1, HC-CDR2, and HC-CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the VH chain region, and the VH chain region has the sequence described in any one of SEQ ID NOs: 123-156 and SEQ ID NOs: 285-301.

[0011] In some embodiments of any of the constructs described above, the antibody portion is a single domain (sdAb) portion. In some embodiments, the sdAb portion that binds to albumin is camelid, chimeric, human, partially humanized, or fully humanized.

[0012] In some embodiments of any of the constructs described above, the antibody portion comprises the amino acid sequence of any of SEQ ID NO: 123-156 and SEQ ID NO: 285-301, or a variant thereof having at least about 80% sequence identity with any of SEQ ID NO: 123-156 and SEQ ID NO: 285-301.

[0013] In some embodiments of any of the constructs described above, the antibody portion comprises a VHH domain or a variant thereof, the VHH domain comprising the amino acid sequence of any of SEQ ID NO: 123-156 and SEQ ID NO: 285-301, the variant comprising up to about 3 (such as 3, 2, 1) amino acid substitutions in the VHH domain.

[0014] In some embodiments of any of the constructs described above, the antibody portion has reduced fucosylation.

[0015] In some embodiments of any of the constructs described above, the construct is a fusion protein further comprising a half-life extending domain. In some embodiments, the half-life extending domain comprises an Fc domain or an albumin binding domain.

[0016] In some embodiments of any of the constructs described above, the construct is a fusion protein further comprising a cytokine. In some embodiments, the cytokine is IL-21 or IL-15. In some embodiments, the half-life extending domain comprises an Fc domain or an albumin binding domain. In some embodiments, the construct comprises a linker between the anti-MSLN antibody portion and the cytokine. In some embodiments, the linker comprises the amino acid sequence of any of SEQ ID NO: 66-74, SEQ ID NO: 267-282, and SEQ ID NO: 307-324. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable.

[0017] In some embodiments of any of the constructs described above, the construct is a fusion protein that further comprises a) a half-life extension domain; and b) a cytokine. In some embodiments, the cytokine is IL-21 or IL-15. In some embodiments, the construct comprises a linker between the anti-MSLN antibody portion and the cytokine. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 66-74, 267-282, and 307-324. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable.

[0018] In some embodiments, the half-life extension domain is fused to the N-terminus of the anti-MSLN antibody portion. In some embodiments, the half-life extension domain is fused to the C-terminus of the anti-MSLN antibody portion. In some embodiments, the cytokine is fused to the N-terminus of the anti-MSLN antibody portion or the half-life extension domain. In some embodiments, the cytokine is fused to the C-terminus of the anti-MSLN antibody portion or the half-life extension domain.

[0019] This application also provides a polynucleotide encoding any of the anti-MSLN constructs described above.

[0020] This application also provides a nucleic acid construct comprising any of the polynucleotides described herein, optionally further comprising a promoter operably linked to the polynucleotide.

[0021] This application also provides a vector comprising any of the nucleic acid constructs described above.

[0022] This application also provides a host cell comprising any of the polynucleotides, nucleic acid constructs, or vectors described above.

[0023] This application also provides a culture medium comprising any of the anti-MSLN constructs or host cells described herein.

[0024] This application also provides a kit comprising: a) any of the anti-MSLN constructs, polynucleotides, nucleic acid constructs, vectors, host cells, or culture media described above; and b) instructions.

[0025] This application also provides a pharmaceutical composition comprising any of the anti-MSLN constructs described above and a pharmaceutically acceptable carrier.

[0026] This application also provides a method of treating a disease or condition in an individual, comprising administering to the individual any of the anti-MSLN constructs described above.

[0027] The present application also provides a method of treating a disease or condition in an individual, comprising administering an anti-MSLN construct, the anti-MSLN construct comprising an anti-MSLN antibody portion that specifically binds to mesothelin, wherein the anti-MSLN antibody portion has reduced fucosylation.

[0028] In some embodiments of any of the methods described above, the disease or condition is cancer. In some embodiments, the cancer is a mesothelin-positive cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is selected from the group consisting of: gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, and breast cancer. In some embodiments, the cancer is gastric cancer or lung cancer.

[0029] In some embodiments of any of the methods described above, the method further comprises administering a second agent to the individual. In some embodiments, the second agent comprises a cytokine. In some embodiments, the cytokine is IL-21 or IL-15. In some embodiments, IL-21 or IL-15 is in the form of a fusion protein comprising a half-life extension domain. In some embodiments, IL-21 comprises an IL-21 variant lacking 1-11 amino acids at the C-terminus of SEQ ID NO:1.

[0030] The present application also provides a method of treating mesothelin-positive cancer in an individual, comprising administering to the individual a) an anti-mesothelin agent; b) an anti-Her2 agent; and c) IL-21. In some embodiments, IL-21 or IL-15 is in the form of a fusion protein comprising a half-life extension domain. In some embodiments, IL-21 comprises an IL-21 variant lacking 1-11 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the anti-Her2 agent is Herceptin. In some embodiments, the anti-mesothelin agent comprises an anti-mesothelin antibody portion comprising an anti-MSLN heavy chain variable region (VH), the anti-MSLN heavy chain variable region comprising: HC-CDR1, HC-CDR2, and HC-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3 within the VH chain region, respectively, the VH chain region having the sequence described in any of SEQ ID NO:123-156 and SEQ ID NO:285-301.

[0031] In some embodiments of any of the methods described above, the individual is human. Brief Description of the Drawings

[0032] Figure 1 Depicts exemplary IL-21 fusion proteins provided herein.

[0033] Figure 2 Depict the assembly of an exemplary IL-21 fusion protein expression vector, where the albumin-binding molecule is an anti-HSA antibody.

[0034] Figure 3 Depict the assembly of an exemplary IL-21 fusion protein expression vector, where the albumin-binding molecule is an ABD that binds to HSA.

[0035] Figures 4A - 4B Depict the binding of an anti-MSLN antibody to human or simian mesothelin (MSLN).

[0036] Figures 5A - 5D Depict the dose-dependent binding of an anti-MSLN antibody-IgG1 Fc fusion protein to the cell surface of cancer cell lines NCI-H226, OVCAR3, NCI-N87, and AsPC-1.

[0037] Figure 6A Depict the number of remaining tumor cells after treating NK cells alone or in combination with anti-mesothelin antibodies MORAb-009, R2G12, R3C7, or R3D5. Figure 6B (NCI-H226), Figure 6C (OVCAR3), Figure 6D (NCI-N87), and Figure 6E (AsPC-1) Depict the NK cell-mediated antibody-dependent cytotoxicity dose-response curves of anti-MSLN antibodies R2G12 and R3C7 against four human cancer cell lines.

[0038] Figure 7 Depict the number of remaining tumor cells after treating NK cells alone or in combination with anti-mesothelin antibodies P303, P303F, or MORAb-009.

[0039] Figures 8A - 8C Depict the number of remaining cells of N87 cells ( Figure 8A ), H226 cells ( Figure 8B ), and CT26 / MSLN cells ( Figure 8C ) after treating NK cells in combination with anti-mesothelin antibodies P303, P303F, P197, and P197F for 48 hours.

[0040] Figure 9A Depict the change in tumor volume in an animal model of SCID mice with N87 tumors after treatment with anti-mesothelin antibodies MORAb, P303F, P303, P197F, or P197. Figure 9B Depict the change in tumor volume in an animal model of NSG mice with N87 tumors after treatment with anti-mesothelin antibody P197. Figure 9CDepict the change in tumor volume in an animal model of BALB / c mice with CT26 / MSLN tumors after treatment with the anti-mesothelin antibody P303F.

[0041] Figure 10A Depict the binding of the anti-HSA antibody AWT-367 (i.e., P367) and its humanized anti-AWT-P494 (i.e., P494) to human, monkey, or mouse albumin. Figure 10B Depict the K of the binding of AWT-P367 or AWT-494 to human, monkey, or mouse albumin D 。

[0042] Figure 11 Depict the remaining cell number of N87 cells after treating NK cells alone or in combination with the investigational drug as shown in the figure.

[0043] Figure 12 Depict the remaining cell number of N87 cells after treating NK cells alone or in combination with a) Herceptin alone, b) P303F alone, c) Herceptin and P303F, or d) Herceptin, P303F, and the IL-21-anti-HSA fusion protein P394.

[0044] Figure 13 Depict the remaining cell number of N87 cells after treating NK cells in combination with a) P303F, b) P303F and recombinant human IL-21 (i.e., rhIL-21), c) P303F and P480, or d) P303F and recombinant human IL-15 (i.e., rhIL-15).

[0045] Figure 14 Depict the remaining cell number of N87 cells after treating NK cells in combination with a) P303F, b) P303F and recombinant human IL-21 (i.e., rhIL-21), c) P431 / P435, or d) P545 / P435.

[0046] Figure 15 Depict the remaining cell number of N87 cells (upper figure) and the IC50 of the three drugs (lower figure) after treating NK cells in combination with a) P480, b) P597, or c) rIL-15.

[0047] Figure 16Depicts the remaining cell numbers of H226 cells after treatment of NK cells in combination with a) anti-mesothelin antibodies P129 (i.e., R2G12) and P126 (i.e., human IL-21-R2G12-IgG1 fusion), b) P129 and IL-21, c) P129 and P107 (human IL-21-IgG1 fusion), d) P129 and P325 (human IL-21-R2D2 fusion), or e) P129 and P286 / 288 (human IL-21-R3C7-IgG1-R2G12).

[0048] Figure 17 Depicts the remaining cell numbers of N87 cells after treatment of NK cells in combination with a) P197 and P390; or b) P197 and P394.

[0049] Figure 18 Depicts the change in tumor volume in an animal model of NSG mice bearing N87 tumors after treatment with a) 25 μg of P394, b) 100 μg of P303F, c) 100 μg of P303F and 25 μg of P394, or d) 100 μg of P303F and 5 μg of P394.

[0050] Figure 19 Depicts the change in tumor volume in an animal model of SCID mice bearing N87 tumors after treatment with 100 μg of P303F alone or in combination with a) 25 μg of P390, b) 5 μg of P390, or c) 2.5 μg of rmIL-21. Detailed Description

[0051] The present invention provides novel fusion proteins comprising IL-21 or variants thereof. The fusion proteins provided herein comprise IL-21 or variants thereof, an albumin-binding molecule, and a binding molecule that binds to an antigen (e.g., a cancer antigen), wherein IL-21 or variants thereof are linked to the albumin-binding molecule via a first linker, and wherein the albumin-binding molecule is linked to the binding molecule via a second linker. Figure 1 Illustrates exemplary fusion proteins provided herein.

[0052] The fusion proteins disclosed herein offer numerous advantages. For example, in some embodiments, the binding molecule that binds to a cancer antigen (e.g., a solid tumor cancer antigen such as MSLN) incorporated into the fusion proteins of the present invention is capable of locally delivering IL-21 near the cancer, resulting in lower off-target toxicity and increased efficacy.

[0053] The fusion proteins of the present invention offer certain advantages due to their tertiary structure and overall configuration design. For example, in some embodiments, by placing IL-21 with an MMP-sensitive linker after a binding molecule targeting a cancer antigen and an ABD (or anti-HSA), the interaction between IL-21 and IL-21R is temporarily blocked because its interaction requires its N-terminus (close to the C-terminus in the tertiary structure). When it binds to the cancer antigen, IL-21 is released from the fusion protein, and it is known that cancer cells secrete various MMPs, which will cleave the linker between IL-21 and the ABD or anti-albumin antibody. The MMP-sensitive linker ensures that cancers, which typically have high MMP activity, have a higher exposure to active IL-21. Thus, unnecessary toxicity and side effects of IL-21 can be avoided. Additionally, in such embodiments, the cancer delivery efficiency of the IL-21 fusion protein can be enhanced by preventing the interaction between IL-21 and IL-21Rα on peripheral immune cells.

[0054] In certain embodiments, the presence of an albumin-binding molecule in the fusion proteins provided herein can increase the circulatory half-life compared to fusion proteins that do not have an albumin-binding molecule, resulting in a higher drug exposure to cancer over a longer period of time.

[0055] In certain embodiments, the use of sdAbs with a relatively small molecular weight in the fusion proteins provided herein can help increase the cancer penetrance of the fusion protein, making it more suitable for treating certain cancers, such as solid tumor cancers.

[0056] This application provides a mesothelin construct comprising an anti-MSLN antibody portion, the anti-MSLN antibody portion comprising a single-domain antibody that specifically binds to mesothelin. In some embodiments, the mesothelin construct (such as a single-domain anti-MSLN antibody) exhibits beneficial effects (such as killing tumor cells, such as treating a tumor in an individual). In some embodiments, the anti-MSLN construct is a single-domain anti-MSLN antibody. In some embodiments, the anti-MSLN construct is a fusion protein comprising an anti-MSLN antibody portion and a second domain. In some embodiments, the second domain is a half-life extension domain. In some embodiments, the second domain is a cytokine. In some embodiments, the anti-MSLN construct comprises a fusion protein comprising a) an anti-MSLN antibody portion as described herein, b) a second domain (such as a half-life extension domain), and c) a third domain (such as a cytokine).

[0057] This application further provides a method of treating a disease or condition (such as cancer) by administering to an individual any of the mesothelin constructs described herein. In some embodiments, the method further comprises administering a second agent, such as a cytokine.

[0058] The present application further provides methods for treating diseases (such as cancer, such as mesothelin-positive cancer, such as gastric cancer), said methods comprising administering a) an anti-mesothelin construct (such as any of the anti-MSLN constructs described herein), b) a cytokine (such as IL-21); c) an anti-Her2 agent. In some embodiments, the combination therapies as described in the above methods exhibit synergy.

[0059] Furthermore, the present invention provides novel antibodies that target cancer antigens (such as MSLN), which, when part of the fusion proteins provided herein, can effectively deliver IL-21 to cancer.

[0060] In certain embodiments, advantages are provided by targeting one or more cancer antigens in the IL-21 fusion proteins provided herein with multiple antibodies. For example, in some embodiments, targeting different domains of a cancer antigen (such as MSLN) with two sdAbs each increases the avidity of cancer cell binding.

[0061] The above-mentioned and other properties make the fusion proteins provided herein favorable candidates for cancer treatment.

[0062] Definitions

[0063] The techniques and procedures described or referenced herein include those that are generally readily understandable and / or generally employed by those skilled in the art using conventional methods, such as, for example, methods widely used and described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An, ed., 2009); Monoclonal Antibodies : Methods and Protocols (Monoclonal Antibodies: Methods and Protocols ) (Albitar, ed., 2010); and Antibody Engineering Volumes 1 and 2 (Kontermann and Dübel, eds., 2nd ed. 2010).

[0064] Unless otherwise defined herein, technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following descriptions of terms will apply and, whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event of any conflict between the descriptions of terms set forth herein and any document incorporated herein by reference, the description of terms set forth below shall prevail.

[0065] The term "binding molecule" refers to a protein that includes a portion that binds to a target or antigen (e.g., one or more binding regions such as CDRs) and optionally a framework or scaffold portion (e.g., one or more framework or scaffold regions) that allows the binding portion to adopt a conformation that promotes binding of the binding protein to a polypeptide, fragment, or epitope. In the context of the present invention, a binding molecule is said to specifically bind or selectively bind to an antigen, e.g., when the dissociation constant (K D ) ≤ 10 -6 M. In some embodiments, the binding molecule may specifically bind to an antigen with a K -6 of from about 10 -12 M to about 10 D M. In certain embodiments, when K D ≤ 10 -7 M or K D ≤ 10 -8 M, the binding molecule can specifically bind to the antigen with high affinity. In one embodiment, the binding molecule may specifically bind to a purified human antigen with a K -8 of from 1×10 -8 M to 10×10 D M, as measured by . In one embodiment, the binding molecule may specifically bind to a purified human antigen with a K -9 of from 1×10 -9 M to 10×10 D M, as measured by . In yet another embodiment, the binding molecule specifically binds to a human antigen expressed on a cell with a K -9 of from 0.1×10 -9 M to 10×10 D M. In certain embodiments, the binding molecule has a K -9 of about 0.1×10 -9 M, about 0.5×10 -9 M, about 1×10 -9 M, about 5×10 -9M or any of its ranges or intervals specifically binds to a human antigen expressed on a cell. The term "binding molecule" includes antibodies and molecules derived from antibodies. As used herein, the term "binding molecule" includes antibody fragments (e.g., single domain antibodies) that have a relatively low affinity for an antigen compared to the parental intact antibody.

[0066] As described below, the terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense and, specifically, encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies, single-chain antibodies, and fragments thereof (e.g., bispecific antibodies, provided they exhibit the desired biological activity). Antibodies can be human, humanized, chimeric, and / or affinity matured, as well as antibodies from other species, such as murine and rabbit, etc. The term "antibody" is intended to include the polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two pairs of identical polypeptide chains, where each pair of polypeptide chains has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region. See, for example Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immuno logy (3rd ed. 1997). In specific embodiments, the specific molecular antigen may be bound by the antibodies provided herein (including polypeptides or epitopes). Antibodies also include (but are not limited to) synthetic antibodies, recombinantly produced antibodies, camelized antibodies or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments), which refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and minibodies. Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing antigen-binding sites that bind to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989);Mol. Biology and Biotechnology: A Comprehensive Desk Reference (compiled by Myers, 1995); Huston et al., 1993, Cell Biophysics 22:189 - 224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497 - 515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonistic antibody or an antagonistic antibody.

[0067] An "antigen" is a structure to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is cell - associated, e.g., present on or in a cell, such as a cancer cell (e.g., a solid tumor cancer cell).

[0068] A "full - length" antibody is an antibody that comprises an antigen - binding site and the CL and at least the heavy - chain constant regions CH1, CH2, and CH3. The constant regions can include human constant regions or amino - acid sequence variants thereof. In certain embodiments, the full - length antibody has one or more effector functions.

[0069] The terms "antigen-binding fragment", "antigen-binding domain", "antigen-binding region", and like terms refer to portions of a binding molecule that contain amino acid residues that interact with an antigen and confer upon the binder its specificity and affinity for the antigen (e.g., CDRs). As used herein, "antigen-binding fragment" includes "antibody fragment", which comprises a portion of a whole antibody, such as the antigen-binding or variable region of a whole antibody. Examples of antibody fragments include (but are not limited to) Fab, Fab', F(ab')2, and Fv fragments; diabodies and bis-diabodies (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2003, Nat. Med. 9:129-34; WO 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAb) (see, e.g., Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA 101:12444-49); and multispecific antibodies formed from antibody fragments.

[0070] As used herein, "single domain antibody" or "sdAb" refers to an antibody in which the complementarity determining region is part of a single domain polypeptide. Examples include (but are not limited to) heavy chain antibodies, antibodies that are naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain architectures other than those derived from antibodies. A single domain antibody can be any single domain antibody in the art, or any single domain antibody in the future. Single domain antibodies can be derived from any species, including (but not limited to) mice, humans, camels, llamas, goats, rabbits, and cows. According to one aspect of the invention, the single domain antibody as used herein is a single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO 9404678. For clarity, in some cases, this variable domain derived from a heavy chain antibody that is naturally devoid of light chains is referred to herein as VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., in camels, dromedaries, llamas, and guanacos). Other species besides camelids can produce heavy chain antibodies that are naturally devoid of light chains; such VHHs are within the scope of the present invention. In some embodiments, the single domain antibodies provided herein have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] The term "binds / binding" refers to an interaction between molecules, including, for example, to form a complex. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The complex can also include two or more molecules bound together by covalent or non-covalent bonds, interactions, or forces. The total non-covalent interaction strength between a single antigen binding site on an antibody and a single epitope of a target molecule (e.g., an antigen) is the affinity of the antibody or functional fragment for the epitope. The dissociation rate (k off ) of a binding molecule (e.g., an antibody) from a monovalent antigen and the association rate (k on ) ratio (k off / k on ) is the dissociation constant K D , which is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The K D value is different for different complexes of an antibody and an antigen, depending on both k on and k off . The dissociation constant K DIt can be determined using any method provided herein or any other method well known to those of skill in the art. The affinity of a binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants (such as a multivalent antigen) contacts an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of a reaction occurring at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is referred to as avidity.

[0072] Terms related to the binding molecules described herein, such as "binds to", "specifically binds to", and similar terms, are also used interchangeably herein and refer to a binding molecule having an antigen-binding domain that specifically binds to an antigen (such as a polypeptide). A binding molecule or antigen-binding domain that binds to or specifically binds to an antigen may cross-react with related antigens. In certain embodiments, a binding molecule or antigen-binding domain that binds to or specifically binds to an antigen does not cross-react with other antigens. A binding molecule or antigen-binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassay, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen when it binds to the antigen with an affinity greater than that of any cross-reactive antigen (such as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)). Typically, a specific or selective reaction will be at least twice the background signal or noise and may exceed 10-fold background. See, for example Fundamental ImmunologyThe discussion on binding specificity in 332 - 36 (edited by Paul, 2nd edition, 1989). In certain embodiments, as determined, for example, by fluorescence - activated cell sorting (FACS) analysis or RIA, the degree of binding of a binding molecule or antigen - binding domain to a "non - target" protein is less than about 10% of the degree of binding of the binding molecule or antigen - binding domain to its specific target antigen. Terms such as "specifically binds", "specifically binds to", or "is specific for" mean binding that is measurably different from non - specific interactions. Specific binding can be measured by comparing the binding of a molecule to the binding of a control molecule, which is typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, if the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled target, it indicates specific binding. A binding molecule or antigen - binding domain that binds to an antigen includes a binding molecule or antigen - binding domain that can bind to the antigen with sufficient affinity such that the binding molecule can be used as, for example, a diagnostic fd - fragment agent targeting the antigen. In certain embodiments, the dissociation constant (K D ) of the binding molecule or antigen - binding domain that binds to an antigen is less than or equal to 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, the binding molecule or antigen - binding domain binds to an antigenic epitope that is conserved among antigens from different species (e.g., between human and macaque species).

[0073] "Binding affinity" generally refers to the strength of the sum of non - covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y can generally be expressed by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Low - affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high - affinity antibodies generally bind antigens more quickly and tend to remain bound for a longer time. A variety of methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present invention. Specific illustrative embodiments include the following. In one embodiment, "K D” or “K D value”. K D can be measured in an RIA, for example, with the Fab form of the antibody of interest and its antigen (Chen et al., 1999, Journal of Molecular Biology 293:865 - 81). K D or K D values can also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays, by using, for example of the system or by using, for example or of to measure. “Association rate” or “rate of association” or “association rate” or “kon” can also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using, for example or systems.

[0074] In certain embodiments, a binding molecule or antigen - binding domain can comprise a “chimeric” sequence, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass and fragments of such antibodies, so long as it exhibits the desired biological activity. (See U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proceedings of the National Academy of Sciences of the United States of America 81:6851 - 55).

[0075] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody comprising a human immunoglobulin (e.g., acceptor antibody), wherein the native CDR residues are replaced with residues of the corresponding CDRs from a non-human species (e.g., donor antibody), such as a mouse, rat, rabbit, or non-human primate, which residues have the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced with the corresponding non-human residues. In addition, a humanized antibody may comprise residues not found in the acceptor antibody or in the donor antibody. These modifications are made to further optimize antibody performance. A humanized heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin, and all or substantially all of the FRs are the FRs of a human immunoglobulin sequence. In certain embodiments, a humanized antibody will comprise at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0076] In certain embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "fully human antibody" or "human antibody," where the terms are used interchangeably herein and refer to an antibody comprising human variable regions and, for example, human constant regions. In a specific embodiment, the term refers to an antibody comprising variable and constant regions of human origin. In certain embodiments, a "fully human" antibody may also encompass an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a somatic variant of a naturally occurring human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes an antibody having variable and constant regions that correspond to, e.g., those described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, the human germline immunoglobulin sequences as described in the fifth edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). "Human antibodies" are antibodies having an amino acid sequence corresponding to the amino acid sequence of antibodies produced by humans and / or antibodies produced by any technique for making human antibodies. This definition of human antibodies specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, Journal of Molecular Biology (J. Mol. Biol.) 227:381; Marks et al., 1991, Journal of Molecular Biology (J. Mol. Biol.) 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). It can also be a method for preparing human monoclonal antibodies, such as described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77(1985); Boerner et al., 1991, Journal of Immunology (J. Immunol.) 147(1):86-95; and van Dijk and van de Winkel, 2001, Current Opinion in Pharmacology (Curr. Opin. Pharmacol.) 5:368-74. Human antibodies can be prepared by injecting an antigen into a transgenic animal (such as a mouse) that has been modified to produce such antibodies in response to an antigen challenge, but whose endogenous loci have been disabled (see, for example, Jakobovits, 1995, Current Opinion in Biotechnology (Curr. Opin. Biotechnol.) 6(5):561-66; Brüggemann and Taussing, 1997, Current Opinion in Biotechnology (Curr. Opin. Biotechnol.) 8(4):455-58; and U.S. Patents Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE TM technology). See also, for example, Li et al., 2006, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 103:3557-62, regarding human antibodies produced via human B cell hybridoma technology.

[0077] In certain specific embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody", where the phrase includes human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic and / or transchromosomal animals (e.g., mice or cows) of the human immunoglobulin genes (see, e.g., Taylor, L.D. et al. (1992) Nucleic Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest , 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using transgenic animals with human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences derived from and related to human germline VH and VL sequences, which may not be present in the human antibody germline repertoire in vivo.

[0078] In certain embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody", where the term as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on an antigen. In specific embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing antibodies. For example, monoclonal antibodies suitable for the present invention can be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or can be prepared using recombinant DNA methods in bacteria or eukaryotic or plant cells (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing the cloned cell lines and monoclonal antibodies thus expressed are well known in the art. See, e.g., Short Protocols inMolecular Biology (eds. Ausubel et al., 5th ed. 2002).

[0079] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H chain and L chain also has regularly spaced intra-chain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. VL aligns with VH and CL aligns with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example Basic and Clinical Immunology 71 (eds. Stites et al., 8th ed. 1994); and Immunobiology (eds. Janeway et al., 5th ed. 2001).

[0080] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgG typically contains two Fab regions, each located on one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region of each of the heavy and light chains. More precisely, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. According to the present invention, VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding ability. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and arranged in a different order, which will be described in detail below.

[0081] The terms "variable region", "variable domain", "V region" or "V domain" refer to a part of the light or heavy chain of an antibody, which is usually located at the amino terminus of the light or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that the sequences of certain segments of the variable regions in each antibody vary widely. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 - amino - acid span of the entire variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches called framework regions of about 15 - 30 amino acids, which are separated by shorter regions called "hypervariable regions" having higher variability (e.g., extreme variability), each of which is about 9 - 12 amino acids long. The variable regions of the heavy and light chains each contain four FRs, mainly in a β - sheet conformation, connected by three hypervariable regions, which form loops connecting the β - sheet structures and in some cases form part of the β - sheet structure. The hypervariable regions in each chain are held closely together by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen - binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant region does not directly participate in binding the antibody to the antigen but exhibits various effector functions, such as the antibody's participation in antibody - dependent cell - mediated cytotoxicity (ADCC) and complement - dependent cytotoxicity (CDC). The variable regions of different antibodies vary widely in sequence. In a specific embodiment, the variable region is a human variable region.

[0082] The term "Kabat numbering of variable region residues" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the compiled heavy chain variable region or light chain variable region of antibodies by Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues of a given antibody can be determined by aligning the homologs in the antibody sequence with the "standard" Kabat numbering sequence. The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is commonly used (e.g., the EU index reported in Kabat et al. (supra)). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0083] When used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, where the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids and the carboxyl-terminal portion includes a constant region. The constant region can be one of five different types (e.g., isotypes) based on the amino acid sequence of the heavy chain constant region, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to the five well-known antibody classes (e.g., isotypes), IgA, IgD, IgE, IgG, and IgM, including four IgG subclasses, namely IgG1, IgG2, IgG3, and IgG4.

[0084] When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, where the amino-terminal portion includes a variable region of approximately 100 to approximately 110 or more amino acids and the carboxyl-terminal portion includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. There are two different types based on the amino acid sequence of the constant domain, called kappa (κ) or lambda (λ).

[0085] As used herein, the terms "hypervariable region", "HVR", "complementary determining region", and "CDR" are used interchangeably. "CDR" refers to one of three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0086] CDR regions are well known to those of skill in the art and have been defined by well-known numbering systems. For example, the Kabat complementary determining regions (CDRs) are sequence-based and the most commonly used (see, e.g., Kabat et al., supra). Chothia actually refers to the positions of structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, then the loop end is at 32; if only 35A is present, then the loop end is at 33; if both 35A and 35B are present, then the loop end is at 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Volume 2 (ed. Kontermann and Dübel, 2nd ed. 2010)). "Contact ( contact ) " hypervariable regions are based on the analysis of available complex crystal structures. Another commonly used numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. In this article, CDRs are referred to based on both the amino acid sequence and their position within the light or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain is conserved across species and exists within loop structures, CDRs and framework residues can be readily identified by sorting the variable domain sequences according to their structural features using a numbering system. This information can be used to graft and replace CDR residues of immunoglobulins from one species into the acceptor framework of a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plückthun, 2001, J. Mol. Biol. 309:657-70. The correspondence between numbering systems, including for example Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). Residues from each of these hypervariable regions or CDRs are labeled below.

[0087] Table 1

[0088]

[0089] The boundaries of a given CDR may vary depending on the discrimination scheme. Thus, unless otherwise stated, the terms "CDR" and "complementary determining region" of a given antibody or its region, such as the variable region and the individual CDRs of an antibody or its region (e.g., "CDR-H1, CDR-H2") should be understood to encompass the complementary determining regions as defined by any of the known schemes described above. In some cases, the discrimination scheme specifying a particular CDR or CDRs, such as those defined by the Kabat, Chothia or Contact methods, is given. In other cases, the specific amino acid sequence of a given CDR is given.

[0090] Hypervariable regions may include "extended hypervariable regions" such as 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

[0091] The terms "constant region" or "constant domain" refer to the carboxy-terminal portions of the light and heavy chains that do not directly participate in binding of the antibody to an antigen but exhibit various effector functions, such as interaction with Fc receptors. The term refers to the portion of the immunoglobulin molecule that has an amino acid sequence that is more conserved relative to the other parts of the immunoglobulin (the variable region, which contains the antigen-binding site). The constant region can contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0092] The terms "framework" or "FR" refer to those variable region residues that flank the CDRs. FR residues are present, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, bispecific antibodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0093] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from the Cys226 position or from the amino acid residue at Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can contain a population of antibodies in which all K447 residues are removed, a population of antibodies in which the K447 residues are not removed, and a population of antibodies that is a mixture of antibodies with and without the K447 residue. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (such as the B cell receptor), etc. Such effector functions generally require the combination of the Fc region with a binding region or binding domain (such as an antibody variable region or domain) and can be evaluated using various assays known to those of skill in the art. A "variant Fc region" contains an amino acid sequence that is different from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification (such as a substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or to the Fc region of a parental polypeptide, for example, from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native sequence Fc region or in the Fc region of the parental polypeptide. A variant Fc region as used herein can have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parental polypeptide, or at least about 90% homology therewith, such as at least about 95% homology therewith.

[0094] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a continuous amino acid of the polypeptide ("linear" epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide ("conformational", "non-linear" or "discontinuous" epitope). One of ordinary skill in the art will appreciate that, generally speaking, a linear epitope may or may not depend on secondary, tertiary or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether it is folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0095] The "percent amino acid sequence identity (%)" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a comparison polypeptide after a sequence alignment in which any conservative substitutions are considered part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of this application, the amino acid sequence identity values % are generated using the sequence comparison computer program MUSCLE (Edgar, R.C. Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0096] As used herein, the term "cytokine" is understood to mean any protein or peptide, analogs or functional fragments thereof, capable of stimulating or inducing a cell-killing immune response against a preselected cell type (e.g., cancer cells or virus-infected cells) in a mammal. Thus, it is contemplated that a variety of cytokines can be incorporated into the present application. Suitable cytokines include, for example, tumor necrosis factor (TNF), interleukin (IL), lymphokine (LL), colony-stimulating factor (CSF), interferon (IFN), including species variants and truncated analogs thereof capable of stimulating or inducing such a cell-killing immune response. Suitable tumor necrosis factors include, for example, TNFα. Suitable lymphokines include, for example, LT. Suitable colony-stimulating factors include, for example, GM-CSF and M-CSF. Suitable interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22 and IL-33. Suitable interferons include, for example, IFN-α, IFN-α and IFN-γ. The term "cytokine" is also understood to encompass any variant of a wild-type cytokine (such as IL-21, IL-7, IL-15, etc.) that contains at least one essential portion (e.g., at least about 50%) that is modified and maintains any of its desired functions.

[0097] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The terms also encompass amino acid polymers modified by natural or intervening means; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids, including (but not limited to) non-natural amino acids, and other modifications known in the art. It is understood that since the polypeptides of the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptide" may occur as a single chain or as two or more related chains.

[0098] The term "vector" refers to a substance for carrying or including a nucleic acid sequence (including, for example, a nucleic acid sequence encoding a binding molecule (such as an antibody) as described herein) for introducing the nucleic acid sequence into a host cell. Vectors suitable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable to stably integrate into the chromosome of the host cell. Additionally, a vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or provide key nutrients not present in the medium. Expression control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., well known in the art. When two or more nucleic acid molecules are to be co-expressed (such as an antibody heavy chain and light chain, or antibody VH and VL), the two nucleic acid molecules may be (for example) inserted into a single expression vector or in separate expression vectors. For single vector expression, the coding nucleic acids may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art may be used to confirm the introduction of a nucleic acid molecule into a host cell. Such methods include, for example, nucleic acid analysis, such as Northern blotting of mRNA or polymerase chain reaction (PCR) amplification, immunoblotting for the expression of the gene product, or other suitable assays for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the amount of nucleic acid molecule expressed is sufficient to produce the desired product, and will further understand that expression levels may be optimized using methods well known in the art to obtain sufficient expression.

[0099] As used herein, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0100] As used herein, the term "host cell" refers to a specific subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such cells. Due to mutations or environmental influences, the progeny of such cells may not be identical to the parental cell transfected with the nucleic acid molecule, said mutations or environmental influences occurring during the production of the nucleic acid molecule or its integration into the host cell genome.

[0101] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from the components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is not associated with all of the other components in its production environment. Contaminant components of its production environment (such as those produced by recombinant transfected cells) are substances that will generally interfere with the research, diagnostic, or therapeutic use of the antibody and can include enzymes, hormones, and other proteins or non-proteinaceous solutes. In a preferred embodiment, the polypeptide will be purified: (1) to greater than 95% by weight antibody, as determined, for example, by the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to an extent sufficient to obtain the N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or preferably silver stain. An isolated antibody (or construct) includes an antibody in situ within a recombinant cell, since at least one component in the antibody's natural environment will not be present. However, generally, an isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0102] "Isolated nucleic acid" is nucleic acid, such as RNA, DNA, or hybrid nucleic acids, that is substantially separated from other genomic DNA sequences and from proteins or complexes (such as ribosomes and polymerases) that naturally accompany the native sequence. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or media when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term includes nucleic acid sequences removed from their natural environment and includes recombinant or cloned DNA isolates and analogs or biosynthetic analogs chemically synthesized by heterologous systems.. A substantially pure molecule can include an isolated form of the molecule.

[0103] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to short, single-stranded, synthetic polynucleotides that are typically (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides. The cells that produce the binding molecules of the present invention can include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which the nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of the nascent RNA transcript added 5' to 3' is referred to as the transcription direction; the sequence region on the DNA strand that has the same sequence as the sequence of the RNA transcript from the 5' end of the RNA transcript to the 5' end of the RNA transcript is called the "upstream sequence"; the sequence region on the DNA strand that has the same sequence as the sequence of the RNA transcript from the 3' end of the RNA transcript to the 3' end of the RNA transcript is called the "downstream sequence".

[0104] As used herein, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or a state government, or United States Pharmacopedia, European Pharmacopedia otherwise listed in other recognized pharmacopoeias and applicable to animals, and more particularly to humans.

[0105] "Excipient" means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption promoters, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilizing agents, sweetening agents, solubilizers, wetting agents and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (such as Freunds' adjuvant (complete or incomplete)) or vehicle.

[0106] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol, sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG), and PLURONICS TM . Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition, 1990).

[0107] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; edited by Rowe et al.; Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition; edited by Ash and Ash; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; edited by Gibson; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to the cells or mammals to which it is exposed at the doses and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffer solution.

[0108] In some embodiments, the excipient is a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary excipient when the composition (e.g., a pharmaceutical composition) is administered intravenously. Aqueous solutions of saline, dextrose and glycerol may also be employed as liquid excipients, particularly for injectable solutions. Excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffering agents. The composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. Oral compositions including the formulation may include standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0109] A composition comprising a pharmaceutical compound may contain, for example, a binding molecule (e.g., an antibody) in isolated or purified form, as well as a suitable amount of excipient.

[0110] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a binding molecule (e.g., an antibody) or a pharmaceutical composition provided herein that is sufficient to produce the desired result.

[0111] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, the subject is a mammal such as a non - primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal diagnosed with a condition or disorder, such as a human. In another embodiment, the subject is a mammal at risk of contracting a condition or disorder, such as a human.

[0112] "Administer / administration" refers to the act of injecting or otherwise physically delivering a substance to a patient when the substance is outside the body, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0113] As used herein, the terms “treat / treatment / treating” refer to a reduction or improvement in the course, severity, and / or duration of a disease or condition caused by the administration of one or more therapies. Treatment can be determined by assessing whether one or more symptoms associated with the underlying condition have been reduced, alleviated, and / or mitigated, such that improvement is observed in the patient, even though the patient may still have the underlying condition. The term “treatment” includes the control and improvement of a disease. The term “manage / managing / management” refers to the beneficial effects obtained by a subject from a therapy that does not necessarily result in a cure of the disease.

[0114] The term “prevent / preventing / prevention” refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or one or more associated symptoms (such as cancer).

[0115] The term “cancer” or “cancer cell” is used herein to refer to tissue or cells found in a neoplasm that have characteristics that distinguish them from normal tissue or tissue cells. Such characteristics include (but are not limited to): degree of anaplasia, irregular shape, abnormal cell contours, nuclear size, alterations in nuclear or cytoplasmic structure, other phenotypic alterations, the presence of cellular proteins indicative of a cancerous or pre-cancerous state, increased mitotic rate, and the ability to metastasize. Words related to “cancer” include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and neuroma, transformed tumor, etc.

[0116] The terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of a given value or range, or less than a given value or range.

[0117] As used in this invention and the claims, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include the plural forms.

[0118] It should be understood that wherever embodiments are described herein using the term “comprising”, additional similar embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided. It should also be understood that wherever embodiments are described herein using the phrase “consisting primarily of”, additional similar embodiments described in terms of “consisting of” are also provided.

[0119] The term “between” as used in phrases such as “between A and B” or “between A - B” refers to a range that includes both A and B.

[0120] The term "and / or" as used in phrases such as "A and / or B" in this text is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0121] IL-21 fusion protein

[0122] IL - 21 and its Variants

[0123] The IL-21 protein in the IL-21 fusion protein provided herein can be a human wild-type IL-21 protein having the amino acid sequence of SEQ ID NO:1 (see below).

[0124]

[0125] In some embodiments, an IL-21 variant can be in the IL-21 fusion protein provided herein. The variation can be a substitution, deletion or insertion of one or more codons encoding the IL-21 polypeptide, which results in an alteration of the amino acid sequence as compared to the human wild-type IL-21 protein. The amino acid substitution can be the result of replacing one amino acid with another amino acid having a similar structure and / or chemical property, such as replacing leucine with serine, for example a conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding the molecules provided herein, including for example site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions. The insertion or deletion can optionally be in the range of about 1 to 10 amino acids. In certain embodiments, the substitution, deletion or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution at one or more predicted non-essential amino acid residues. The allowed variations can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the activity of the resulting variants exhibited by the full-length or mature native sequence.

[0126] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions in the length range from one residue to multiple residues, as well as internal insertions of single or multiple amino acid residues.

[0127] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the biological activity of the resulting mutants (e.g., binding to the IL-21 receptor or IL-21R) can be screened to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the activity of the protein can be determined.

[0128] Conservative substitutions (e.g., within amino acid groups having similar properties and / or side chains) can be made so as to maintain or not significantly alter the properties of the IL-21 protein. Amino acids can be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry pp. 73-75 (2nd ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0129] Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0130] Thus, in one embodiment, the IL-21 variants provided herein comprise an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of IL-21 having SEQ ID NO:1 described herein.

[0131] Mutations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23) or other known techniques can be performed on the cloned DNA to generate polypeptides.

[0132] In some embodiments, the IL-21 variant lacks one or more amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any one amino acid between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any two amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any three amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any four amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any five amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any six amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any seven amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any eight amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any nine amino acids between S124 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any ten amino acids between S124 and S133 at the C-terminus.

[0133] In some embodiments, the IL-21 variant lacks 11 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 10 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 9 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 8 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 7 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 6 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 5 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 4 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 3 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 2 amino acids at the C-terminus of SEQ ID NO:1. In some embodiments, the IL-21 variant lacks 1 amino acid at the C-terminus of SEQ ID NO:1.

[0134] In a specific embodiment, the IL-21 variant provided herein has the amino acid sequence of SEQ ID NO:2, which lacks 10 amino acids at the C-terminus and represents the sequence of Q1 to L123 of SEQ ID NO:1.

[0135] Albumin - Binding Molecule

[0136] Albumin (e.g., human serum albumin or HSA) has been used to increase the serum half-life of biopharmaceuticals. See Dennis et al., The Journal of Biological Cheminstry, 2002, 277(38):35035-35043; Adams et al., MABS, 2016, 8(7):1336-1346. For example, human serum albumin (HSA) has been utilized. HSA is the most abundant protein in blood, is widely distributed in tissues, and has non-acute functions. It has a half-life of 19 days. Thus, in some embodiments, among other advantages, albumin (e.g., HSA) can be used herein to increase the half-life of the fusion proteins provided herein. Albumin can be used in several ways. One exemplary method is to directly include an albumin domain (e.g., HSA) genetically or chemically in the fusion proteins provided herein. Another exemplary method is to use an albumin-binding domain (ABD) or an anti-albumin antibody.

[0137] In some embodiments, the fusion proteins provided herein comprise albumin-binding molecules. In some embodiments, the albumin-binding molecules provided herein are albumin-binding domains (ABDs). In some embodiments, the ABD can bind to human serum albumin (HSA). In other embodiments, the ABD can bind to mouse serum albumin (MSA).

[0138] In some embodiments, the ABD binds to HSA with a K between 1 - 1000 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 900 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 800 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 700 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 600 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 500 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 400 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 300 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 200 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 100 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 50 nM D In some embodiments, the ABD binds to HSA with a K between 1 - 25 nM D In some embodiments, the ABD binds to HSA with a K between 0.1 - 1 nM D In other embodiments, the ABD binds to HSA with a K between 10 - 800 nM D In some embodiments, the ABD binds to HSA with a K between 20 - 500 nM D In other embodiments, the ABD binds to HSA with a K between 50 - 300 nM D In other embodiments, the ABD binds to HSA with a K between 100 - 200 nM D binds to HSA.

[0139] In some specific embodiments, the ABD has the amino acid sequence of LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO: 3), and its K for binding to HSAD is about 1.2 nM.

[0140] In some embodiments, an ABD having a relatively low affinity for HSA as compared to the ABD of SEQ ID NO:3 is preferred. Accordingly, variants of SEQ ID NO:3 having a lower affinity for HSA are included in the present invention.

[0141] The variation can be a substitution, deletion or insertion of one or more codons encoding the ABD polypeptide of SEQ ID NO:3, which results in a change in the amino acid sequence. An amino acid substitution can be the result of replacing one amino acid with another amino acid having similar or different structure and / or chemical properties. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that results in amino acid substitutions. The insertions or deletions can optionally be in the range of about 1 to 15 amino acids. In certain embodiments, relative to the original molecule of SEQ ID NO:3, the substitutions, deletions or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions or fewer than 2 amino acid substitutions. The allowed variations can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the activity of the resulting variants, and in some embodiments, variants having a lower affinity for HSA are selected. Some such variants are exemplified in Table 7 in Example 1 below.

[0142] In some specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:4. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:5. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:6. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:7. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:8. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:9. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:10. In other specific embodiments, the ABD has the amino acid sequence of SEQ ID NO:11.

[0143] According to the present invention, the albumin-binding molecule can also be an anti-albumin antibody or an antigen-binding fragment thereof. In some embodiments, the anti-albumin antibody or an antigen-binding fragment thereof is an anti-HSA antibody or an antigen-binding fragment thereof.

[0144] Several isotypes of HSA are listed in Table 2 below (see UniProtKB - P02768 (ALBU_HUMAN))

[0145] Table 2

[0146]

[0147]

[0148] The anti - HSA antibodies provided herein can bind to any of these isotypes or fragments thereof. In some embodiments, the anti - HSA antibodies provided herein bind to SEQ ID NO:260 or a fragment thereof. In some embodiments, the anti - HSA antibodies provided herein bind to SEQ ID NO:261 or a fragment thereof. In other embodiments, the anti - HSA antibodies provided herein bind to SEQ ID NO:262 or a fragment thereof.

[0149] The anti - HSA antibodies provided herein can be (but are not limited to) synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single - chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies or their humanized forms, Fab fragments, F(ab') fragments, disulfide - linked Fv (sdFv), anti - idiotypic (anti - Id) antibodies, and epitope - binding fragments of any of the above.

[0150] Specifically, the anti - HSA antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen - binding sites that immunospecifically bind to HSA. The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. In a specific embodiment, the anti - HSA antibody provided herein is an IgG antibody, such as an IgG1 antibody.

[0151] The present invention also includes variants and derivatives of the antibody, which include antibody fragments that retain the ability to specifically bind to an epitope of HSA. Exemplary fragments include Fab fragments (antibody fragments containing an antigen-binding domain and comprising a light chain and a portion of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single antigen-binding domain, the antigen-binding domain comprising a Fab and an additional portion of the heavy chain through the hinge region); F(ab')2 (two Fab' molecules joined by an interchain disulfide bond in the hinge region of the heavy chain; the Fab' molecules can be directed to the same or different epitopes); bispecific Fab (a Fab molecule having two antigen-binding domains, each of which can be directed to a different epitope); a single-chain Fab chain containing variable regions, also known as sFv (the variable antigen-binding determinants of a single light and heavy chain of an antibody joined together by a 10-25 amino acid chain); disulfide-linked Fv or dsFv (the variable antigen-binding determinants of a single light and heavy chain of an antibody joined together by a disulfide bond); camelized VH (the variable antigen-binding determinant of a single heavy chain of an antibody, where some of the amino acids at the VH interface are those found in the heavy chain of a naturally occurring camel antibody); bispecific sFv (an sFv or dsFv molecule having two antigen-binding domains, each of which can be directed to a different epitope); bifunctional antibody (a dimerized sFv formed when the VH domain of a first sFv is assembled with the VL domain of a second sFv and the VL domain of the first sFv is assembled with the VH domain of the second sFv; the two antigen-binding regions of the bifunctional antibody can be directed to the same or different epitopes); and trifunctional antibody (a trimerized sFv formed in a manner similar to the bifunctional antibody, but where three antigen-binding domains are produced in a single complex; the three antigen-binding domains can be directed to the same or different epitopes). Derivatives of the antibody also include one or more CDR sequences of the antibody combining site. When two or more CDR sequences are present, the CDR sequences can be joined together architecturally. In certain embodiments, the anti-HSA antibodies provided herein comprise single-chain Fv ("scFv"). An scFv is an antibody fragment containing the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide also contains a polypeptide linker between the VH and VL domains, enabling the scFv to form the required structure for antigen binding. For a review of scFv, see Pluckthun , The Pharmacology of Monoclonal Antibodies , Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994).

[0152] In certain instances, there are advantages to using anti-HSA antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in better access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson et al., 2003, Nature Med 9:129-34.

[0153] A variety of techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived via proteolytic digestion of whole antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys., Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed and secreted in E. coli or yeast cells, thus allowing for easy production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab' fragments with increased in vivo half-life are described in U.S. Patent No. 5,869,046, which includes salvage receptor binding epitope residues. Other techniques for the production of antibody fragments will be apparent to the skilled artisan. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, for example, WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458). Fv and scFv have a complete combining site without a constant region; thus, they may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to produce effector protein fusions at the amino or carboxyl terminus of the scFv (see, for example, Borrebaeck, ed., supra). Antibody fragments can also be, for example, "linear antibodies" as described in the references cited above. Such linear antibodies can be monospecific or multispecific, such as bispecific. In a particular embodiment, the antibody fragment is a single-domain antibody. )2

[0154] ​The anti-HSA antibodies provided herein can be from any animal source, including birds and mammals (e.g., humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In certain embodiments, the antibodies provided herein are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequences of human immunoglobulins and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.

[0155] In certain embodiments, the anti-HSA antibody is a fully human antibody, such as a fully human antibody that immunospecifically binds to a cancer antigen. Such fully human antibodies will be superior to fully murine (or other fully or partially non-human species antibodies), humanized antibodies, or chimeric antibodies in minimizing the development of unwanted side effects (such as an immune response against non-fully human antibodies when administered to a subject).

[0156] The anti-HSA antibodies provided herein can be monospecific, bispecific, trispecific, or more specific. Multispecific antibodies can be specific for different epitopes of a polypeptide or can be specific for both a polypeptide and a heterologous epitope, such as a heterologous polypeptide or a solid support. In some embodiments, the antibodies provided herein are monospecific for a given epitope of a polypeptide and do not immunospecifically bind to other epitopes.

[0157] The anti-HSA antibodies provided herein can be monoclonal antibodies or derived from monoclonal antibodies. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).

[0158] In the hybridoma method, a mouse or other suitable host animal (such as a hamster) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and fused with a myeloma cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice (Monoclonal Antibodies:Principles and Practice)》 59-103 (1986)).

[0159] The hybridoma cells thus prepared are inoculated and grown in a suitable medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of non-fused parental myeloma cells (also referred to as fusion partners). For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), then the selective medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0160] Exemplary fusion partner myeloma cells are those that can fuse efficiently, can support a stable high level of antibody production by the selected antibody-producing cells, and are sensitive to the selective medium directed against the unfused parental cells. Exemplary myeloma cell lines are murine myeloma lines such as SP-2 and derivatives, e.g., X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, VA), and those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA). Human myelomas and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, 1984, Immunol 133:3001-05; and Brodeur et al., 《Monoclonal Antibody Production Techniques and Applications)》 51-63 (1987)).

[0161] The production of monoclonal antibodies against the antigen is detected in the medium in which the hybridoma cells are growing. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as RIA or ELISA. The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis as described by Munson et al., 1980, Anal. Biochem. 107:220-39.

[0162] Once hybridoma cells that produce anti-HSA antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown as ascites tumors in animals, e.g., by intraperitoneal injection of the cells into mice.

[0163] Monoclonal antibodies secreted by subclones are appropriately isolated from culture media, ascites fluid, or serum by conventional antibody purification procedures such as affinity chromatography (e.g., using protein A or protein G-agarose), or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0164] DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of murine antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells that do not otherwise produce the antibody protein, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize the monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., 1993, Curr. Opinion in Immunol. 5:256-62 and Plückthun, 1992, Immunol. Revs. 130:151-88.

[0165] In another embodiment, the monoclonal antibody or antibody fragment can be obtained from using, for example 《Antibody Phage Display:Methods and Protocols)》Isolation of antibody phage libraries generated by the techniques described in (O'Brien and Aitken, eds., 2002). In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Examples of phage display methods that can be used to prepare the antibodies described herein include Brinkman et al., 1995, Journal of Immunological Methods 182:41-50; Ames et al., 1995, Journal of Immunological Methods 184:177-186; Kettleborough et al., 1994, European Journal of Immunology 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Application No. PCT / GB91 / 01134; International Publications WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patents Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0166] In principle, synthetic antibody clones are selected by screening a phage library containing phages that display various fragments of the antibody variable region (Fv) fused to the phage coat protein. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments that are able to bind to the desired antigen are adsorbed to the antigen, thereby separating them from the unbound clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0167] Variable domains can be functionally displayed on phage, either as single-chain Fv (scFv) fragments where VH and VL are covalently linked via a short flexible peptide, or as Fab fragments where they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0168] Libraries of VH and VL genes can be cloned separately by PCR and randomly recombined in a phage library, and antigen-binding clones can then be searched for as described by Winter et al. above. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive libraries can be cloned to provide a single source of human antibodies to a wide range of non-self and self antigens without any immunization, as described by Griffiths et al., 1993, EMBO J. 12:725-34. Finally, naive libraries can also be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 region and achieve rearrangement in vitro, as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-88.

[0169] Screening of libraries can be achieved by various techniques known in the art. For example, HSA polypeptide or fragments thereof can be used to coat the wells of a binding plate, expressed on host cells attached to a binding plate or for cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning a display library. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by using methods such as long washes and monovalent phage display as described by Bass et al., 1990, Proteins 8:309-14 and WO 92 / 09690, and low antigen coating density as described by Marks et al., 1992, Biotechnol. 10:779-83.

[0170] Antibodies can be obtained by designing appropriate antigen screening procedures to select clones of the phage of interest, followed by constructing full-length antibody clones using VH and / or VL sequences (e.g., Fv sequences) or various CDR sequences from the VH and VL sequences, such as described in Kabat et al., supra, from the phage clone of interest and appropriate constant region (e.g., Fc) sequences.

[0171] The anti-HSA antibodies described herein may also include, for example, chimeric antibodies. A chimeric antibody is a molecule in which different parts of the antibody are from different immunoglobulin molecules. For example, a chimeric antibody may contain the variable region of a murine or rat monoclonal antibody fused to the constant region of a human antibody. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

[0172] Anti-HSA antibodies or antigen-binding fragments produced using the techniques described herein can be isolated using standard, well-known techniques. For example, the antibody or antigen-binding fragment can be suitably isolated from, for example, culture medium, ascites fluid, serum, cell lysates, synthetic reaction materials, etc. by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. As used herein, an "isolated" or "purified" antibody is substantially free of cellular material or other proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0173] In some more specific embodiments, the anti-albumin antibody or its antigen-binding fragment is an sdAb that binds to HSA. In some embodiments, the sdAb is a V H H single-domain antibody.

[0174] In some embodiments, the sdAb binds to HSA with a K between 1-1000 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-900 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-800 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-700 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-600 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-500 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-400 nM. D In some embodiments, the sdAb binds to HSA with a K between 1-300 nM.D Binds to HSA. In some embodiments, the sdAb has a K between 1 - 200 nM D Binds to HSA. In some embodiments, the sdAb has a K between 1 - 100 nM D Binds to HSA. In some embodiments, the sdAb has a K between 1 - 50 nM D Binds to HSA. In some embodiments, the sdAb has a K between 1 - 25 nM D Binds to HSA. In some embodiments, the sdAb has a K between 0.1 - 1 nM D Binds to HSA. In other embodiments, the sdAb has a K between 10 - 800 nM D Binds to HSA. In some embodiments, the sdAb has a K between 20 - 500 nM D Binds to HSA. In other embodiments, the sdAb has a K between 50 - 300 nM D Binds to HSA. In other embodiments, the sdAb has a K between 100 - 200 nM D Binds to HSA.

[0175] Cancer Antigen-Binding Molecules

[0176] In some embodiments, the binding molecule that binds to the antigen in the fusion protein provided herein can bind to a cancer antigen and thereby facilitate the targeting or delivery of IL-21 or a variant thereof to cancer cells, such as solid tumor cancer cells.

[0177] In some embodiments, the binding molecule is an antibody or an antigen-binding fragment thereof that binds to an antigen expressed on cancer cells. In some embodiments, the cancer cells are solid tumor cancer cells.

[0178] In some embodiments, the antibody or an antigen-binding fragment thereof provided herein can immunospecifically bind to a polypeptide, polypeptide fragment, or epitope of an antigen expressed on cancer cells. In one embodiment, the antibody binds to a human cancer antigen. In some embodiments, the antibody or an antigen-binding fragment thereof provided herein binds to the extracellular domain (ECD) of a cancer antigen. In certain embodiments, the antibody binds to an epitope in the ECD of a cancer antigen. In some embodiments, the cancer antigen is expressed on solid tumor cancer cells.

[0179] Antibodies that bind to the cancer antigens provided herein can be, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFvs) (e.g., including monospecific, bispecific, etc.), camelized antibodies or their humanized variants, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the foregoing.

[0180] Specifically, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that immunospecifically bind to cancer antigens (e.g., solid tumor cancer antigens). The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. In a specific embodiment, the antibodies provided herein are IgG antibodies, such as IgG1 antibodies.

[0181] The invention also includes variants and derivatives of the antibodies, which include antibody fragments that retain the ability to specifically bind to an epitope of a cancer antigen. Exemplary fragments include Fab fragments; Fab'; F(ab')2; bispecific Fab; single-chain Fab chains that contain variable regions, also known as sFvs; disulfide-linked Fvs or dsFvs; camelized VHs; bispecific sFvs; bifunctional antibodies; and trifunctional antibodies. Derivatives of the antibodies also include one or more CDR sequences of the antibody combining site. When two or more CDR sequences are present, the CDR sequences can be joined together architecturally. In certain embodiments, the antibodies provided herein comprise single-chain Fvs (“scFvs”). A variety of techniques have been developed for producing antibody fragments, as briefly described in the foregoing section.

[0182] In some embodiments, single domain antibodies (sdAbs) that bind to cancer antigens are preferred. Certain types of organisms, such as camels and cartilaginous fish, have single V-like domains with high affinity, and the V-like domains are mounted on an Fc-equivalent domain structure as part of their immune system. (Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA 101:12444-49). The V-like domains (referred to as VhH in camels and V-NAR in sharks) typically display long loop surfaces that allow penetration of cavities in the target antigen. It also stabilizes the isolated VH domain by masking hydrophobic surface patches.

[0183] These VhH and V-NAR domains have been used to engineer sdAbs. Selection using phage libraries and other methods has been designed to generate stable, high-binding VL- and VH-derived domains. In a specific embodiment, the antibody that binds to the cancer antigen is an sdAb. In one embodiment, the sdAb is a V H H single domain antibody.

[0184] The antibodies provided herein can be from any animal source, including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). In certain embodiments, the antibodies provided herein are human or humanized monoclonal antibodies. As used herein, a "human" antibody includes an antibody having the amino acid sequence of a human immunoglobulin and includes antibodies isolated from a human immunoglobulin library or from a mouse expressing an antibody from a human gene.

[0185] In certain embodiments, the antibody is a fully human antibody, such as a fully human antibody that immunospecifically binds to a cancer antigen. Such fully human antibodies would be preferred over fully murine (or other fully or partially non-human species antibodies), humanized antibodies, or chimeric antibodies to minimize the development of unwanted side effects (such as an immune response against non-fully human antibodies when administered to a subject).

[0186] The antibodies provided herein can be monospecific, bispecific, trispecific, or more specific. Multispecific antibodies can be specific for different epitopes of a polypeptide or can be specific for both a polypeptide and a heterologous epitope, such as a heterologous polypeptide or a solid support. In some embodiments, the antibodies provided herein are monospecific for a given epitope of a polypeptide and do not immunospecifically bind to other epitopes.

[0187] Antibodies that bind to the cancer antigens provided herein can be monoclonal antibodies or derived from monoclonal antibodies. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or can be prepared by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). The production of monoclonal antibodies was briefly described in the above section.

[0188] The antibodies described herein can also include, for example, chimeric antibodies. Antibodies or antigen-binding fragments produced using the techniques described herein can be isolated using standard, well-known techniques.

[0189] In certain embodiments, the antigen-binding molecule in the IL-21 fusion proteins provided herein comprises multiple antibodies linked by a linker, each antibody (e.g., sdAb) binding to the antigen. In some embodiments, the antigen-binding molecule comprises two antibodies or fragments thereof. In some embodiments, the antigen-binding molecule comprises two sdAbs. In some embodiments, the antigen-binding molecule comprises two sdAbs that each bind to a different epitope of the antigen.

[0190] In some more specific embodiments, the antigen-binding molecule comprises two sdAbs that each bind to a different epitope of MSLN. For example, in some embodiments, one sdAb targeting amino acids 296 to 359 (N-terminus) of MSLN and one sdAb targeting amino acids 538 to 622 (C-terminus) of MSLN are used in the IL-21 fusion proteins provided herein. Certain advantages can be provided by targeting specific functional domains of the cancer antigen in the IL-21 fusion protein with one or more antibodies. Targeting the N-terminus of MSLN with an sdAb disrupts the MSLN-MUC16 interaction and reduces cancer cell metastasis. Targeting the C-terminus of MSLN with an sdAb reduces the shedding of MSLN from the cancer cell surface.

[0191] In some embodiments, the cancer antigen is mesothelin (MSLN). In some embodiments, the binding molecule is an sdAb that binds to MSLN (such as those described herein).

[0192] Mesothelin (anti-MSLN) construct

[0193] This application provides mesothelin-specific anti-mesothelin constructs. The anti-mesothelin construct contains an antibody portion of an anti-MSLN heavy chain variable region (VH). In some embodiments, the anti-MSLN VH binds to an antigen comprising the sequence of SEQ ID NO:283. In some embodiments, the anti-MSLN VH binds to an antigen comprising the sequence of SEQ ID NO:284.

[0194] In some embodiments, the anti-mesothelin construct comprises an anti-MSLN antibody portion, and the anti-MSLN antibody portion comprises a single-domain anti-mesothelin antibody as described herein.

[0195] In some embodiments, the anti-MSLN constructs described herein comprise, from the N-terminus to the C-terminus, in the order of a) an anti-MSLN antibody portion, b) a second domain. In some embodiments, the anti-MSLN constructs described herein comprise, from the N-terminus to the C-terminus, in the order of a) a second domain, b) an anti-MSLN antibody portion.

[0196] In some embodiments, the anti-MSLN constructs described herein comprise, from the N-terminus to the C-terminus, in the order of a) an anti-MSLN antibody portion, b) a second domain, c) a third domain. In some embodiments, the anti-MSLN constructs described herein comprise, from the N-terminus to the C-terminus, in the order of a) a second domain, b) an anti-MSLN antibody portion, c) a third domain. In some embodiments, the anti-MSLN constructs described herein comprise, from the N-terminus to the C-terminus, in the order of a) a second domain, b) a third domain, c) an anti-MSLN antibody portion.

[0197] In some embodiments, the second domain or the third domain is a half-life extending domain. In some embodiments, the second domain or the third domain is a cytokine.

[0198] In some embodiments, the construct is a fusion protein that further comprises a half-life extending domain (such as an Fc domain or an albumin binding domain). In some embodiments, the construct is a fusion protein that further comprises a cytokine (such as IL-21, IL-15). In some embodiments, the construct is a fusion protein that further comprises a) a half-life extending domain; and b) a cytokine. Exemplary fusion proteins are described herein.

[0199] In some embodiments, the half-life extending domain is fused to the N-terminus or the C-terminus of the anti-MSLN antibody portion. In some embodiments, the cytokine is fused to the N-terminus of the anti-MSLN antibody portion or the half-life extending domain. In some embodiments, the half-life extending domain is fused to the N-terminus or the C-terminus of the cytokine.

[0200] In some embodiments, the construct comprises a linker between the anti-MSLN antibody portion and the second domain or the third domain (such as a cytokine). Exemplary linkers are described herein. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 66-74 and SEQ ID NOs: 267-282. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable.

[0201] In some embodiments, the anti-MSLN construct comprises the amino acid sequence of any one of SEQ ID NOs: 123-156 and SEQ ID NOs: 285-331, or a variant thereof having at least about 80% (e.g., about 85%, 90%, 95%, 98% or 99%) sequence identity to any one of SEQ ID NOs: 123-156 and SEQ ID NOs: 285-331.

[0202] Single domain antibody that binds to mesothelin (MSLN)

[0203] The present invention provides single domain antibodies that specifically bind to mesothelin. In some embodiments, the sdAbs provided herein bind to human MSLN. Several isotypes of human MSLN are listed in Table 3 below (see UniProtKB-Q13421 (MSLN_HUMAN)). Isotype 2 of SEQ ID NO: 264 is the major human MSLN isotype.

[0204] Table 3

[0205]

[0206]

[0207] The anti-MSLN antibodies (e.g., sdAbs) provided herein can bind to any one of the isotypes listed in the table above or any fragment thereof. In some embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 263 or a fragment thereof. In some embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 264 or a fragment thereof. In other embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 265 or a fragment thereof. In other embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 266 or a fragment thereof. In some embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 283. In some embodiments, the anti-MSLN antibodies provided herein bind to SEQ ID NO: 284.

[0208] Thus, in one aspect, the present invention provides single domain antibodies (sdAbs) that bind to mesothelin (MSLN) and comprise the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from Tables 4 and 10 below.

[0209] More specifically, the present disclosure provides single domain antibodies (sdAbs) that bind to mesothelin (MSLN) and have the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: (i) CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, and SEQ ID NO:120;(ii) The CDR2 has an amino acid sequence selected from the group consisting of: SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118 and SEQ ID NO:121; and / or (iii) The CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:119 and SEQ ID NO:122.;

[0210] In some embodiments, provided herein is a single-domain antibody that binds to mesothelin (anti-MSLN sdAb) and that comprises a VH that comprises CDR1, CDR2, and CDR3 of any one of the antibodies anti-MSLN-1, anti-MSLN-2, anti-MSLN-3, anti-MSLN-4, anti-MSLN-5, anti-MSLN-6, anti-MSLN-7, anti-MSLN-8, anti-MSLN-9, anti-MSLN-10, anti-MSLN-11, anti-MSLN-12, anti-MSLN-13, anti-MSLN-14, anti-MSLN-15, anti-MSLN-16, anti-MSLN-17, anti-MSLN-18, anti-MSLN-19, anti-MSLN-20, anti-MSLN-21, anti-MSLN-22, anti-MSLN-23, anti-MSLN-24, anti-MSLN-25, anti-MSLN-26, anti-MSLN-27, anti-MSLN-28, anti-MSLN-29, anti-MSLN-30, anti-MSLN-31, anti-MSLN-32, anti-MSLN-33, and anti-MSLN-34 set forth in Table 9.

[0211] In some embodiments, CDR1 has SEQ ID NO:12, CDR2 has SEQ ID NO:13, and CDR3 has SEQ ID NO:14.

[0212] In some embodiments, CDR1 has SEQ ID NO:15, CDR2 has SEQ ID NO:16, and CDR3 has SEQ ID NO:17.

[0213] In some embodiments, CDR1 has SEQ ID NO:18, CDR2 has SEQ ID NO:19, and CDR3 has SEQ ID NO:20.

[0214] In some embodiments, CDR1 has SEQ ID NO:21, CDR2 has SEQ ID NO:22, and CDR3 has SEQ ID NO:23.

[0215] In some embodiments, CDR1 has SEQ ID NO:24, CDR2 has SEQ ID NO:25, and CDR3 has SEQ ID NO:26.

[0216] In some embodiments, CDR1 has SEQ ID NO:27, CDR2 has SEQ ID NO:28, and CDR3 has SEQ ID NO:29.

[0217] In some embodiments, CDR1 has SEQ ID NO:30, CDR2 has SEQ ID NO:31, and CDR3 has SEQ ID NO:32.

[0218] In some embodiments, CDR1 has SEQ ID NO:33, CDR2 has SEQ ID NO:34, and CDR3 has SEQ ID NO:35.

[0219] In some embodiments, CDR1 has SEQ ID NO:36, CDR2 has SEQ ID NO:37, and CDR3 has SEQ ID NO:38.

[0220] In other embodiments, CDR1 has SEQ ID NO:39, CDR2 has SEQ ID NO:40, and CDR3 has SEQ ID NO:41.

[0221] In other embodiments, CDR1 has SEQ ID NO:42, CDR2 has SEQ ID NO:43, and CDR3 has SEQ ID NO:44.

[0222] In other embodiments, CDR1 has SEQ ID NO:45, CDR2 has SEQ ID NO:46, and CDR3 has SEQ ID NO:47.

[0223] In other embodiments, CDR1 has SEQ ID NO:48, CDR2 has SEQ ID NO:49, and CDR3 has SEQ ID NO:50.

[0224] In other embodiments, CDR1 has SEQ ID NO:51, CDR2 has SEQ ID NO:52, and CDR3 has SEQ ID NO:53.

[0225] In other embodiments, CDR1 has SEQ ID NO:54, CDR2 has SEQ ID NO:55, and CDR3 has SEQ ID NO:56.

[0226] In other embodiments, CDR1 has SEQ ID NO:57, CDR2 has SEQ ID NO:58, and CDR3 has SEQ ID NO:59.

[0227] In still other embodiments, CDR1 has SEQ ID NO:60, CDR2 has SEQ ID NO:61, and CDR3 has SEQ ID NO:62.

[0228] In other additional embodiments, CDR1 has SEQ ID NO:63, CDR2 has SEQ ID NO:64, and CDR3 has SEQ ID NO:65.

[0229] In other additional embodiments, CDR1 has SEQ ID NO:75, CDR2 has SEQ ID NO:76, and CDR3 has SEQ ID NO:77.

[0230] In other additional embodiments, CDR1 has SEQ ID NO:78, CDR2 has SEQ ID NO:79, and CDR3 has SEQ ID NO:80.

[0231] In other additional embodiments, CDR1 has SEQ ID NO:81, CDR2 has SEQ ID NO:82, and CDR3 has SEQ ID NO:83.

[0232] In other additional embodiments, CDR1 has SEQ ID NO:84, CDR2 has SEQ ID NO:85, and CDR3 has SEQ ID NO:86.

[0233] In other additional embodiments, CDR1 has SEQ ID NO:87, CDR2 has SEQ ID NO:88, and CDR3 has SEQ ID NO:89.

[0234] In other additional embodiments, CDR1 has SEQ ID NO:90, CDR2 has SEQ ID NO:91, and CDR3 has SEQ ID NO:92.

[0235] In other additional embodiments, CDR1 has SEQ ID NO:93, CDR2 has SEQ ID NO:94, and CDR3 has SEQ ID NO:95.

[0236] In other additional embodiments, CDR1 has SEQ ID NO:96, CDR2 has SEQ ID NO:97, and CDR3 has SEQ ID NO:98.

[0237] In other additional embodiments, CDR1 has SEQ ID NO:99, CDR2 has SEQ ID NO:100, and CDR3 has SEQ ID NO:101.

[0238] In still other embodiments, CDR1 has SEQ ID NO:102, CDR2 has SEQ ID NO:103, and CDR3 has SEQ ID NO:104.

[0239] In still other embodiments, CDR1 has SEQ ID NO:105, CDR2 has SEQ ID NO:106, and CDR3 has SEQ ID NO:107.

[0240] In still other embodiments, CDR1 has SEQ ID NO:108, CDR2 has SEQ ID NO:109, and CDR3 has SEQ ID NO:110.

[0241] In still other embodiments, CDR1 has SEQ ID NO:111, CDR2 has SEQ ID NO:112, and CDR3 has SEQ ID NO:113.

[0242] In still other embodiments, CDR1 has SEQ ID NO:114, CDR2 has SEQ ID NO:115, and CDR3 has SEQ ID NO:116.

[0243] In still other embodiments, CDR1 has SEQ ID NO:117, CDR2 has SEQ ID NO:118, and CDR3 has SEQ ID NO:119.

[0244] In still other embodiments, CDR1 has SEQ ID NO:120, CDR2 has SEQ ID NO:121, and CDR3 has SEQ ID NO:122.

[0245] Table 4

[0246]

[0247]

[0248]

[0249]

[0250] In some embodiments, provided herein is a single-domain antibody that binds to mesothelin (anti-MSLN sdAb) and that comprises one or more CDR regions from any one of the antibodies anti-MSLN-1, anti-MSLN-2, anti-MSLN-3, anti-MSLN-4, anti-MSLN-5, anti-MSLN-6, anti-MSLN-7, anti-MSLN-8, anti-MSLN-9, anti-MSLN-10, anti-MSLN-11, anti-MSLN-12, anti-MSLN-13, anti-MSLN-14, anti-MSLN-15, anti-MSLN-16, anti-MSLN-17, anti-MSLN-18, anti-MSLN-19, anti-MSLN-20, anti-MSLN-21, anti-MSLN-22, anti-MSLN-23, anti-MSLN-24, anti-MSLN-25, anti-MSLN-26, anti-MSLN-27, anti-MSLN-28, anti-MSLN-29, anti-MSLN-30, anti-MSLN-31, anti-MSLN-32, anti-MSLN-33, and anti-MSLN-34 as set forth in Table 9.

[0251] In some embodiments, the anti-MSLN sdAb provided herein has one or more CDR regions from anti-MSLN-1.

[0252] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 123. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 123. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 123. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 123. CDR sequences can be determined according to well-known numbering systems. As described above, CDR regions are well-known to those skilled in the art and have been defined by well-known numbering systems. Residues from each of these hypervariable regions or CDRs are labeled in Table 1 above. In some embodiments, the CDR is numbered according to Kabat. In some embodiments, the CDR is numbered according to AbM. In other embodiments, the CDR is numbered according to Chothia. In other embodiments, the CDR is numbered according to Contact. In some embodiments, the CDR is numbered according to IMGT.

[0253] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 12. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 13. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 14. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 12 and a CDR2 of SEQ ID NO: 13. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 12 and a CDR3 of SEQ ID NO: 14. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 13 and a CDR3 of SEQ ID NO: 14. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 12, a CDR2 of SEQ ID NO: 13, and a CDR3 of SEQ ID NO: 14.

[0254] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-2.

[0255] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 124. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 124. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 124. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 124. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 124. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 124. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 124. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0256] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 15. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 16. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 17. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 15 and a CDR2 of SEQ ID NO: 16. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 15 and a CDR3 of SEQ ID NO: 17. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 16 and a CDR3 of SEQ ID NO: 17. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 15, a CDR2 of SEQ ID NO: 16, and a CDR3 of SEQ ID NO: 17.

[0257] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-3.

[0258] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 125. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 125. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 125. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 125. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0259] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 18. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 19. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 20. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 18 and a CDR2 of SEQ ID NO: 19. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 18 and a CDR3 of SEQ ID NO: 20. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19, and a CDR3 of SEQ ID NO: 20.

[0260] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-4.

[0261] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 126. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 126. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 126. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 126. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0262] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 21. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 22. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 23. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 21 and a CDR2 of SEQ ID NO: 22. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 21 and a CDR3 of SEQ ID NO: 23. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 22 and a CDR3 of SEQ ID NO: 23. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 21, a CDR2 of SEQ ID NO: 22, and a CDR3 of SEQ ID NO: 23.

[0263] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-5.

[0264] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 127. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 127. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 127. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 127. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 127. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 127. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 127. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0265] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 24. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 25. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 26. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 24 and a CDR2 of SEQ ID NO: 25. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 24 and a CDR3 of SEQ ID NO: 26. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 25 and a CDR3 of SEQ ID NO: 26. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 24, a CDR2 of SEQ ID NO: 25, and a CDR3 of SEQ ID NO: 26.

[0266] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-6.

[0267] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:128. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:128. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:128. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:128. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:128. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:128. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:128. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0268] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:27. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:28. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:29. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:27 and a CDR2 of SEQ ID NO:28. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:27 and a CDR3 of SEQ ID NO:29. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:28 and a CDR3 of SEQ ID NO:29. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:27, a CDR2 of SEQ ID NO:28, and a CDR3 of SEQ ID NO:29.

[0269] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-7.

[0270] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 129. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 129. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 129. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 129. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 129. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 129. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 129. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0271] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 30. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 31. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 32. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 30 and a CDR2 of SEQ ID NO: 31. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 30 and a CDR3 of SEQ ID NO: 32. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 31 and a CDR3 of SEQ ID NO: 32. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 30, a CDR2 of SEQ ID NO: 31, and a CDR3 of SEQ ID NO: 32.

[0272] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-8.

[0273] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 130. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 130. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 130. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 130. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0274] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 33. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 34. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 35. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 33 and a CDR2 of SEQ ID NO: 34. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 33 and a CDR3 of SEQ ID NO: 35. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 34 and a CDR3 of SEQ ID NO: 35. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 33, a CDR2 of SEQ ID NO: 34, and a CDR3 of SEQ ID NO: 35.

[0275] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-9.

[0276] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 131. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 131. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 131. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 131. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 131. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 131. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 131. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0277] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 36. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 37. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 38. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 36 and a CDR2 of SEQ ID NO: 37. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 36 and a CDR3 of SEQ ID NO: 38. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 37 and a CDR3 of SEQ ID NO: 38. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38.

[0278] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-10.

[0279] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO:132. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 as set forth in SEQ ID NO:132. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 as set forth in SEQ ID NO:132. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as set forth in SEQ ID NO:132. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as set forth in SEQ ID NO:132. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as set forth in SEQ ID NO:132. In some embodiments, the sdAb has a CDR1, a CDR2 and a CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:132. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0280] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:39. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:40. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:41. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:39 and a CDR2 of SEQ ID NO:40. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:39 and a CDR3 of SEQ ID NO:41. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:40 and a CDR3 of SEQ ID NO:41. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:39, a CDR2 of SEQ ID NO:40 and a CDR3 of SEQ ID NO:41.

[0281] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-11.

[0282] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO:133. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 as set forth in SEQ ID NO:133. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 as set forth in SEQ ID NO:133. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as set forth in SEQ ID NO:133. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as set forth in SEQ ID NO:133. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as set forth in SEQ ID NO:133. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO:133. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0283] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:42. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:43. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:44. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:42 and a CDR2 of SEQ ID NO:43. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:42 and a CDR3 of SEQ ID NO:44. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:43 and a CDR3 of SEQ ID NO:44. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:42, a CDR2 of SEQ ID NO:43, and a CDR3 of SEQ ID NO:44.

[0284] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-12.

[0285] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 134. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 134. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 134. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 134. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 134. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 134. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 134. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0286] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 45. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 46. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 47. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 45 and a CDR2 of SEQ ID NO: 46. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 45 and a CDR3 of SEQ ID NO: 47. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 46 and a CDR3 of SEQ ID NO: 47. In specific embodiments, the sdAb has a CDR1 of SEQ ID NO: 45, a CDR2 of SEQ ID NO: 46, and a CDR3 of SEQ ID NO: 47.

[0287] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-13.

[0288] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:135. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:135. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:135. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:135. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0289] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:48. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:49. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:50. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:48 and a CDR2 of SEQ ID NO:49. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:48 and a CDR3 of SEQ ID NO:50. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:49 and a CDR3 of SEQ ID NO:50. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:48, a CDR2 of SEQ ID NO:49, and a CDR3 of SEQ ID NO:50.

[0290] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-14.

[0291] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 136. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 136. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 136. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 136. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 136. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 136. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 136. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0292] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 51. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 52. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 53. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 51 and a CDR2 of SEQ ID NO: 52. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 51 and a CDR3 of SEQ ID NO: 53. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 52 and a CDR3 of SEQ ID NO: 53. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 51, a CDR2 of SEQ ID NO: 52, and a CDR3 of SEQ ID NO: 53.

[0293] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-15.

[0294] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:137. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:137. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:137. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:137. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:137. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:137. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:137. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0295] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:54. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:55. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:56. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:54 and a CDR2 of SEQ ID NO:55. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:54 and a CDR3 of SEQ ID NO:56. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:55 and a CDR3 of SEQ ID NO:56. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:54, a CDR2 of SEQ ID NO:55, and a CDR3 of SEQ ID NO:56.

[0296] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-16.

[0297] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO:138. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 as set forth in SEQ ID NO:138. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 as set forth in SEQ ID NO:138. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as set forth in SEQ ID NO:138. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as set forth in SEQ ID NO:138. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as set forth in SEQ ID NO:138. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO:138. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0298] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:57. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:58. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:59. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:57 and a CDR2 of SEQ ID NO:58. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:57 and a CDR3 of SEQ ID NO:59. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:58 and a CDR3 of SEQ ID NO:59. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:57, a CDR2 of SEQ ID NO:58, and a CDR3 of SEQ ID NO:59.

[0299] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-17.

[0300] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO: 139. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 as set forth in SEQ ID NO: 139. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 as set forth in SEQ ID NO: 139. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as set forth in SEQ ID NO: 139. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as set forth in SEQ ID NO: 139. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as set forth in SEQ ID NO: 139. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 139. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0301] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 60. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 61. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 62. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 60 and a CDR2 of SEQ ID NO: 61. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 60 and a CDR3 of SEQ ID NO: 62. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 61 and a CDR3 of SEQ ID NO: 62. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61, and a CDR3 of SEQ ID NO: 62.

[0302] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-18.

[0303] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 140. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 140. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 140. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 140. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0304] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 63. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 64. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 65. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 63 and a CDR2 of SEQ ID NO: 64. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 63 and a CDR3 of SEQ ID NO: 65. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 64 and a CDR3 of SEQ ID NO: 65. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQ ID NO: 65.

[0305] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-19.

[0306] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 141. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 141. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 141. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 141. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 141. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 141. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 141. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0307] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 75. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 76. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 77. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 75 and a CDR2 of SEQ ID NO: 76. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 75 and a CDR3 of SEQ ID NO: 77. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 76 and a CDR3 of SEQ ID NO: 77. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 75, a CDR2 of SEQ ID NO: 76, and a CDR3 of SEQ ID NO: 77.

[0308] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-20.

[0309] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 142. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 142. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 142. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 142. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 142. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 142. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 142. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0310] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 78. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 79. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 80. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 78 and a CDR2 of SEQ ID NO: 79. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 78 and a CDR3 of SEQ ID NO: 80. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 79 and a CDR3 of SEQ ID NO: 80. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 78, a CDR2 of SEQ ID NO: 79, and a CDR3 of SEQ ID NO: 80.

[0311] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-21.

[0312] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:143. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:143. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:143. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:143. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:143. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:143. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:143. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0313] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:81. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:82. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:83. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:81 and a CDR2 of SEQ ID NO:82. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:81 and a CDR3 of SEQ ID NO:83. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:82 and a CDR3 of SEQ ID NO:83. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:81, a CDR2 of SEQ ID NO:82, and a CDR3 of SEQ ID NO:83.

[0314] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-22.

[0315] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 144. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 144. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 144. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 144. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0316] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 84. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 85. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 86. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 84 and a CDR2 of SEQ ID NO: 85. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 84 and a CDR3 of SEQ ID NO: 86. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 85 and a CDR3 of SEQ ID NO: 86. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 84, a CDR2 of SEQ ID NO: 85, and a CDR3 of SEQ ID NO: 86.

[0317] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-23.

[0318] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:145. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:145. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:145. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:145. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:145. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:145. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:145. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0319] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:87. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:88. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:89. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:87 and a CDR2 of SEQ ID NO:88. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:87 and a CDR3 of SEQ ID NO:89. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:88 and a CDR3 of SEQ ID NO:89. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:87, a CDR2 of SEQ ID NO:88, and a CDR3 of SEQ ID NO:89.

[0320] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-24.

[0321] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 146. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 146. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 146. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 146. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0322] In some embodiments, the sdAb has the CDR1 of SEQ ID NO: 90. In some embodiments, the sdAb has the CDR2 of SEQ ID NO: 91. In other embodiments, the sdAb has the CDR3 of SEQ ID NO: 92. In some embodiments, the sdAb has the CDR1 of SEQ ID NO: 90 and the CDR2 of SEQ ID NO: 91. In some embodiments, the sdAb has the CDR1 of SEQ ID NO: 90 and the CDR3 of SEQ ID NO: 92. In some embodiments, the sdAb has the CDR2 of SEQ ID NO: 91 and the CDR3 of SEQ ID NO: 92. In a specific embodiment, the sdAb has the CDR1 of SEQ ID NO: 90, the CDR2 of SEQ ID NO: 91, and the CDR3 of SEQ ID NO: 92.

[0323] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-25.

[0324] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 147. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 147. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 147. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 147. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0325] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 93. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 94. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 95. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 93 and a CDR2 of SEQ ID NO: 94. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 93 and a CDR3 of SEQ ID NO: 95. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 94 and a CDR3 of SEQ ID NO: 95. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 93, a CDR2 of SEQ ID NO: 94, and a CDR3 of SEQ ID NO: 95.

[0326] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-26.

[0327] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 148. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 148. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 148. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 148. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 148. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 148. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 148. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0328] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 96. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 97. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 98. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 96 and a CDR2 of SEQ ID NO: 97. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 96 and a CDR3 of SEQ ID NO: 98. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 97 and a CDR3 of SEQ ID NO: 98. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 96, a CDR2 of SEQ ID NO: 97, and a CDR3 of SEQ ID NO: 98.

[0329] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-27.

[0330] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:149. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:149. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:149. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:149. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:149. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:149. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:149. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0331] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:99. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:100. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:101. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:99 and a CDR2 of SEQ ID NO:100. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:99 and a CDR3 of SEQ ID NO:101. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:100 and a CDR3 of SEQ ID NO:101. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:99, a CDR2 of SEQ ID NO:100, and a CDR3 of SEQ ID NO:101.

[0332] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-28.

[0333] In some embodiments, the sdAb has a CDR1 having an amino acid sequence of CDR1 as set forth in SEQ ID NO:150. In some embodiments, the sdAb has a CDR2 having an amino acid sequence of CDR2 as set forth in SEQ ID NO:150. In other embodiments, the sdAb has a CDR3 having an amino acid sequence of CDR3 as set forth in SEQ ID NO:150. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of CDR1 and CDR2 as set forth in SEQ ID NO:150. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of CDR1 and CDR3 as set forth in SEQ ID NO:150. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of CDR2 and CDR3 as set forth in SEQ ID NO:150. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO:150. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0334] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:102. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:103. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:104. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:102 and a CDR2 of SEQ ID NO:103. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:102 and a CDR3 of SEQ ID NO:104. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:103 and a CDR3 of SEQ ID NO:104. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:102, a CDR2 of SEQ ID NO:103, and a CDR3 of SEQ ID NO:104.

[0335] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-29.

[0336] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:151. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:151. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:151. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:151. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:151. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:151. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:151. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0337] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:105. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:106. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:107. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:105 and a CDR2 of SEQ ID NO:106. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:105 and a CDR3 of SEQ ID NO:107. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:106 and a CDR3 of SEQ ID NO:107. In one specific embodiment, the sdAb has a CDR1 of SEQ ID NO:105, a CDR2 of SEQ ID NO:106, and a CDR3 of SEQ ID NO:107.

[0338] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-30.

[0339] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 152. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 152. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 152. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 152. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 152. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 152. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 152. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0340] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 108. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 109. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 110. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 108 and a CDR2 of SEQ ID NO: 109. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 108 and a CDR3 of SEQ ID NO: 110. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 109 and a CDR3 of SEQ ID NO: 110. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 108, a CDR2 of SEQ ID NO: 109, and a CDR3 of SEQ ID NO: 110.

[0341] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-31.

[0342] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 153. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 153. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 153. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 153. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 153. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 153. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 153. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0343] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 111. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 112. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 113. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 111 and a CDR2 of SEQ ID NO: 112. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 111 and a CDR3 of SEQ ID NO: 113. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 112 and a CDR3 of SEQ ID NO: 113. In one specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113.

[0344] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-32.

[0345] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:154. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:154. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:154. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:154. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:154. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:154. In some embodiments, the sdAb has a CDR1, a CDR2 and a CDR3 having the amino acid sequences of CDR1, CDR2 and CDR3 set forth in SEQ ID NO:154. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0346] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:114. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:115. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:116. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:114 and a CDR2 of SEQ ID NO:115. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:114 and a CDR3 of SEQ ID NO:116. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:115 and a CDR3 of SEQ ID NO:116. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:114, a CDR2 of SEQ ID NO:115 and a CDR3 of SEQ ID NO:116.

[0347] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-33.

[0348] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 155. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 155. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 155. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 155. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 155. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 155. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 155. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0349] In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 117. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 118. In other embodiments, the sdAb has a CDR3 of SEQ ID NO: 119. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 117 and a CDR2 of SEQ ID NO: 118. In some embodiments, the sdAb has a CDR1 of SEQ ID NO: 117 and a CDR3 of SEQ ID NO: 119. In some embodiments, the sdAb has a CDR2 of SEQ ID NO: 118 and a CDR3 of SEQ ID NO: 119. In one specific embodiment, the sdAb has a CDR1 of SEQ ID NO: 117, a CDR2 of SEQ ID NO: 118, and a CDR3 of SEQ ID NO: 119.

[0350] In some embodiments, the anti-MSLN sdAbs provided herein have one or more CDR regions from anti-MSLN-34.

[0351] In some embodiments, the sdAb has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO:156. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO:156. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO:156. In some embodiments, the sdAb has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO:156. In some embodiments, the sdAb has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO:156. In some embodiments, the sdAb has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO:156. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO:156. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT.

[0352] In some embodiments, the sdAb has a CDR1 of SEQ ID NO:120. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:121. In other embodiments, the sdAb has a CDR3 of SEQ ID NO:122. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:120 and a CDR2 of SEQ ID NO:121. In some embodiments, the sdAb has a CDR1 of SEQ ID NO:120 and a CDR3 of SEQ ID NO:122. In some embodiments, the sdAb has a CDR2 of SEQ ID NO:121 and a CDR3 of SEQ ID NO:122. In a specific embodiment, the sdAb has a CDR1 of SEQ ID NO:120, a CDR2 of SEQ ID NO:121, and a CDR3 of SEQ ID NO:122.

[0353] In some embodiments of the various sdAbs of MSLN provided herein, the sdAb further comprises one or more FR sequences of the following antibodies: anti-MSLN-1, anti-MSLN-2, anti-MSLN-3, anti-MSLN-4, anti-MSLN-5, anti-MSLN-6, anti-MSLN-7, anti-MSLN-8, anti-MSLN-9, anti-MSLN-10, anti-MSLN-11, anti-MSLN-12, anti-MSLN-13, anti-MSLN-14, anti-MSLN-15, anti-MSLN-16, anti-MSLN-17, anti-MSLN-18, anti-MSLN-19, anti-MSLN-20, anti-MSLN-21, anti-MSLN-22, anti-MSLN-23, anti-MSLN-24, anti-MSLN-25, anti-MSLN-26, anti-MSLN-27, anti-MSLN-28, anti-MSLN-29, anti-MSLN-30, anti-MSLN-31, anti-MSLN-32, anti-MSLN-33, and anti-MSLN-34, as set forth in Table 9.

[0354] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 123. In some embodiments, the sdAb provided herein comprises an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 123. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 123.

[0355] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:124. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:124. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:124.

[0356] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1 and FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1 and FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1 and FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR2 and FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR2 and FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR3 and FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR1, FR3 and FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 125. In some embodiments, the sdAb provided herein comprises FR2, FR3 and FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 125. In a specific embodiment, the sdAb provided herein comprises FR1, FR2, FR3 and FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 125.

[0357] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 126. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 126. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 126.

[0358] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:127. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:127. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:127.

[0359] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1 and FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1 and FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1 and FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR2 and FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR2 and FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR3 and FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1, FR2, and FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1, FR2, and FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR1, FR3, and FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 128. In some embodiments, the sdAb provided herein comprises FR2, FR3, and FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 128. In a specific embodiment, the sdAb provided herein comprises FR1, FR2, FR3, and FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 128.

[0360] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:129. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:129. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:129.

[0361] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 130. In some embodiments, the sdAb provided herein comprises an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 130. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 130.

[0362] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 as set forth in SEQ ID NO:131. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 as set forth in SEQ ID NO:131. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 as set forth in SEQ ID NO:131.

[0363] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 132. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 132. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 132.

[0364] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:133. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:133. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:133.

[0365] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:134. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:134. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:134.

[0366] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:135. In some embodiments, the sdAb provided herein comprises an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:135. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:135.

[0367] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:136. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:136. In one specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:136.

[0368] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:137. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:137. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:137.

[0369] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 138. In some embodiments, the sdAb provided herein comprises an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 138. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 138.

[0370] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1 and FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1 and FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1 and FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR2 and FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR2 and FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR3 and FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR1, FR3 and FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 139. In some embodiments, the sdAb provided herein comprises FR2, FR3 and FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 139. In a specific embodiment, the sdAb provided herein comprises FR1, FR2, FR3 and FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 139.

[0371] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 140. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 140. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 140.

[0372] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 as set forth in SEQ ID NO: 141. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 as set forth in SEQ ID NO: 141. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 as set forth in SEQ ID NO: 141.

[0373] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:142. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:142. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:142.

[0374] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 143. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 143. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 143.

[0375] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 144. In some embodiments, the sdAb provided herein comprises an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 144. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 144.

[0376] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 as set forth in SEQ ID NO: 145. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 as set forth in SEQ ID NO: 145. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 as set forth in SEQ ID NO: 145.

[0377] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 146. In some embodiments, the sdAb provided herein comprises an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 146. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 146.

[0378] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 147. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 147. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 147.

[0379] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:148. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:148. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:148.

[0380] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO: 149. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO: 149. In one specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO: 149.

[0381] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1 and FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1 and FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1 and FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR2 and FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR2 and FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR3 and FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1, FR2 and FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR1, FR3 and FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:150. In some embodiments, the sdAb provided herein comprises FR2, FR3 and FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:150. In a specific embodiment, the sdAb provided herein comprises FR1, FR2, FR3 and FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:150.

[0382] In some embodiments, the sdAb provided herein comprises an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 151. In some embodiments, the sdAb provided herein comprises an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 151. In a specific embodiment, the sdAb provided herein comprises an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 151.

[0383] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:152. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:152. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:152.

[0384] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR3 having the amino acid sequences of FR1, FR2 and FR3 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2 and an FR4 having the amino acid sequences of FR1, FR2 and FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3 and an FR4 having the amino acid sequences of FR1, FR3 and FR4 set forth in SEQ ID NO:153. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3 and an FR4 having the amino acid sequences of FR2, FR3 and FR4 set forth in SEQ ID NO:153. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3 and an FR4 having the amino acid sequences of FR1, FR2, FR3 and FR4 set forth in SEQ ID NO:153.

[0385] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 154. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 154. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 154.

[0386] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO:155. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO:155. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO:155.

[0387] In some embodiments, the sdAbs provided herein comprise an FR1 having the amino acid sequence of FR1 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR2 having the amino acid sequence of FR2 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR3 having the amino acid sequence of FR3 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR4 having the amino acid sequence of FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR2 having the amino acid sequences of FR1 and FR2 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR3 having the amino acid sequences of FR1 and FR3 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1 and an FR4 having the amino acid sequences of FR1 and FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR3 having the amino acid sequences of FR2 and FR3 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR2 and an FR4 having the amino acid sequences of FR2 and FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR3 and an FR4 having the amino acid sequences of FR3 and FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR3 having the amino acid sequences of FR1, FR2, and FR3 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1, an FR2, and an FR4 having the amino acid sequences of FR1, FR2, and FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR1, an FR3, and an FR4 having the amino acid sequences of FR1, FR3, and FR4 set forth in SEQ ID NO: 156. In some embodiments, the sdAbs provided herein comprise an FR2, an FR3, and an FR4 having the amino acid sequences of FR2, FR3, and FR4 set forth in SEQ ID NO: 156. In a specific embodiment, the sdAbs provided herein comprise an FR1, an FR2, an FR3, and an FR4 having the amino acid sequences of FR1, FR2, FR3, and FR4 set forth in SEQ ID NO: 156.

[0388] In some embodiments, the sdAbs provided herein are humanized sdAbs that include one or more humanized FR sequences in Table 11. A more detailed description of the humanized sdAbs provided herein is provided below.

[0389] The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, for example, Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues in the variable region that surround the CDRs in an N-terminal to C-terminal fashion: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues from the N-terminus to the CDR1 amino acid residues, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the amino acid residues from the C-terminus to the CDR3 amino acid residues, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0390] In certain more specific embodiments, the sdAb includes the VH (or VHH) sequence set forth in Table 9 in the following examples.

[0391] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 123.

[0392] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 124.

[0393] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 125.

[0394] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 126.

[0395] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 127.

[0396] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 128.

[0397] In some embodiments, the sdAb includes the amino acid sequence of SEQ ID NO: 129.

[0398] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 130.

[0399] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 131.

[0400] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 132.

[0401] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 133.

[0402] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 134.

[0403] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 135.

[0404] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 136.

[0405] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 137.

[0406] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 138.

[0407] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 139.

[0408] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 140.

[0409] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 150.

[0410] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 151.

[0411] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 152.

[0412] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 153.

[0413] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 154.

[0414] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 155.

[0415] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 156.

[0416] In some embodiments, the sdAb binds to MSLN with a K between 0.01 - 100 nM. D In some embodiments, the sdAb binds to MSLN with a K between 0.1 - 100 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 100 nM. D In other embodiments, the sdAb binds to MSLN with a K between 1 - 90 nM. D In other embodiments, the sdAb binds to MSLN with a K between 1 - 80 nM. D In other embodiments, the sdAb binds to MSLN with a K between 1 - 70 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 60 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 50 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 40 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 30 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 20 nM. D In some embodiments, the sdAb binds to MSLN with a K between 1 - 10 nM. D In some embodiments, the sdAb binds to MSLN with a K between 0.1 - 1 nM. D In some embodiments, the sdAb binds to MSLN with a K between 0.01 - 1 nM. D In some embodiments, the sdAb binds to MSLN with a K between 0.01 - 0.1 nM. D binds to MSLN.

[0417] In some embodiments, a binding molecule that binds to a cancer antigen comprises two antibodies or antigen-binding fragments thereof that each bind to an antigen expressed on a cancer cell. In some embodiments, the cancer cell is a solid tumor cancer cell. In some embodiments, the two antibodies or antigen-binding fragments thereof are two sdAbs. In some embodiments, the two sdAbs are V HH single-domain antibody. In some embodiments, the two sdAbs each bind to two different epitopes of MSLN. In some embodiments, one epitope is on the N-terminus of MSLN and the other epitope is on the C-terminus of MSLN.

[0418] In some embodiments, each of the two sdAbs has a K between 0.01 - 100 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 0.1 - 100 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 1 - 100 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 1 - 90 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 1 - 80 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 1 - 70 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 1 - 60 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 1 - 50 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 1 - 40 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 1 - 30 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 1 - 20 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 1 - 10 nM D and binds to MSLN. In other embodiments, each of the two sdAbs has a K between 0.1 - 1 nM D and binds to MSLN. In some embodiments, each of the two sdAbs has a K between 0.01 - 0.1 nM D and binds to MSLN.

[0419] In other embodiments, the two antibodies or their antigen-binding fragments are linked by a third linker.

[0420] In yet another aspect, the present disclosure provides antibodies that compete with one of the single domain antibodies that bind to MSLN described above. Such antibodies may also bind to the same epitope, or an overlapping epitope, as one of the single domain antibodies mentioned above. Antibodies and fragments that compete with or bind to the same epitope as the antibodies described above are expected to exhibit similar functional properties. Exemplary antigen-binding proteins and fragments include antigen-binding proteins and fragments having the VH regions and CDRs described herein, including the antigen-binding proteins and fragments in Tables 5 and 7.

[0421] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an amino acid sequence having a specific percent identity relative to any of the following antibodies: anti-MSLN-1, anti-MSLN-2, anti-MSLN-3, anti-MSLN-4, anti-MSLN-5, anti-MSLN-6, anti-MSLN-7, anti-MSLN-8, anti-MSLN-9, anti-MSLN-10, anti-MSLN-11, anti-MSLN-12, anti-MSLN-13, anti-MSLN-14, anti-MSLN-15, anti-MSLN-16, anti-MSLN-17, anti-MSLN-18, anti-MSLN-19, anti-MSLN-20, anti-MSLN-21, anti-MSLN-22, anti-MSLN-23, anti-MSLN-24, anti-MSLN-25, anti-MSLN-26, anti-MSLN-27, anti-MSLN-28, anti-MSLN-29, anti-MSLN-30, anti-MSLN-31, anti-MSLN-32, anti-MSLN-33, and anti-MSLN-34, as set forth in Table 9.

[0422] The determination of "percent identity" between two sequences, such as amino acid sequences or nucleic acid sequences, can be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used for iterative searches that detect distant relationships between molecules (id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information, NCBI, on the world wide web at ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0423] The percent identity between two sequences can be determined using techniques similar to those described above, whether or not gaps are permitted. In computing the percent identity, only exact matches are usually counted.

[0424] In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a VH domain having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 123, wherein the antibody immunospecifically binds to MSLN. In certain embodiments, the antibody or antigen-binding fragment thereof described herein comprises a VH domain having at least 80%, at...

Claims

1. An anti-mesothelin (anti-MSLN) antibody, the antibody comprising an anti-MSLN heavy chain variable region (VH), the anti-MSLN heavy chain variable region comprising: 1) HC-CDR1 having the amino acid sequence shown in SEQ ID NO:27, HC-CDR2 having the amino acid sequence shown in SEQ ID NO:28, and HC-CDR3 having the amino acid sequence shown in SEQ ID NO:29; or 2) HC-CDR1 having the amino acid sequence shown in SEQ ID NO:36, HC-CDR2 having the amino acid sequence shown in SEQ ID NO:37, and HC-CDR3 having the amino acid sequence shown in SEQ ID NO:

38.

2. The anti-MSLN antibody according to claim 1, wherein the anti-MSLN heavy chain variable region (VH) has the sequence of any one of SEQ ID NO:128 and SEQ ID NO:291-295.

3. The anti-MSLN antibody according to claim 1, wherein the antibody is a single domain (sdAb) antibody.

4. The anti-MSLN antibody according to claim 1, wherein the antibody comprises a VHH domain, the VHH domain comprising the amino acid sequence of any one of SEQ ID NO:128 and SEQ ID NO:291-295.

5. The anti-MSLN antibody according to claim 3, wherein the sdAb antibody that binds to the mesothelin is camel, human, partially humanized or fully humanized.

6. A polynucleotide encoding the anti-MSLN antibody according to any one of claims 1 to 5.

7. A kit, comprising: a) The anti-MSLN antibody according to any one of claims 1 to 5, or the polynucleotide according to claim 6, and b) Instructions.

8. A pharmaceutical composition, comprising the anti-MSLN antibody according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

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