Anti-MSLN antibodies and methods of use

Abstract

The present disclosure relates to antibodies and antibody derivatives that bind to MSLN and methods of using same, in certain embodiments, the antibodies or antibody derivatives disclosed herein comprise single domain antibodies that bind to MSLN.

Description

[Technical field]

[0001] The present disclosure relates to antibodies and antibody derivatives that bind to MSLN and methods of their use. [Background technology]

[0002] Mesothelin (MSLN) is a 40 kDa glycosylphosphatidylinositol-anchored cell surface protein expressed in mesothelial cells. Although the exact function of MSLN is unclear, it is well known as a tumor differentiation antigen and has been found to be overexpressed in many types of tumors, such as epithelial mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, cholangiocarcinoma, and certain squamous cell carcinomas. Previous studies have shown that aberrant MSLN expression in tumor cells promoted tumor cell proliferation and invasion by activating key cell signaling pathways, such as the NFκB, MAPK, and PI3K pathways. Clinical studies have also shown that increased MSLN expression levels are associated with increased tumor burden and poor prognosis. Therefore, there is a need in the art for the development of therapeutic molecules and methods that target MSLN for cancer treatment. Summary of the Invention

[0003] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to MSLN with high affinity, including monospecific anti-MSLN antibodies and multispecific antibodies that bind to MSLN and one or more other targets. In certain embodiments, the antibodies or antibody derivatives disclosed herein include single domain antibodies that bind to MSLN. The present disclosure further provides methods of making and using the antibodies and antibody derivatives disclosed herein, as well as pharmaceutical compositions comprising them, for example, to treat diseases and disorders, such as cancer. The present invention is based, in part, on the discovery of novel single domain antibodies that bind to MSLN, which can target tumor cells and / or increase the immune response against tumor cells, thereby resulting in improved anti-tumor effects.

[0004] The present disclosure provides antibodies that bind to MSLN, including single domain antibodies that bind to MSLN. In certain embodiments, the single domain antibodies are at least 1×10 -7 In certain embodiments, the single domain antibody binds to MSLN with a KD of 5×10 -8 In certain embodiments, the single domain antibody binds to MSLN with a KD of 1×10 -8 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -10 M ~ approx. 5×10 -8 It binds to MSLN with a KD of M. In certain embodiments, the single domain antibody comprises a VHH. In certain embodiments, the single domain antibody or VHH comprises a heavy chain variable region (VH).

[0005] In certain embodiments, the single domain antibody comprises a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18, e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42;

[0006] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31 or 36, or a variant thereof comprising up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32 or 37, or a variant thereof comprising up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33 or 38, or a variant thereof comprising up to about three amino acid substitutions.

[0007] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain are comprised in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, and 39.

[0008] In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:38.

[0009] In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, and 39. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the single domain antibody comprises a human antibody.

[0010] In certain embodiments, the antibody comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L; S239D, A330L, and I332E; or L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L. In certain embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L.

[0011] In certain embodiments, the heavy chain variable region is linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 78. In certain embodiments, the antibody comprises a full-length immunoglobulin, a single chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), (dsFv)2, a VHH, a VHH-Fc fusion, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, a tetrabody, or any combination thereof.

[0012] In certain embodiments, the antibody is comprised in a multispecific antibody, e.g., a bispecific antibody, in which the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In certain embodiments, the second antigen is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PDL1, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGFR), and / or EGFR-associated antigen (EGFR-associated antigen). P2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2,3,4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), B7H3, L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), tyrosine protein kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2, and any combination thereof. In certain embodiments, the second antigen is an immune checkpoint regulator. In certain embodiments, the immune checkpoint regulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof. In certain embodiments, the second antigen is an immune co-stimulatory molecule or a subunit of the T cell receptor / CD3 complex.In certain embodiments, the immune co-stimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, and CD40, and any combination thereof. In certain embodiments, the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof.

[0013] The present disclosure provides an immunoconjugate comprising any of the antibodies disclosed herein linked to a therapeutic agent or label. In certain embodiments, the therapeutic agent is a cytotoxin or a radioisotope. In certain embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0014] The present disclosure further provides an antigen recognition receptor comprising an extracellular antigen binding domain comprising an antibody disclosed herein. In certain embodiments, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a recombinant T cell receptor. In certain embodiments, the antigen recognition receptor is a CAR. In certain embodiments, the antibody comprised in the extracellular antigen binding domain comprises a VHH.

[0015] The present disclosure provides an immune response cell comprising the antigen recognition receptor disclosed herein. In certain embodiments, the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells. In certain embodiments, the immune response cell is a T cell. The present disclosure provides a pharmaceutical composition comprising: a) any of the antibodies disclosed herein, any of the immunoconjugates disclosed herein, or any of the immune response cells disclosed herein, and b) a pharma- ceutically acceptable carrier.

[0016] The disclosure further provides nucleic acids encoding any of the antibodies disclosed herein, vectors comprising any of the nucleic acids disclosed herein, and host cells comprising any of the nucleic acids or vectors disclosed herein.

[0017] The present disclosure provides methods of preparing the antibodies disclosed herein, comprising expressing the antibody in a host cell disclosed herein and isolating the antibody from the host cell. The present disclosure further provides a method of reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of an antibody disclosed herein, an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates the tumor in the subject. In certain embodiments, the tumor exhibits high microsatellite instability (MSI). In certain embodiments, the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, cholangiocarcinoma, head and neck cancer, hematological cancer, and combinations thereof.

[0018] The present disclosure provides a method of treating and / or preventing cancer or prolonging the survival of a subject with cancer. In certain embodiments, the method comprises administering to the subject an effective amount of an antibody disclosed herein, an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein. In certain embodiments, the cancer exhibits high microsatellite instability (MSI). In certain embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, cholangiocarcinoma, head and neck cancer, hematological cancer, and combinations thereof.

[0019] The present disclosure further provides any of the antibodies and / or pharmaceutical compositions disclosed herein for use as a medicament. The present disclosure further provides any of the antibodies and / or pharmaceutical compositions disclosed herein for use in treating cancer. In certain embodiments, the cancer exhibits high microsatellite instability (MSI). In certain embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumors, cholangiocarcinoma, head and neck cancer, hematological cancer, and combinations thereof.

[0020] The present disclosure provides kits comprising an antibody disclosed herein, an immunoconjugate disclosed herein, a pharmaceutical composition disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or an immune response cell disclosed herein, in certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm. [Brief description of the drawings]

[0021] [Figure 1A] Figure 1 shows the binding ability of representative anti-MSLN VHH clones to human MSLN (1A) and cynomolgus MSLN (1B) as assessed by ELISA. A reference anti-MSLN antibody, Ab237 analog, was used as a positive control. An anti-PD1 antibody, h1G4, was used as a negative control. [Figure 1B] Figure 1 shows the binding ability of representative anti-MSLN VHH clones to human MSLN (1A) and cynomolgus MSLN (1B) as assessed by ELISA. A reference anti-MSLN antibody, Ab237 analog, was used as a positive control. An anti-PD1 antibody, h1G4, was used as a negative control. [Diagram 2] Figure 1 shows the binding activity of anti-MSLN VHH-Fc to N87 cells assessed by FACS. The Ab237 analog was used as a positive control. [Diagram 3]FIG. 1 shows the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-MSLN VHH-Fc antibodies measured by percent cell lysis using NK92-CD16 cells as effector cells and N87 cells as target cells. [Figure 4] Figure 1 shows the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-MSLN VHH-Fc antibodies measured by percent cell lysis using human PBMCs as effector cells and N87 cells as target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to MSLN with high affinity, including monospecific anti-MSLN antibodies and multispecific antibodies that bind to MSLN and one or more other targets. In certain embodiments, the antibodies or antibody derivatives disclosed herein include single domain antibodies that bind to MSLN. The present disclosure further provides methods of making and using the antibodies and antibody derivatives disclosed herein, as well as pharmaceutical compositions comprising them, for example, to treat diseases and disorders, such as cancer. The present invention is based, in part, on the discovery of novel single domain antibodies that bind to MSLN, which can target tumor cells and / or increase the immune response against tumor cells, thereby resulting in improved anti-tumor effects.

[0023] For purposes of clarity, and not of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections: 1. Definition, 2. Antibodies and antibody derivatives, 3. How to use; 4. Pharmaceutical preparations, and 5. Products.

[0024] 1. Definition

[0025] As mentioned herein, the term "antibody" includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). An "antibody" can be a separate molecule or part of an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multispecific antibodies (e.g., bispecific antibodies), antigen-recognizing receptors (e.g., chimeric antigen receptors), antibody complexes that contain additional proteinaceous or non-proteinaceous moieties (e.g., antibody-drug conjugates or polymer-coated antibodies), and other multifunctional molecules that include antibodies.

[0026] "Full-length antibody," "intact antibody," and "whole antibody" refer to an antibody having a heavy chain that resembles a native antibody structure or contains an Fc region as defined herein. In certain embodiments, a full-length antibody comprises two heavy chains and two light chains. In certain embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions of both chains generally contain three highly variable loops, referred to as complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein can be defined or identified by well-known conventions, such as those of Kabat, Chothia, MacCallum, IMGT, and AHo, as described below. The three CDRs of a heavy or light chain are inserted between flanking stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, the full-length antibody is glycosylated. In certain embodiments, a full-length antibody comprises a glycan linked to its Fc region, hi certain embodiments, a full-length antibody comprises a branched glycan.

[0027] As used herein, the terms "antigen-binding portion," "antibody fragment," and "antibody portion" refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single domain antibodies, VHH, VHH-Fc, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment of an antibody that binds to an antigen or combinations thereof. "VHH" refers to a single domain antibody isolated from a camelid. In certain embodiments, the VHH comprises the variable region of the heavy chain of a camelid heavy chain antibody. In certain embodiments, the VHH has a size not exceeding about 25 kDa. In certain embodiments, the VHH has a size not exceeding about 20 kDa. In certain embodiments, the VHH has a size not exceeding about 15 kDa.

[0028] An "antibody that cross-competes for binding" with a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody that blocks the antibody from binding to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0029] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment is composed of a dimer of one heavy and one light chain variable region in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops in each of the heavy and light chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind to an antigen, although in some cases with a lower affinity than the entire binding site.

[0030] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv consisting of a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment that contains an antibody domain. H and V L It further comprises a polypeptide linker between the domains. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0031] For purposes herein, an "acceptor human framework" or "human framework" is a framework that comprises the amino acid sequence of a light chain variable region (VL) framework or a heavy chain variable region (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can contain amino acid sequence changes. In certain embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In certain embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0032] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by methods well known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0033] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more CDRs or hypervariable regions (HVRs), compared to a parent antibody that does not possess such alterations, which alterations result in an improvement in the affinity of the antibody for antigen. The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In certain embodiments, a chimeric antibody disclosed herein comprises a murine heavy chain variable region and a human Fc region. In certain embodiments, a chimeric antibody disclosed herein comprises a camelid heavy chain variable region and a human Fc region.

[0034] As used herein, the term "CDR" or "complementarity determining region" is intended to mean non-contiguous antigen-binding sites within the variable regions of the heavy and / or light chains. These specific regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc The CDRs are described by MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and when compared with each other, the definitions include overlapping or subsets of amino acid residues. Nevertheless, it is intended to be within the scope of the term as defined and used herein to apply any one of the definitions to refer to the CDRs of an antibody or grafted antibody, or variants thereof. The amino acid residues encompassing the CDRs as defined in each of the above cited references are set forth below in Table 1 for comparison. CDR prediction algorithms and interfaces are well known in the art and include, for example, Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The contents of the references cited in this section are incorporated herein by reference in their entirety for use in this application and as may be included in one or more claims herein.

[0035] Table 1: CDR definition

[0036] [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al., supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra. 3 Residue numbering follows the nomenclature of MacCallum et al., supra. 4 Residue numbering follows the nomenclature of Lefranc et al., supra. 5 Residue numbering follows the nomenclature of Honegger and Pluckthun et al., supra.

[0037] The phrases "Kabat variable region residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of or insertion into the FRs or CDRs of the variable domain. For example, a heavy chain variable region may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence.

[0038] In certain embodiments, the amino acid residues encompassing the CDRs of a single domain antibody are defined according to the IMGT nomenclature in Lefranc et al., supra. In certain embodiments, the amino acid residues encompassing the CDRs of a full-length antibody are defined according to the Kabat nomenclature in Kabat et al., supra. In certain embodiments, the numbering of residues in an immunoglobulin heavy chain, e.g., in an Fc region, is the EU index numbering as in Kabat et al., supra. "EU index as in Kabat" refers to the residue numbering of a human IgG1 EU antibody.

[0039] "Framework" or "FR" refers to those variable domain residues other than the CDR residues as herein defined.

[0040] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs / HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises at least one, and typically two, variable domains, in which all or substantially all of the HVRs / CDRs correspond to the HVRs / CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0041] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or an antibody made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques well known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to immunized transgenic animals, e.g., xenomouse, that have been modified to produce such antibodies in response to antigen challenge, but that are deficient at the endogenous locus (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE® technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), e.g., regarding human antibodies generated by human B cell hybridoma technology.

[0042] "Percent amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percent amino acid residues in a candidate sequence that are identical to the amino acid residues in the compared polypeptide after alignment of the sequences, taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0043] "Homology" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed by aligning the two sequences to obtain maximum homology.

[0044] The "light chains" of antibodies (e.g., immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0045] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin that contains the antigen binding site, i.e., the variable domain. The constant domain contains the CH1, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CL domain of the light chain. In certain embodiments, the "CH1 domain" (also called "C1" for "H1" domain) extends from about amino acid 118 to about amino acid 215 (EU numbering system). In certain embodiments, the "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain S-S bonds in the same positions. In certain embodiments, the "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in intact native IgG molecules. It has been speculated that the carbohydrates may provide a substitute for domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985). In certain embodiments, the "CH3 domain" (also referred to as the "C2" domain) comprises residues between the CH2 domain and the C-terminus of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 for IgG).

[0046] The term "Fc region" or "fragment crystallizable region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions, or dimers thereof. In certain embodiments, a human IgG Fc region extends from Cys226 to its carboxyl terminus. In certain embodiments, a human IgG Fc region extends from Pro231 to its carboxyl terminus. In certain embodiments, a human IgG Fc region comprises a CH2 domain and a CH3 domain. In certain embodiments, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by genetically modifying the nucleic acid encoding the heavy chain of the antibody. In certain embodiments, a composition of intact antibodies may include an antibody population having all K447 residues removed, an antibody population in which the K447 residue has not been removed, or a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4 Fc regions. "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, and FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including FcRs identified in the future, are encompassed by the term "FcR" herein.

[0047] As used herein, the term "epitope" refers to a particular group of atoms or amino acids on an antigen to which an antibody or antibody derivative binds. Two antibodies or antigen-binding portions can bind to the same epitope in an antigen if they exhibit competitive binding to the antigen.

[0048] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody molecule, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which can be an epitope) is an antibody or antibody portion that binds to this target with higher affinity, higher affinity, higher rapidity, and / or longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≦10 -6 M, ≦10 -7 M, ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, ≦10 -11 M or ≦10 -12 Dissociation constant of M (K D ). In some embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but need not necessarily include, exclusive binding. The binding specificity of an antibody or antigen binding domain can be determined experimentally by methods well known in the art. Such methods include Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE test, and the like. TM These include, but are not limited to, peptide scans, and peptide scans.

[0049] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In certain embodiments, an isolated polypeptide is free or substantially free from association with all other components from its production environment.

[0050] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. In certain embodiments, an isolated nucleic acid is free or substantially free from association with all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide and antibody described herein is in a form other than the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid encoding a polypeptide and antibody described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. The term "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0051] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

[0052] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that act as autonomously replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0053] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid, including the primary subject cell and its progeny.

[0054] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0055] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the subject is a human. An "effective amount" of an agent refers to an amount effective to achieve a desired therapeutic or prophylactic result, at the dosage and for the period of time required. The specific dosage may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system by which it is delivered. A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present application may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the substance / molecule, agonist, or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0056] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or mitigating the progression of the disease, improving the condition, providing remission (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving or improving the quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also includes reduction in the pathological consequences of cancer (e.g., tumor volume, etc.). The methods of the present application contemplate any one or more of these aspects of treatment. "Treatment" does not necessarily mean that the condition being treated is cured.

[0057] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0058] As used herein, the term "about" or "approximately" means that a particular value determined by one of ordinary skill in the art is within an acceptable error range, which depends in part on how the value is measured or determined, i.e., limited by the measurement system. In certain embodiments, "about" can mean within 3 standard deviations, or more than 3 standard deviations, according to the practice of the art. In certain embodiments, "about" can mean within a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. In certain embodiments, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold, or within 2-fold.

[0059] As used herein, the term "modulation" means to alter in a positive or negative direction. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0060] As used herein, the term "increase" means a change in a positive direction of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0061] As used herein, the term "reduce" means to alter in a negative direction by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0062] As used herein, the term "about X to Y" has the same meaning as "about X to about Y." As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0063] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0064] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0065] The term "pharmaceutical formulation" refers to a formulation that is in a form such that the biological activity of the active ingredients contained therein is effective, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation may be administered.

[0066] As used herein, a "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0067] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. In certain embodiments, the heavy and light chain variable domains of a natural antibody (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 61 ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated from an antibody that binds to that antigen using the VH or VL domain to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0068] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immune response cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen-recognizing receptors include natural and modified T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0069] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule that comprises an extracellular antigen-binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an antibody or an antibody fragment, such as a VHH or scFv. In certain embodiments, an antibody (e.g., a VHH or scFv) is fused to a transmembrane domain, which is fused to an intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for an antigen. By "immune response cell" is meant a cell that functions in the immune response, or a precursor or progeny thereof.

[0070] As used herein, "MSLN," "MSLN protein," or "MSLN polypeptide" refers to any MSLN polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), or any fragment thereof, and can optionally include up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, additions, and / or deletions. The term encompasses full-length unprocessed MSLN, as well as any form of MSLN that results from processing in a cell. The term also encompasses naturally occurring variants of MSLN, such as splice variants or allelic variants. In certain embodiments, the MSLN polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a sequence having NCBI reference number: NP_001170826.1, NP_005814.2, or NP_037536.2 (homology herein can be determined using standard software such as BLAST or FASTA). In certain embodiments, the MSLN polypeptide comprises or has an amino acid sequence that is the entire or continuous portion of SEQ ID NO: 41, 42, or 43.

[0071] The term "ECD of MSLN" refers to the extracellular domain of MSLN. In certain embodiments, the ECD of an exemplary MSLN polypeptide can include the amino acid sequence set forth in SEQ ID NO:43.

[0072] The terms "anti-MSLN antibody" and "antibody that binds to MSLN" refer to an antibody that is capable of binding to MSLN with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent for targeting MSLN. In one embodiment, the extent of binding of an anti-MSLN antibody to an unrelated non-MSLN protein is less than about 10% of the binding of the antibody to MSLN, e.g., as measured by a BIACORE® surface plasmon resonance assay. In certain embodiments, an antibody that binds to MSLN has an affinity of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -12 M, for example, 10 -9 M~10 -10 MSLN has a dissociation constant (KD) of 100 M. In certain embodiments, the anti-MSLN antibody binds to an epitope of MSLN that is conserved among MSLNs from different species. In certain embodiments, the anti-MSLN antibody binds to an epitope on MSLN that is within the ECD of the protein.

[0073] 2. Antibodies and Antibody Derivatives

[0074] The present disclosure provides isolated monoclonal antibodies and antibody derivatives, including monospecific anti-MSLN antibodies and multispecific antibodies that bind to MSLN and one or more other targets. In certain embodiments, the antibodies or antibody derivatives disclosed herein include single domain antibodies that bind to MSLN. In certain embodiments, the present disclosure is based in part on the discovery of single domain antibodies that bind to MSLN, which can be used in anti-tumor therapy, where the antibodies can selectively target tumor cells and / or inhibit signal pathways mediated by MSLN, thereby inducing beneficial anti-tumor effects on tumor cells. In certain embodiments, the single domain antibodies disclosed herein are antagonistic antibodies, which inhibit MSLN function. In certain embodiments, the single domain antibodies can enhance anti-tumor immune responses against tumor cells expressing MSLN protein. In certain embodiments, the single domain antibodies include camelid antibodies or VHH antibodies. In certain embodiments, the single domain antibody has improved tissue infiltration capability due to its small size compared to conventional antibodies in the form of IgG, Fab, and / or scFv. In certain embodiments, the anti-MSLN antibody exhibits anti-tumor effects in subjects. In certain embodiments, the anti-MSLN antibody exhibits superior anti-tumor effects compared to reference antibodies, such as Ab237 analogs. Ab237 is an anti-MSLN fab antibody disclosed in International Publication No. WO2014004549A2.

[0075] In certain embodiments, the antibodies of the present disclosure can be or include monoclonal antibodies, including chimeric, humanized, or human antibodies. In certain embodiments, the antibodies disclosed herein include human antibodies. In certain embodiments, the antibodies include an acceptor human framework, such as a human immunoglobulin framework, or a human consensus framework.

[0076] In certain embodiments, an antibody of the present disclosure can be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In certain embodiments, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, or other antibody class or isotype as defined herein. In certain embodiments, an antibody or antibody derivative of the present disclosure can incorporate any of the features described herein, e.g., as detailed in Sections 2.1-2.12 herein, either alone or in combination. The antibodies and antibody derivatives of the present disclosure are useful, for example, for the diagnosis or treatment of neoplasms or cancers. In certain embodiments, neoplasms and cancers whose growth can be inhibited using the antibodies of the present disclosure include neoplasms and cancers that normally respond to immunotherapy. In certain embodiments, neoplasms and cancers include breast cancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other examples of cancers treatable using the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharyngeal cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer, These include gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, breast cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, breast cancer, pelvic cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, including those induced by asbestos, e.g., mesothelioma, and combinations of the above cancers.

[0077] 2.1 Exemplary Anti-MSLN Antibodies

[0078] The present disclosure provides an isolated antibody that binds to MSLN protein.In certain embodiments, the anti-MSLN antibody of the present disclosure binds to the ECD of MSLN.In certain embodiments, the ECD comprises the amino acid sequence shown in SEQ ID NO: 43.In certain embodiments, the anti-MSLN antibody binds to the same epitope as the anti-MSLN antibody described herein. In certain embodiments, the anti-MSLN antibodies disclosed herein can function as antagonists of MSLN-based signaling pathways. In certain embodiments, the anti-MSLN antibodies can block or reduce signaling pathways that depend on MSLN protein. In certain embodiments, the anti-MSLN antibodies can reduce the activity of the signaling pathway by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%. In certain embodiments, treatment with the anti-MSLN antibodies shows an anti-tumor effect in the subject, thereby reducing tumor growth and / or prolonging the survival of the subject. In certain embodiments, the anti-MSLN antibodies increase the immune response and / or anti-tumor effect of immune cells, such as T cells and / or NK cells, against tumor cells expressing MSLN. In certain embodiments, anti-MSLN antibodies, including single domain antibodies (e.g., VHHs), have a smaller molecular size compared to full-length antibodies due to the smaller size of single domain antibodies compared to the Fab domain of full-length antibodies, which can result in superior tissue penetration, for example at tumor sites, compared to full-length antibodies. In certain embodiments, treatment with anti-MSLN antibodies shows superior anti-tumor effects compared to treatment with full-length anti-MSLN antibodies.

[0079] In certain embodiments, the anti-MSLN antibody comprises a single domain antibody that binds to MSLN. In certain embodiments, the single domain antibody comprises a VHH. In certain embodiments, the single domain antibody comprises a heavy chain variable region (VH). In certain embodiments, the single domain antibody is linked to an Fc region. In certain embodiments, the single domain antibody is not linked to an Fc region.

[0080] In certain embodiments, the single domain antibody is about 1×10 -7 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -8 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 5×10 -9 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -9 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -10 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -11 M ~ approx. 1×10 -7 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -10 M ~ approx. 1×10 -7 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -10 M ~ approx. 1×10 -8 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -11 M ~ approx. 1×10 -9 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 2×10 -10 M ~ approx. 5×10 -9 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -9 M ~ approx. 5×10 -8 In certain embodiments, the single domain antibody binds to MSLN with a KD of about 1×10 -10 M ~ approx. 1×10 -9 Binds to MSLN at the KD of M.

[0081] In certain embodiments, the single domain antibody comprises a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18, e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42;

[0082] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31 or 36, or a variant thereof comprising up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32 or 37, or a variant thereof comprising up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33 or 38, or a variant thereof comprising up to about three amino acid substitutions.

[0083] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain are comprised in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, and 39.

[0084] In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:31, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:38.

[0085] In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34 and 39. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34 and 39. In certain embodiments, a single domain antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34 and 39.

[0086] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:19. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:24. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:29. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:34. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:39. In certain embodiments, the single domain antibody comprises a human antibody. In certain embodiments, any one of the amino acid sequences contained in the heavy chain variable region can contain up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, deletions, and / or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.

[0087] In certain embodiments, the single domain antibody comprises a human framework. In certain embodiments, the human framework comprises the framework sequence of the heavy chain variable region sequence shown in SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, or 39. In certain embodiments, the anti-MSLN antibody does not comprise an Fc region. In certain embodiments, the anti-MSLN antibody further comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises one or more mutations that modify antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E.

[0088] In certain embodiments, a single domain antibody comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35 and 40. In certain embodiments, a single domain antibody comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35 and 40. In certain embodiments, a single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35 and 40.

[0089] In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:10. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:15. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:20. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:25. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:30. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:35. In certain embodiments, the anti-MSLN antibody comprises the amino acid sequence set forth in SEQ ID NO:40.

[0090] In certain embodiments, the heavy chain variable region is linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises about 4 to about 15 amino acids. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 78. In certain embodiments, the anti-MSLN antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, a VHH-Fv fusion, a diabody, a tribody, a tetrabody, or any combination thereof.

[0091] In certain embodiments, the antibody is included in a larger molecule that is an antibody derivative. In certain embodiments, the antibody derivative is a multispecific antibody, e.g., a bispecific antibody, in which the multispecific antibody comprises a second antibody portion that specifically binds to a second antigen. In certain embodiments, the second antigen is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is Her-2, EGFR, PD-L1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EGF), or a combination thereof. protein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), tyrosine protein kinase transmembrane receptor type 1 (ROR1), PVR, PVRL2, and any combination thereof. In certain embodiments, the second antigen is an immune checkpoint regulator. In certain embodiments, the immune checkpoint regulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.In certain embodiments, the binding of the antibody derivative or multispecific antibody to the second antigen inhibits an immune checkpoint regulator. In certain embodiments, the second antigen is an immune costimulatory molecule or a subunit of the T cell receptor / CD3 complex. In certain embodiments, the immune costimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, CD40, and any combination thereof. In certain embodiments, the binding of the antibody derivative or multispecific antibody to the second antigen activates the immune costimulatory molecule. In certain embodiments, the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof. In certain embodiments, the binding of the antibody derivative or multispecific antibody to the second antigen activates the T cell receptor / CD3 complex.

[0092] In certain embodiments, the anti-MSLN antibody is linked to the second antigen-binding moiety via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises about 4 to about 15 amino acids. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-78. In certain embodiments, the anti-MSLN antibody is conjugated to a therapeutic agent or label, hi certain embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0093] 2.2 Antibody affinity

[0094] In certain embodiments, the antibodies or antibody derivatives disclosed herein have high binding affinity for their target antigens. In certain embodiments, the antibodies or antibody derivatives have a binding affinity of about 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 5×10-9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 It binds to the target with a KD of less than or equal to M.

[0095] In certain embodiments, the antibody or antibody derivative is about 1×10 -11 M ~ approx. 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -11 M ~ approx. 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 2×10 -10 M ~ approx. 5×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 M ~ approx. 5×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -9 It binds to the target with a KD of M.

[0096] The KD of an antibody or antibody derivative may be determined by methods well known in the art, including, but not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, Octet-BIACORE® test, and peptide scan.

[0097] In certain embodiments, KD can be measured using a BIACORE® surface plasmon resonance assay. For example, but not limited to, an assay using a BIACORE®-2000 or BIACORE® 3000 (Biacore, Inc., Piscataway, NJ) is performed at 25° C. with immobilized antigen CMS chips immobilized at about 10 response units (RU). In certain embodiments, a carboxymethylated dextran biosensor chip (CMS, Biacore, Inc.) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / mL (about 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μL / min to obtain about 10 response units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (KD) can be calculated as the ratio of koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate by the surface plasmon resonance assay is 10 6 M -l s -1If the on-rate exceeds , the on-rate may be determined by a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations, measured with a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette.

[0098] 2.3 Antibody fragments

[0099] In certain embodiments, the antibodies of the present disclosure include antigen-binding or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, VHH, Fv, and scFv fragments, as well as other fragments described herein. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthin in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-31 5 (1994), and also WO 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab)2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0100] In certain embodiments, the antibodies of the present disclosure can be diabodies. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01 161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are further described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0101] In certain embodiments, an antibody of the present disclosure may comprise a single domain antibody. A single domain antibody is an antibody fragment that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 Bl). In certain embodiments, a single domain antibody is a camelid single domain antibody. In certain embodiments, a single domain antibody is a VHH. In certain embodiments, a single domain antibody is humanized. In certain embodiments, a single domain antibody comprises or is a human antibody.

[0102] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0103] 2.4 Chimeric and humanized antibodies

[0104] In certain embodiments, the antibody of the present disclosure is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In certain embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In certain embodiments, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from those of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0105] In certain embodiments, the antibody of the present disclosure can be a humanized antibody. Generally, humanizing a non-human antibody reduces its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and one or more frameworks (FRs) (or any portion thereof) are derived from a human antibody sequence. A humanized antibody can optionally also comprise at least a portion of a human constant region. In certain embodiments, certain FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. Humanized antibodies and methods for making them are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing dSDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0106] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0107] 2.5 Human antibodies

[0108] In certain embodiments, the antibodies of the present disclosure can be human antibodies (e.g., human domain antibodies, or human DAbs). Human antibodies can be produced using a variety of techniques well known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are well known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0109] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, e.g., XENOMOUSE, J. Immunol. 2004, 143:1117-1125 (2005). TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, describing HuMab® technology, U.S. Patent No. 5,770,429, describing HuMab® technology, U.S. Patent No. 7,041,870, describing KM MOUSE® technology, and U.S. Patent Publication No. US2007 / 0061900, describing VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with a different human constant region.

[0110] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0111] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0112] 2.6 Library-derived antibodies

[0113] Antibodies of the present disclosure can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, various methods are well known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single domain antibody libraries are described, for example, in U.S. Patent No. 7,371,849.

[0114] In certain phage display methods, V H and V LThe repertoires of genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in phage libraries, and then screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol, 12:433-455 (1994). Phages typically display antibody fragments, either as scFv fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide single-source antibodies to a broad range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene fragments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and perform rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0115] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0116] 2.7 Antibody variants The present disclosure further provides amino acid sequence variants of the disclosed antibodies. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, but are not limited to, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final antibody, i.e., the modified antibody, has the desired properties, e.g., antigen binding.

[0117] 2.7.1 Substitution, Insertion, and Deletion Mutants

[0118] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the subject antibody, and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0119] Table 2. Amino acid substitutions

[0120] [Table 2]

[0121] Amino acids can be grouped according to 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 that affect chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe. In certain embodiments, non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0122] In certain embodiments, one type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a certain biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have a certain biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR (or CDR) residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0123] Modifications (e.g., substitutions) can be made in the HVRs (or CDRs), for example, to improve antibody affinity. Such modifications can be made in HVR (or CDR) "hot spots", i.e., residues encoded by codons that undergo high frequency of mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting from secondary libraries is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In certain embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity includes HVR (or CDR)-directed approaches, in which several HVR (or CDR) residues (e.g., 4-6 residues at a time) are randomized. HVR (or CDR) residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0124] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs (or CDRs) so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in an HVR (or CDR). Such modifications may be in HVR (or CDR) "hot spots" or outside of the CDRs. In certain embodiments of the above-described variant VH sequences, each HVR (or CDR) is either unaltered or has no more than one, two, or three amino acid substitutions.

[0125] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The mutants may be screened to determine whether they have the desired properties.

[0126] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0127] 2.7.2 Glycosylation variants

[0128] In certain embodiments, the antibody is altered to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed. If the antibody comprises an Fc region (e.g., scFv-Fc), the carbohydrate linked thereto can be altered. Natural antibodies produced by mammalian cells typically have the carbohydrate of the Fc region linked thereto by an N-linkage. H The biantennary oligosaccharides comprise a branched, biantennary oligosaccharide that is generally linked to Asn297 of the 2 domain. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In certain embodiments, modifications of the oligosaccharides in the antibody can be made to generate antibody variants with certain improved properties.

[0129] In certain embodiments, the antibody has a carbohydrate structure that lacks fucose linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) linked to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region, however, Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294-300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004), and Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1; Presta, L, and WO 2004 / 056312 A1; Adams et al.), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0130] In certain embodiments, the antibody has a bisected oligosaccharide, e.g., a biantennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0131] 2.7.3 Fc Region Variants

[0132] In certain embodiments, the Fc region of the presently disclosed antibodies or antibody derivatives can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid modification (e.g., a substitution) at one or more amino acid positions. In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody moiety (e.g., an scFv-Fc or a VHH-Fc), thereby generating an Fc region variant.

[0133] In certain embodiments, the Fc region possesses some, but not all, effector functions, making it a desirable candidate for applications in which the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI® Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods well known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0134] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0135] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In certain embodiments, the Fc region comprises one or more mutations according to EU numbering of residues. In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In certain embodiments, the Fc region is an IgG2 or IgG4 Fc region. In certain embodiments, the Fc region is an IgG4 Fc region comprising an F234A mutation and / or an L235A mutation.

[0136] In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises one or more mutations that modify antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L. In certain embodiments, the IgG1 Fc region comprises substitutions at Fc region positions 298, 333, and / or 334. In certain embodiments, the IgG1 Fc region comprises the following mutations: S267E and L328F.

[0137] In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the IgG4 Fc region comprises an S228P mutation.

[0138] In certain embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0139] In certain embodiments, the antibody (e.g., scFv-Fc or VHH-Fc) variants comprise variant Fc regions that contain one or more amino acid substitutions that alter half-life and / or modify binding to the fetal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to the fetal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise Fc regions with one or more substitutions that modify binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the Fc region residues, for example, a substitution at Fc region residue 434 (US Pat. No. 7,371,826).

[0140] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 relating to other examples of Fc region variants.

[0141] 2.7.4 Cysteine ​​Engineered Antibody Variants

[0142] In certain embodiments, it is desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties, e.g., drug moieties, or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibody moieties may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0143] 2.8 Antibody derivatives In certain embodiments, the antibodies described herein may be further modified into antibody derivatives, including other proteins or non-protein moieties that are well known and readily available in the art. Suitable non-protein moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for diagnosis under limited conditions, etc. In certain embodiments, the antibody may be further modified into an antibody derivative that includes one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biological activity" or "biologically active" means to exhibit biological activity to perform a specific function in the body. For example, it can mean to bind to a specific biomolecule, such as, for example, a protein, DNA, and then promote or inhibit the activity of such a biomolecule. In certain embodiments, biologically active proteins, or fragments thereof, include proteins and polypeptides administered to a patient as an active drug substance for the prevention or treatment of a disease or condition, and proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and polypeptides administered to a patient to prevent a disease, such as a vaccine.

[0144] 2.9 Production Method

[0145] The antibodies and antibody derivatives disclosed herein can be produced using any technique available or known in the art. For example, but not limited to, the antibodies and antibody derivatives can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. Detailed procedures for producing the antibodies and antibody derivatives are described in the Examples below.

[0146] The presently disclosed subject matter further provides an isolated nucleic acid encoding an antibody or antibody derivative disclosed herein. For example, the isolated nucleic acid can encode an amino acid sequence comprising the VL of the antibody, and / or an amino acid sequence comprising the VH of the antibody, e.g., the light chain and / or the heavy chain of the antibody.

[0147] In certain embodiments, the nucleic acid can be present in one or more vectors, e.g., expression vectors. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA segment can be ligated. Another type of vector is a viral vector, into which another DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are thereby replicated along with the host genome. Furthermore, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors of utility in recombinant DNA technology are often in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0148] The various portions of the antibody or antibody derivative disclosed herein can be assembled into a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Exemplary elements that generate polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES), as well as cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Also suitable is the combination of a retroviral vector with an appropriate packaging line, where the capsid protein is functional to infect human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902), and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, such as pseudotyped particles having a VSVG, RD114, or GALV envelope, and any others known in the art.

[0149] In certain embodiments, a nucleic acid encoding an antibody or antibody derivative of the present disclosure and / or one or more vectors comprising the nucleic acid can be introduced into a host cell. In certain embodiments, the introduction of the nucleic acid into the cell can be performed by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing virus or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, the host cell can include, for example, a host cell that has been transformed with a vector comprising a nucleic acid encoding a single domain antibody and / or an amino acid sequence comprising the VH of the single domain antibody. In certain embodiments, the host cell can include, for example, a host cell that has been transformed with (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In certain embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, YO, NSO, Sp20 cell).

[0150] In certain embodiments, methods of making an antibody or antibody derivative disclosed herein can include culturing a host cell into which nucleic acid encoding the antibody or antibody derivative has been introduced under conditions suitable for expression of the antibody or antibody derivative, and, optionally, recovering the antibody or antibody derivative from the host cell and / or host cell medium. In certain embodiments, the antibody or antibody derivative is recovered from the host cell by chromatographic techniques.

[0151] For recombinant production of the antibody or antibody derivative of the present disclosure, for example, nucleic acids encoding the antibody or antibody derivative described above can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody or antibody derivative). Suitable host cells for cloning or expressing vectors encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, antibodies or antibody derivatives can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody or antibody derivative can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0152] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies or antibody derivatives with partial or complete human glycosylation patterns. See Gemgross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:21 0-215 (2006). Suitable host cells for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. In certain embodiments, plant cell cultures can be utilized as host cells. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES® technology for producing antibodies in transgenic plants). In certain embodiments, vertebrate cells can also be used as hosts, for example, but not limited to, mammalian cell lines adapted to suspension growth may be useful. Non-limiting examples of useful mammalian host cells include monkey kidney CV1 line transformed with SY40 (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep 02), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC 5 cells, and FS4 cells as described in NYAcad.Sci.383:44-68 (1982). Other useful mammalian host cells include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad.Sci. USA 77:42 I6 (1980)), and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cells suitable for the production of antibodies or antibody derivatives, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0153] In certain embodiments, techniques for making bispecific and / or multispecific antibodies include, but are not limited to, recombinant expression of two immunoglobulin heavy-light chain pairs with the same specificity, where one or two of the heavy or light chains are fused to an antigen-binding moiety with different specificity (e.g., a single domain antibody, e.g., VHH), recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983)), PCT Patent Application No. WO 93 / 08829, and Traunecker et al., EMBO J 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Bispecific antibodies can also be engineered using electrostatic steering effects (WO 2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and see, e.g., Tutt et al. J Immunol. Antibodies can also be made by the preparation of trispecific antibodies as described in J. Immunol. 147:60 (1991).

[0154] Bispecific and multispecific molecules of the present disclosure can also be made using chemical techniques (see, e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" techniques (see, e.g., U.S. Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific and multispecific molecules of the presently disclosed subject matter can also be prepared by conjugating the binding specificities of the constituent moieties, e.g., the binding specificities of a first epitope and a second epitope, using methods well known in the art and described herein. For example, and without limitation, each binding specificity of the bispecific and multispecific molecules can be produced together by recombinant fusion protein technology or can be produced separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins.Mitt.(1985) No.78,1 18-132, Brennan(1985) Science 229:81-83), Glennie(1987) J Immunol.139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge region can be modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[0155] In certain embodiments, both binding specificities of a bispecific antibody can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are MAb x MAb, MAb x Fab, Fab x F(ab')2, or ligand x Fab fusion proteins. In certain embodiments, the bispecific antibodies of the present disclosure can be single chain molecules, such as single chain bispecific antibodies, single chain bispecific molecules comprising one single chain antibody and a binding determinant cluster, or single chain bispecific molecules comprising two binding determinant clusters. Bispecific and multispecific molecules can also be single chain molecules or can comprise at least two single chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858. Engineered antibodies having three or more functional antigen binding sites (e.g., epitope binding sites), including "octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576 A1).

[0156] In certain embodiments, an animal system can be used to produce the antibodies or antibody derivatives of the present disclosure. One animal system for preparing hybridomas is the murine system.

[0157] Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes are well known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York).

[0158] 2.10 Assay

[0159] The antibodies and antibody derivatives of the present disclosure provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays well known in the art and provided herein.

[0160] In certain embodiments, the antigen-binding activity of an antibody or antibody derivative of the present disclosure can be tested by well-known methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay. Each of these assays typically detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, an antibody or antibody derivative can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody or antibody derivative. Alternatively, an antibody or antibody derivative can be detected using any of a variety of other immunoassays. For example, an antibody or antibody derivative can be radioactively labeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). The radioactive isotope can be detected by such methods as the use of a Geiger counter or a scintillation counter or by autoradiography.

[0161] In certain embodiments, a competitive assay can be used to identify antibodies or antibody derivatives that compete with the antibodies of the present disclosure for binding to MSLN. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as the antibodies disclosed herein bind. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0162] In a non-limiting example of a competitive assay, immobilized MSLN can be incubated in a solution containing a first labeled antibody or antibody derivative that binds to MSLN and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to MSLN. The second antibody may be present in a hybridoma supernatant. As a control, immobilized MSLN is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to MSLN, excess unbound antibody is removed and the amount of label associated with immobilized MSLN is measured. If the amount of label associated with immobilized MSLN is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to MSLN. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0163] The present disclosure provides assays for identifying anti-MSLN antibodies, or antibody derivatives thereof, that have biological activity, such as activating immune cells or immune activation reporters, such as NFAT reporters or NF-κB reporters. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0164] 2.11 Immune complexes

[0165] The presently disclosed subject matter further provides immunoconjugates comprising an antibody or antibody derivative disclosed herein conjugated to one or more detection probes and / or a cytotoxic agent, e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioisotope. For example, an antibody or antigen-binding portion of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or otherwise) to one or more other binding molecules, e.g., another antibody, an antibody fragment, a peptide, or a binding mimetic.

[0166] In certain embodiments, the immunoconjugate comprises a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer 53:3336-3342 (1993); and Lode et al., Cancer 53:3336-3342 (1993). Res. 58:2925-2928 (1998)), anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Pat. No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and antibody-drug conjugates (ADCs) in which an antibody is conjugated to one or more drugs, including, but not limited to, CC1065. In certain embodiments, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Abrasiongum protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0167] In certain embodiments, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When a radioconjugate is used for detection, it can contain a radioactive atom for scintigraphy studies, such as tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0168] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-4 labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) can be used. The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0169] 2.12 Antigen Recognition Receptors

[0170] The presently disclosed subject matter further provides an antigen recognition receptor comprising the antibody or antibody fragment disclosed herein. An antigen recognition receptor is a receptor that can activate, stimulate, or inhibit an immune response cell (e.g., a T cell) in response to binding to an antigen. Non-limiting examples of antigen recognition receptors include natural and recombinant T cell receptors (TCRs), chimeric costimulatory receptors (CCRs), chimeric antigen receptors (CARs), or inhibitory CARs (iCARs). The design and use of antigen-recognizing receptors are well known in the art and have been described in the literature, for example, in International Publications WO 2018 / 027155, WO 2019 / 099483, WO 2019 / 157454, WO 2019 / 133969, WO 2019 / 099993, WO 2015 / 142314, WO 2018 / 027197, and WO 2014055668.

[0171] In certain embodiments, the presently disclosed subject matter provides a chimeric antigen receptor (CAR) comprising an antibody or antibody fragment disclosed herein. A CAR is an engineered receptor that can graft or confer a specificity of interest onto immune effector cells. In certain embodiments, a CAR can be used to graft the specificity of a monoclonal antibody onto a T cell, the transfer of the coding sequence of which is facilitated by a vector. In certain embodiments, the CAR is a "first generation" CAR, which is typically composed of an extracellular antigen binding domain (e.g., scFv, Fab, or VHH) fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular signaling domain. A "first generation" CAR provides de novo antigen recognition and can trigger activation of immune response cells, e.g., CD4+ and CD8+ T cells, via the signaling domain of the CD3z chain in a single fusion molecule, without relying on HLA-mediated antigen presentation. In certain embodiments, the CAR is a "second generation" CAR, which further comprises an intracellular signaling domain from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) to the cytoplasmic tail of the CAR to provide additional signals to immune response cells, thereby "second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3z). In certain embodiments, the CAR is a "third generation" CAR, which comprises multiple costimulatory domains (e.g., CD28 and 4-1BB) and activation (CD3z). In certain embodiments, the CAR is a second generation CAR. In certain embodiments, the CAR comprises an extracellular antigen binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, where the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the CAR further comprises a hinge / spacer region between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the extracellular antigen-binding domain comprises an antibody or antibody fragment disclosed herein. In certain embodiments, the antibody or antibody fragment comprises a VHH or an scFv.

[0172] In certain embodiments, the presently disclosed subject matter provides a recombinant TCR comprising an antibody or antibody fragment disclosed herein. A native TCR is a protein complex comprising a disulfide-linked heterodimeric protein composed of two variable chains expressed as part of a complex with a CD3 chain molecule. A native TCR is found on the surface of T cells and is responsible for the recognition of antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a native TCR comprises an α chain and a β chain (encoded by the TRA and TRB genes, respectively). In certain embodiments, a TCR comprises a γ chain and a δ chain (encoded by the TRG and TRD genes, respectively). Each of the α, β, γ, and δ chains comprises two extracellular domains: a variable (V) region and a constant (C) region. The constant region is near the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each variable region has three complementarity determining regions (CDRs). In certain embodiments, the TCR comprises a receptor complex with CD3δ, CD3γ, CD3ε, and CD3ζ. When the TCR complex binds to its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0173] In certain embodiments, the recombinant TCR is a non-naturally occurring TCR. In certain embodiments, the recombinant TCR comprises a recombinant alpha chain and / or a recombinant b chain, where a portion or the entire variable region of the recombinant alpha chain and / or the recombinant b chain is replaced by an antibody or antibody fragment disclosed herein. In certain embodiments, the antibody or antibody fragment comprises a VHH, a VH, a VL, or a scFv. In certain embodiments, the antibody or antibody fragment comprises a VHH. In certain embodiments, the recombinant TCR binds to an antigen of interest in an MHC / HLA independent manner. In certain non-limiting embodiments, antigen binding can activate an immune response cell comprising the recombinant TCR.

[0174] The presently disclosed subject matter provides immune response cells comprising an antigen recognition receptor (e.g., CAR or TCR) as disclosed herein. In certain embodiments, the antigen recognition receptor can activate the immune response cell. The immune response cells of the presently disclosed subject matter can be cells of lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune response cells of lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and multipotent stem cells (e.g., from which lymphoid cells can differentiate). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and gd T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introduction of an antigen recognition receptor, e.g., CAR or TCR. In certain embodiments, the immune response cells are T cells. The T cells may be CD4+ T cells or CD8+ T cells. In certain embodiments, the T cells are CD4+ T cells. In certain embodiments, the T cells are CD8+ T cells. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to carry out a cytotoxic effect on target cells.The types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, such as those described in Sadelain, M. et al., 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes, including a and b heterodimers), Panelli, MC et al., 2000 J Immunol 164:495-504, Panelli, MC et al., 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) of tumor biopsies), and Dupont, J. et al., 2005 Cancer Res. 65:5417-5427; Papanicolaou, GA et al., 2003 Blood 102:2498-2505 (disclosing selective in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells). In certain embodiments, immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0175] 3. How to use

[0176] The presently disclosed subject matter further provides methods of using the disclosed antibodies and antibody derivatives. In certain embodiments, the methods relate to therapeutic uses of the presently disclosed antibodies or antibody derivatives. In certain embodiments, the methods relate to diagnostic uses of the presently disclosed antibodies or antibody derivatives.

[0177] 3.1 Treatment method

[0178] The present disclosure provides methods and uses of the antibodies or antibody derivatives disclosed herein for treating diseases and disorders or for increasing immune responses. In certain embodiments, the antibodies, antibody derivatives, or pharmaceutical compositions comprising the same disclosed herein can be administered to a subject (e.g., a mammal, such as a human) to treat diseases and disorders or increase immune responses. In certain embodiments, the diseases and disorders involve immune checkpoint inhibition and / or abnormal MSLN activity. In certain embodiments, diseases and disorders treatable by the antibodies or antibody derivatives disclosed herein include, but are not limited to, neoplasms, such as cancer.

[0179] In certain embodiments, the present disclosure provides an antibody or antibody derivative (or a fragment thereof) described herein for use in the manufacture of a medicament. In certain embodiments, the present disclosure provides an antibody or antibody derivative (or a fragment thereof) described herein for use in the manufacture of a medicament for treating cancer. In certain embodiments, the present disclosure provides an antibody or antibody derivative (or a fragment thereof) described herein for use in the treatment of cancer in a subject. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody derivative (or a fragment thereof) provided herein for use in the treatment of cancer in a subject. In certain embodiments, the cancer can be a blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable carcinomas further include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and liver metastases thereof, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatoma, cholangiocarcinoma, synovium, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminodal carcinoma, pulmonary arterial ... The cancers may include any of the well-known carcinomas in the field of oncology, including tumors of the breast such as ductal and lobular adenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine and ovarian epithelial carcinoma, prostate cancer, transitional squamous cell carcinoma of the bladder, B and T cell lymphoma (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0180] In certain embodiments, the cancer can be melanoma, NSCLC, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0181] In certain embodiments, the subject to be treated is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the subject is a human. In certain embodiments, the subject is suspected of having or at risk of having cancer, or has been diagnosed with cancer or any other disease with aberrant MSLN expression or activity.

[0182] Many diagnostic methods for cancer or any other disease that shows abnormal MSLN activity and clinical description of these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, and fluorescent in situ hybridization (FISH). Further details regarding diagnostic methods for abnormal MSLN activity or expression are described, for example, in Gupta et al., (2009) Mod Pathol. 22(1): 128-133, Lopez-Rios et al., (2013) J Clin Pathol. 66(5): 381-385, Ellison et al., (2013) J Clin Pathol 66(2): 79-89, and Guha et al., (2013) PLoS ONE 8(6): e67782.

[0183] Administration can be by any suitable route, including, for example, intravenous, intramuscular, or subcutaneous. In some embodiments, an antibody or antibody derivative (or fragment thereof), and / or composition provided herein is administered in combination with a second, third, or fourth agent (e.g., an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder associated with aberrant MSLN activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, injectable fluorouracil, leucovorin, and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, Imprime PGG, and erlotinib. In some embodiments, the antibody or antibody derivative (or fragment thereof) is conjugated to another agent.

[0184] In certain embodiments, the antibodies or antibody derivatives (or fragments thereof) and / or compositions provided herein are administered in combination with one or more additional therapies, such as radiation therapy, surgery, chemotherapy, and / or targeted therapy. In certain embodiments, the antibodies, antibody derivatives (or fragments thereof) and / or compositions provided herein are administered in combination with radiation therapy. In certain embodiments, the combination of the antibodies, antibody derivatives (or fragments thereof) and / or compositions provided herein with radiation therapy is used to treat a neoplasm or cancer as disclosed herein.

[0185] Depending on the indication being treated and dosing factors well known to those of skill in the art, the antibodies or antibody derivatives provided herein are administered in a dose effective to treat the indication while minimizing toxicity and side effects. For the treatment of cancer, a typical dose can be in the range of, for example, 0.001 to 1000 μg, although doses below or above this exemplary range are within the scope of the invention. Daily dosages can be about 0.1 μg / kg to about 100 mg / kg total body weight, about 0.1 μg / kg to about 100 μg / kg total body weight, or about 1 μg / kg to about 100 μg / kg total body weight. As described above, therapeutic or prophylactic efficacy can be monitored by periodic evaluation of treated patients. For repeated administration over several days or more, depending on the circumstances, treatment is repeated until disease symptoms are suppressed as desired. However, other dosing regimens may be useful and are within the scope of the invention. The desired dose can be delivered by administration of a single bolus of the composition, by multiple boluses of the composition, or by continuous infusion of the composition.

[0186] A pharmaceutical composition comprising an antibody or antibody derivative disclosed herein can be administered once, twice, three times, or four times a day. The composition can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The composition can also be administered in a sustained release formulation, such as in an implant that gradually releases the composition for use over a period of time, allowing for less frequent administration of the administered composition, for example, once a month, once every two to six months, once a year, or even a single administration. Sustained release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microparticles, etc.) can be administered by injection or surgically implanted at various locations.

[0187] Cancer treatment can be evaluated, for example, but not limited to, by tumor regression, shrinkage of tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches to determine efficacy of therapy can be used, including, for example, measuring response by radiological imaging.

[0188] In certain embodiments, efficacy of treatment is measured by percent tumor growth inhibition (% TGI) and is calculated using the equation 100-(T / Cx100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In certain embodiments, %TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

[0189] 3.2 Diagnostic and Imaging Methods

[0190] Labeled antibodies or antibody derivatives can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or disorders associated with MSLN expression, abnormal expression, and / or activity. For example, the antibodies and antibody derivatives provided herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic or imaging assays. A method for detecting the expression of MSLN polypeptide includes (a) assaying the expression of the polypeptide in cells (e.g., tissues) or body fluids of an individual using one or more antibodies or antibody derivatives, and (b) comparing the gene expression level with a standard gene expression level, where an increase or decrease in the assayed gene expression level compared to the standard expression level indicates abnormal expression.

[0191] Another embodiment provided herein includes a method for diagnosing a disease or disorder associated with expression or aberrant expression of MSLN in an animal (e.g., a mammal, such as a human). The method includes detecting MSLN molecules in the mammal. In certain embodiments, the diagnosis includes (a) administering an effective amount of a labeled antibody or antibody derivative to the mammal, (b) waiting a period of time after administration to allow the labeled antibody or antibody derivative to preferentially concentrate where the MSLN molecule is expressed (and unbound labeled molecule is cleared to background levels), (c) measuring the background level, and (d) detecting the labeled molecule in the subject, such that detection of the labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with expression or aberrant expression of MSLN. The background level can be determined by a variety of methods, including comparing the amount of the detected labeled molecule to a standard value previously determined for a particular system.

[0192] The antibodies and antibody derivatives provided herein can be used to assay protein levels in biological samples using classical immunohistological methods well known to those of skill in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting gene expression of proteins include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are well known in the art and include glucose oxidase, radioisotopes, e.g., iodine ( 131 I, 125 I, 123 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 115m In, 113m In, 112 In,111 In), and Technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Molybdenum ( 99 Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 These include Ru, luminol, as well as fluorescent labels, such as fluorescein and rhodamine, and enzyme labels, such as biotin.

[0193] Techniques well known in the art can be applied to the labeled antibodies (or fragments thereof) provided herein, including, but not limited to, the use of bifunctional conjugating agents (see, e.g., U.S. Patent Nos. 5,756,065, 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139, 5,342,604, 5,274,119, 4,994,560, and 5,808,003).

[0194] Alternatively, or in addition, levels of nucleic acid or mRNA encoding an MSLN polypeptide in cells can be measured by, for example, fluorescent in situ hybridization (FISH, see WO98 / 45479 published October 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR) using a nucleic acid-based probe corresponding to a nucleic acid encoding MSLN, or its complement. It is also possible to study overexpression of MSLN, for example by measuring shed antigens in biological fluids such as serum using antibody-based assays (see, for example, U.S. Pat. No. 4,933,294, published June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, published March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Apart from the above assays, various in vivo and ex vivo assays are available to the skilled practitioner. For example, cells within the body of a mammal can be exposed to an antibody that is optionally labeled with a detectable label, e.g., a radioisotope, and binding of the antibody to the somatic cells can be assessed by analyzing a sample (e.g., a biopsy or other biological sample) taken from the mammal previously exposed to the antibody, for example by external scanning for radioactivity.

[0195] 4. Pharmaceutical Preparations

[0196] The presently disclosed subject matter further provides pharmaceutical formulations containing an antibody or antibody derivative disclosed herein and a pharma- ceutically acceptable carrier. In certain embodiments, a pharmaceutical composition can include a combination of multiple (e.g., two or more) antibodies and / or antibody derivatives of the presently disclosed subject matter.

[0197] In certain embodiments, the disclosed pharmaceutical formulations can be prepared by combining an antibody or antibody derivative having a desired degree of purity in the form of a lyophilized formulation or an aqueous solution with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). For example, but not limited to, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations can include those described in U.S. Pat. No. 6,171,586, and WO2006 / 044908, the latter formulations including histidine acetate buffers. In certain embodiments, the antibody or antibody derivative can be greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9% pure.

[0198] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, for example, buffers of phosphate, citrate, and other organic acid salts, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum alcohols, and the like. Examples of suitable pharmacokinetic and / or pharmacokinetically acceptable carriers include, but are not limited to, proteins such as bufin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacokinetically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In certain embodiments, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0199] The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, e.g., anti-MSLN antibody, can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0200] The pharmaceutical compositions of the present disclosure may also be administered in combination therapy, i.e., combined with other agents. In certain embodiments, the pharmaceutical compositions disclosed herein may also contain more than one active ingredient for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other, if necessary. In certain embodiments, the pharmaceutical formulation may contain a second active ingredient for treating the same disease as that being treated by the first therapeutic agent. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. For example, and without limitation, the formulations of the present disclosure may also contain more than one active ingredient for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other, if necessary. For example, it may be desirable to further provide a second therapeutic agent useful for treating the same disease. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

[0201] The compositions of the present disclosure can be administered by various methods well known in the art. The route and / or method of administration varies depending on the desired results. The active compound can be prepared with a carrier that protects the compound from rapid release, for example, a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are described, for example, by Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical compositions are manufactured under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0202] Sustained release preparations containing the antibody or antibody derivative disclosed herein can also be prepared.Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody derivative, which matrices are in the form of shaped articles such as films or microcapsules.In certain embodiments, the active ingredient can also be embedded in colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsions, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively.Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0203] To administer the antibody or antibody derivative of the present disclosure by a certain route of administration, it may be necessary to coat the compound with a material that prevents its inactivation or to co-administer the compound with a material that prevents its inactivation.For example, the compound can be administered to a subject in a suitable carrier, such as liposomes or diluents.Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) J Neuroimmunol.7:27).

[0204] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0205] Therapeutic compositions must usually be sterile, substantially isotonic, and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high-concentration doses. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols (for example, mannitol, sorbitol), or sodium chloride in the composition. Absorption of injectable compositions can be delayed by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0206] Sterile injectable solutions can be prepared by incorporating one or more of the antibodies or antibody derivatives disclosed herein in the required amount in a suitable solvent with one or a combination of the above-listed ingredients as required, followed by sterilization precision filtration, for example, by filtration through a sterile filtration membrane. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of the active ingredient plus additional desired ingredients from the solution that has been previously sterile-filtered.

[0207] The therapeutic composition can also be administered using medical devices known in the art.For example, the therapeutic composition of the present disclosure can be administered by needleless hypodermic injection device, such as the device disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Implants and modules useful in the present disclosure include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate, U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin, U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates, U.S. Patent No. 4,447,224, which discloses an implantable variable flow infusion device for continuous drug delivery, U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments, and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0208] With respect to therapeutic compositions, formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of antibody or antibody derivative that can be combined with the carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of antibody or antibody derivative that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient. Dosage forms for topical or transdermal administration of the compositions of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0209] The phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0210] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, prolonged absorption of injectable pharmaceutical formulations can be brought about by the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0211] In certain embodiments, when the antibodies or antibody derivatives of the present disclosure are administered to humans and animals as pharmaceuticals, they can be administered alone or in combination with a pharma- ceutically acceptable carrier, for example, as a pharmaceutical composition containing about 0.01% to about 99.5% (or about 0.1% to about 90%) of the antibody or antibody derivative.

[0212] 5. Manufactured products

[0213] The presently disclosed subject matter further provides articles of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders.

[0214] In certain embodiments, the article of manufacture includes a container and a label or package insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, IV solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The containers can hold the composition by itself or in combination with other compositions effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (e.g., the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0215] In certain embodiments, at least one active agent in the composition is an antibody or antibody derivative of the present disclosure. The label or package insert can indicate that the composition is used for treating the condition of choice.

[0216] In certain embodiments, the article of manufacture can include (a) a first container containing a composition, the composition comprising an antibody or antibody derivative of the present disclosure, and (b) a second container containing a composition, the composition comprising an additional cytotoxic or another therapeutic agent. In certain embodiments, the article of manufacture can further include a package insert indicating that the composition can be used to treat a particular condition.

[0217] Alternatively, or in addition, the article of manufacture may comprise an additional container, e.g., a second or third container, containing a pharma- ceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0218] array

[0219] [Table 3]

[0220] [Table 4]

[0221] [Table 5]

[0222] [Table 6]

[0223] [Table 7]

[0224] [Table 8]

[0225] [Table 9]

[0226] The following examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way. EXAMPLES

[0227] Example 1. Generation and screening of anti-MSLN VHH antibodies

[0228] To discover VHH antibodies targeting a given antigen, a synthetic VHH library was designed and generated based on a sequencing analysis of the diversity found in natural llama VHHs. Briefly, the lengths of CDR1 and CDR2 were fixed at 8 amino acids, five lengths of CDR3 (15, 16, 17, 19, and 21 amino acids) were selected, and random amino acids (excluding cysteine ​​and methionine) were introduced at each position. To generate this library with a humanized scaffold, human germline IGHV3-30 was introduced as a framework, while several key residues were maintained in framework 2 (FR2) as either human-like or llama-like (50 / 50). The phage library was prepared by standard methods and passed standard quality control analysis.

[0229] To identify anti-MSLN specific VHH antibodies, the antigen of recombinant human MSLN extracellular domain (ECD) protein (residues 296-580) fused to a C-terminal polyhistidine tag was purchased from ACROBiosystems (MSN-H5223). Prior to panning, human MSLN-His was biotinylated with EZ-Link Sulfo-NHS-SS-Biotin, No-Weigh format (ThermoFisher #A39258) at a 2:1 ratio of biotin:protein. Biotinylated human MSLN-His was coated with streptavidin-conjugated Dynabeads M280 (ThermoFisher #11206D) and incubated with library phages. After three rounds of panning, eluted phages showing binders of MSLN from the third round were used to infect SS320 cells. Colonies of SS320 cells were selected and cultured in 2xYT medium, and 1 mM IPTG was added to secrete VHH antibodies. Bacterial supernatants containing VHH antibodies were screened by ELISA assay, and positive human MSLN binders were selected for sequencing. Antibody clones with various sequences were selected and fused to human IgG1 Fc to form bivalent full-length VHH-Fc antibodies for further evaluation. The resulting constructs were expressed in the Expi-CHO transient system and purified in-house. Ab237 is an anti-MSLN fab antibody disclosed in International Publication No. WO2014004549A2. For comparison with the anti-MSLN VHH-Fc antibody, a bivalent IgG1 form of Ab237 was generated in-house as a reference anti-MSLN antibody. Cross-species activity of purified VHH-Fc antibodies was measured by ELISA assay using recombinant human MSLN-His pre-coated plates at 2 μg / mL and detected with HRP-conjugated goat anti-human Ig Fc antibody (Southern Biotech #2047-05) in 1:8,000x dilution, followed by addition of TMB substrate. Figures 1A-1B show representative anti-MSLN VHH-Fc antibodies showing specific binding to human and cynomolgus MSLN. The CDR and VHH sequences of the top 8 clones (1G7, 2B9, 2C5, 7H1, 8D3, 11E6, 12D12, and 13G5) are shown in SEQ ID NOs: 1-40.

[0230] Example 2. In vitro characterization of anti-MSLN VHH antibodies

[0231] The whole cell binding activity of selected anti-MSLN VHH-Fc was measured by flow cytometry against the NCI-N87 human gastric cancer cell line (purchased from ATCC), which endogenously expresses MSLN. Briefly, NCI-N87 cells were grown as adherent monolayers in Roswell Park Memorial Institute medium (RPMI) 1640 medium supplemented with 10% FBS. Cells were rinsed twice with 1x PBS (Gibco) and incubated with pre-warmed (37 °C) 0.05% trypsin-EDTA solution for 5-7 min. Once the cells had detached, trypsin was neutralized by adding 4x volume of complete growth medium containing 10% FBS. Cells were then plated at 2 x 10 6 Gently resuspend in FACS buffer (2% FBS / PBS) at 2 × 10 cells / mL per well. 5 Cells were seeded at a cell density of 1000 x 1000 cells. Purified VHH-Fc antibodies were prepared in 3-fold serial dilutions (8 dilutions in total), starting from a concentration of 100 nM, and incubated with the cells in FACS buffer for 30 min on ice. After washing, Alexa fluor488-conjugated anti-human IgG Fc antibody (1:500) (Alexa Fluor488 AffiniPure goat anti-human IgG, Fcγ fragment specific, Jackson labs) was added and incubated on ice for 30 min. After removing the supernatant by centrifugation at 1500 rpm for 3 min, the cells were resuspended in 100 μL of FACS buffer and run through flow cytometry with CytoFlex (Beckman Coulter). Antibody binding activity to human MSLN present on the cell surface was calculated by GraphPad Prism. As shown in FIG. 2, anti-MSLN VHH-Fc showed strong binding to NCI-N87 cells and greater maximal binding to NCI-N87 cells compared to the Ab237 analog.

[0232] Furthermore, the ability of anti-MSLN VHH-Fc to induce antibody-dependent cellular cytotoxicity (ADCC) against human NCI-N87 human gastric carcinoma was evaluated. Briefly, human NK cell line, CD16-positive NK92 cells (NK92-CD16 cells), were used as effector cells. Target cells, NCI-N87 cells, were incubated at 37°C for 30 min at 1 × 10 6 Cells were labeled with BATDA bis(acetoxymethyl) 2,2':6',2''-terpyridine-6,6''-dicarboxylate) DELFIA reagent (Dissociation Enhanced Lanthanide Fluorescence Immunoassay, Perkin Elmer) at a density of 10 cells / mL, then washed with growth medium and incubated at 5 × 10 3 Target cells were co-cultured with NK92-CD16 cells at a 5-fold effector-target (ET) ratio. After 3 h of incubation at 37°C, target cell lysis was measured by time-resolved fluorescence (TRF) using a Varioskan LUX (Thermo Fisher Scientific). As shown in Figure 3, five of the eight VHH-Fc antibodies (13G5, 12D12, 2B9, 8D3, and 2C5) showed dose-dependent tumor lysis. The reference anti-MSLN antibody (an Ab237 analog) did not show any dose-dependent tumor lysis.

[0233] The ability of anti-MSLN VHH-Fc to induce antibody-dependent cellular cytotoxicity (ADCC) against human NCI-N87 human gastric cancer was further evaluated using human peripheral blood mononuclear cells (hPBMCs). Briefly, hPBMCs were isolated from heparinized blood samples by gradient centrifugation. Target cells, NCI-N87 cells, were incubated at 37°C for 30 min at 1 × 10 6 Cells were labeled with BATDA bis(acetoxymethyl) 2,2':6',2''-terpyridine-6,6''-dicarboxylate) DELFIA reagent (Dissociation Enhanced Lanthanide Fluorescence Immunoassay, Perkin Elmer) at a density of 10 cells / mL, then washed with growth medium and incubated at 5 × 10 3The target cells were co-cultured with hPBMCs from seven healthy donors at an effector-target (ET) ratio of 40. After 3 h of incubation at 37°C, target cell lysis was measured by time-resolved fluorescence (TRF) using a Varioskan LUX (Thermo Fisher Scientific). As shown in Figure 4, seven VHH-Fc antibodies (1G7, 2B9, 7H1, 8D3, 11E6, 12D12, and 13G5) and the Ab237 analog showed dose-dependent tumor lysis, with the VHH-Fc antibodies showing a much lower EC50 compared to the Ab237 analog. The results indicate that the anti-MSLN VHH antibodies have superior antitumor efficacy compared to the Ab237 analog.

[0234] In addition to the various embodiments described and claimed, the disclosed subject matter also relates to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0235] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter includes modifications and variations that come within the scope of the appended claims and their equivalents.

[0236] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties.

[0237] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 082932, filed March 25, 2022, the entire contents of which are incorporated by reference and priority thereto is claimed.

Claims

1. An antibody that binds to MSLN, The aforementioned antibody includes a single-domain antibody, The aforementioned single-domain antibody is a) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 3 b) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 6, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 7, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 8, c) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 11, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 12, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 13 d) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 16, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 17, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO:

18. e) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 21, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 23 f) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 26, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 27, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO:

28. g) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 33, or h) Heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 36, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO:

38. An antibody containing a heavy chain variable region.

2. The antibody according to claim 1, wherein the single-domain antibody comprises a heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 31, a heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 32, and a heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO:

33.

3. The antibody according to claim 1 or 2, wherein the single-domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, and 39.

4. The antibody according to claim 1 or 2, wherein the single-domain antibody comprises a heavy chain variable region including the amino acid sequence shown in SEQ ID NO:

34.

5. The antibody according to claim 1 or 2, wherein the antibody includes an Fc region.

6. The antibody according to claim 5, wherein the Fc region includes an Fc region selected from the group consisting of Fc regions of IgG1, IgG2, IgG3, and IgG4.

7. The antibody according to claim 6, wherein the Fc region includes an IgG1 Fc region.

8. The antibody according to claim 7, wherein the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

9. The antibody according to claim 7, wherein the IgG1 Fc region includes mutations L235V, F243L, R292P, and Y300L, mutations S239D, A330L, and I332E, or mutations L235V, F243L, R292P, Y300L, and P396L.

10. An immune complex comprising the antibody described in claim 1, which is linked to a therapeutic agent or label.

11. The immune complex according to claim 10, wherein the therapeutic agent is a cytotoxin or a radioisotope.

12. The immunocomplex according to claim 10, wherein the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.

13. An antigen-recognition receptor comprising an extracellular antigen-binding domain containing the antibody described in claim 1.

14. The antigen-recognizing receptor according to claim 13, which is a chimeric antigen receptor (CAR) or a recombinant T cell receptor.

15. An antigen-recognizing receptor according to claim 13 or 14, which is a CAR.

16. The antigen-recognition receptor according to claim 13 or 14, wherein the antibody comprises VHH.

17. An immune response cell comprising the antigen-recognizing receptor described in claim 13.

18. The immune response cells according to claim 17, wherein the immune response cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells.

19. The immune response cell according to claim 18, wherein the immune response cell is a T cell.

20. a) an antibody according to claim 1, an immune complex according to claim 10, or an immune response cell according to claim 17, and b) a pharmaceutically acceptable carrier, comprising a pharmaceutical composition.

21. One or more nucleic acids encoding the antibody described in claim 1.

22. One or more vectors comprising the nucleic acid described in claim 21.

23. A host cell comprising the nucleic acid described in claim 21 or the vector described in claim 22.

24. A method for preparing an antibody, comprising expressing the antibody in a host cell according to claim 23, and isolating the antibody from the host cell.

25. An antibody according to claim 1 or 2, for use as a pharmaceutical agent.

26. An antibody according to claim 1 or 2, for use in the treatment of cancer.

27. A pharmaceutical composition according to claim 20, for use as a pharmaceutical agent.

28. A pharmaceutical composition according to claim 20 for use in the treatment of cancer.

29. The pharmaceutical composition according to claim 28, wherein the cancer exhibits high microsatellite instability (MSI).

30. The pharmaceutical composition according to claim 28, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, gliablastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

31. A kit comprising the antibody according to claim 1, the immune complex according to claim 10, the nucleic acid according to claim 21, the vector according to claim 22, or the immune response cell according to claim 17.

32. The kit according to claim 31, further comprising written instructions for treating and / or preventing neoplasms.

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