Antibodies targeting mesothelin and uses thereof

Abstract

The presently disclosed subject matter provides antibodies or antigen-binding fragments thereof that bind to mesothelin and methods of using such antibodies or antigen-binding fragments thereof. The presently disclosed subject further provides immunoconjugates comprising anti-mesothelin antibodies or antigen-binding fragments thereof and methods of using such immunoconjugates.

Description

[0001] ANTIBODIES TARGETING MESOTHELIN AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 657,306, filed June 7, 2024, the content of which is incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on June 4, 2025, is entitled “0727341770_SL” and is 33,234 bytes in size.

[0006] TECHNICAL FIELD

[0007] The presently disclosed subject matter relates to antibodies and antigen-binding fragments thereof that bind to mesothelin, immunoconjugates comprising such antibodies, and methods of using such antibodies and immunoconjugates.

[0008] BACKGROUND

[0009] Immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells may be modified or regulated to target tumor antigen. However, translating immunotherapy to solid tumors poses several obstacles that must be overcome to achieve clinical benefit. Malignant cells adapt to generate an immunosuppressive microenvironment to protect themselves from immune recognition and elimination. This tumor microenvironment poses a challenge to methods of treatment involving stimulation of an immune response, such as immunotherapies. Solid tumors may also be restricted within anatomical compartments that impede efficient T cell trafficking, lack expression of agonistic costimulatory ligands and / or express negative regulators of T cell function. The successful elimination of solid tumors thus requires effective tumor infiltration and overcoming tumor- induced immunosuppression. In addition, solid tumors pose a challenge for selecting optimal immune targets - antigens whose targeting would enable tumor eradication by potent T cells, with minimal or tolerable toxicity to non-tumor tissues.

[0010] Thus, new therapeutic approaches are needed to target solid tumors with minimal toxicity. SUMMARY

[0011] The presently disclosed subject matter provides antibodies or antigen-binding fragments thereof that specifically bind to mesothelin, immunoconjugates comprising such antibodies, and methods of using the antibodies or antigen-binding fragments thereof.

[0012] In certain non-limiting embodiments, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen -binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

[0013] In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0014] In certain embodiments, (a) the heavy chain variable region comprising 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12; or (b) the light chain variable region comprising 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% at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the heavy chain variable region comprising 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%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprising 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%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0015] In certain embodiments, the antibody comprises a heavy chain constant region and / or a light chain constant region. In certain embodiments, the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or the light chain constant region comprises 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% at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0016] In certain embodiments, the antibody or antigen-fragment thereof comprises a human variable region framework region. In certain embodiments, the antibody or antigen-fragment thereof is a fully human or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-fragment thereof is a chimeric antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-fragment thereof is a humanized antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab')2, variable fragment (Fv), or single chain variable region (scFv). In certain embodiments, the antigen-binding fragment is an scFv.

[0017] In certain non-limiting embodiments, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0018] In certain non-limiting embodiments, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof comprising a single chain variable region (scFv) comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0019] In certain non-limiting embodiments, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0020] In certain non-limiting embodiments, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof comprising the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0021] In certain non-limiting embodiments, the presently disclosed subject matter provides an antibody or an antigen-binding fragment thereof, which cross-competes for binding to mesothelin with an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein. In certain non-limiting embodiments, the presently disclosed subject matter provides an antibody or an antigen-binding fragment thereof, which binds to the same epitope region on mesothelin with an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein.

[0022] In certain non-limiting embodiments, the presently disclosed subject matter provides an immunoconjugate comprising the anti-mesothelin antibody or antigen-binding fragment thereof disclosed herein, linked to a therapeutic agent. In certain embodiments, the therapeutic agent is a drug, a cytotoxin, or a radioactive isotope.

[0023] In certain non-limiting embodiments, the presently disclosed subject matter provides a multi-specific molecule comprising the antibody or antigen-binding fragment thereof disclosed herein, linked to one or more functional moieties. In certain embodiments, the one or more functional moieties have a different binding specificity than the antibody or antigen binding fragment thereof.

[0024] In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising the antibody, the immunoconjugate, or the multi-specific molecule disclosed herein. In certain embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier. In certain non-limiting embodiments, the presently disclosed subject matter provides a nucleic acid that encodes an antibody or antigen-binding fragment thereof disclosed herein, a heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, or a light chain variable region of an antibody or antigen-binding fragment thereof disclosed herein. In certain non-limiting embodiments, the presently disclosed subject matter provides a vector comprising the nucleic acid disclosed herein. In certain embodiments, the vector is a low copy number vector. In certain non-limiting embodiments, the presently disclosed subject matter provides a host cell comprising the vector disclosed herein.

[0025] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for detecting mesothelin in a whole cell, a tissue, or a blood sample, comprising (a) contacting a cell, tissue or blood sample with the antibody or antigen-binding fragment thereof disclosed herein, wherein the antibody or antigen-binding fragment thereof comprises a detectable label; and determining the amount of the labeled antibody or antigen-binding fragment thereof bound to the cell, tissue or blood sample by measuring the amount of detectable label associated with said cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of mesothelin in the cell, tissue or blood sample.

[0026] In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating or ameliorating a disease or disorder associated with mesothelin in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor, the method comprising administering to the subject the antibody or antigen-binding fragment thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein. In certain embodiments, the disease or disorder is a tumor. In certain embodiments, the method reduces the number of the tumor cells, reduces the tumor size, and / or eradicates the tumor in the subject. In certain embodiments, the method reduces or eradicates tumor burden in the subject. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma. In certain embodiments, the subject is a human.

[0027] In certain non-limiting embodiments, the presently disclosed subject matter provides a kit comprising the antibody or antigen-binding fragment thereof, the immunoconjugate, the multi-specific molecule, the composition, the nucleic acid, the vector, or the host cell disclosed herein. In certain embodiments, the kit further comprises written instructions for using the antibody or antigen-binding fragment thereof, immunoconjugate, multi-specific molecule, composition, the nucleic acid, the vector, or the host cell. In certain embodiments, the kit disclosed herein is for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] The following Detailed Description, given by way of example, but not intended to limit the presently disclosed subject matter to specific embodiments described, may be understood in conjunction with the accompanying drawings.

[0030] Figure 1 shows activity of the R6P1-B1 clone against human mesothelin expressing cells detected by flow cytometry.

[0031] Figures 2A-2E illustrate the structure and interaction of the m912 scFv and R6P1-B1 scFv. Figure 2A shows B-factor view of the m912 scFv and R6P1-B1 scFv. Figure 2B shows the distribution of electrostatic forces of the m912 scFv and R6P1-B1 scFv. Figure 2C shows the lipophilicity of the m912 scFv and R6P1-B1 scFv. Figure 2D shows a summary of the properties of the m912 scFv and R6P1-B1 scFv and the predicted binding pocket. Figure 2E shows the interaction of m912 scFv and R6P1-B1 scFv with human mesothelin.

[0032] DETAILED DESCRIPTION

[0033] The present disclosure provides uses of antibodies and antigen-binding fragments thereof that specifically target mesothelin for treatments, e.g., for treating neoplasia. The present disclosure is based, in part, on unexpected findings demonstrating that the antibodies and antigen-binding fragments disclosed herein exhibit significantly higher expression levels in cell systems (e.g., host cells) compared to other known anti-mesothelin antibodies and their antigen-binding fragments. Without being bound by theory, the inventors believe that this enhanced expression is attributable to the unique antigen-binding domain of the disclosed antibodies and fragments.

[0034] Non-limiting embodiments of the present disclosure are described by the present specification and Examples.

[0035] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0036] 1. Definitions; 2. Mesothelin;

[0037] 3. Anti -mesothelin Antibodies;

[0038] 4. Immunoconjugates;

[0039] 5. Nucleic Acids encoding the Antibodies or Antigen-binding Fragments;

[0040] 6. Pharmaceutical Compositions and Methods of Treatment;

[0041] 7. Diagnostic and Prognostic Methods;

[0042] 8. Kits; and

[0043] 9. Exemplary Embodiments.

[0044] / . Definitions

[0045] In the description that follows, certain conventions will be followed as regards the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art.

[0046] “Antibody” and “antibodies” as those terms are known in the art refer to antigen binding proteins of the immune system. The term “antibody” as referred to herein includes whole, full length antibodies having an antigen-binding region, and any fragment thereof in which the “antigen-binding fragment” or “antigen-binding region” is retained, or single chains, for example, single chain variable fragment (scFv), thereof. A naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system.

[0047] The term “antigen-binding fragment” or “antigen-binding region” of an antibody, as used herein, refers to that region or fragment of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., mesothelin). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigenbinding fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; aF(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 1989;341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).

[0048] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g., Bird et al., Science (1988);242:423-426; and Huston et al., Proc Natl Acad Sci (I998);85:5879-5883. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0049] The term “human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the presently disclosed subject matter may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0050] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0051] The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0052] The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0053] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0054] By “specifically binds” or “specifically binds to” or “specifically target” is meant a polypeptide or a fragment thereof that recognizes and / or binds to a biological molecule of interest (e.g., a polypeptide, e.g., a mesothelin polypeptide), but which does not substantially recognize and / or bind to other molecules in a sample, for example, a biological sample, which naturally includes a presently disclosed polypeptide (e.g., a mesothelin polypeptide).

[0055] An “antibody that competes for binding” or “antibody that cross-competes for binding” with a reference antibody for binding to an antigen, e.g., mesothelin, refers to an antibody that blocks binding of the reference antibody to the antigen (e.g., msothelin) in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to the antigen (e.g., mesothelin) in a competition assay by 50% or more. An exemplary competition assay is described in “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0056] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0057] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term” an antibody which binds specifically to an antigen (e.g., mesothelin).”

[0058] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: :VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0059] Non-limiting examples of linkers, e.g., for use in generating an scFv, are disclosed in Shen et al., Anal Chem (2008);80(6): 1910-1917 and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. In certain embodiments, the linker is a G4S linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, which is provided below: GGGGSGGGGSGGGGS [ SEQ ID NO : 1 ]

[0060] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 2, which is provided below:

[0061] GGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 2 ]

[0062] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 3, which is provided below:

[0063] GGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 3 ] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 4, which is provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 4 ]

[0064] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Set. USA, 1988;85:5879-5883). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008;27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., JImunol 2009; 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007;97(6):955-63; Fife eta., J Clin Invst 2006;l 16(8):2252-61; Brocks et al., Immunotechnology 1997;3(3):173-84; Moosmayer et al., Ther Immunol 1995; 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bio. Chem 2003; 25278(38):36740-7; Xie et al., Nat Biotech 1997; 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997; 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003; 1638(3):257- 66).

[0065] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0066] As used herein, “F(ab’)2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab’) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab’)2” fragment can be split into two individual Fab' fragments.

[0067] As used herein, the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.

[0068] “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e. g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987), or IMGT numbering system (Lefranc, The Immunologist (1999);7: 132-136; Lefranc et al., Dev. Comp. Immunol. (2003); 27:55-77). The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and / or form structurally defined loops (“hypervariable loops”) and / or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope region. In certain embodiments, the CDRs are identified according to the Kabat system.

[0069] The terms “isolated” denotes a degree of separation from original source or surroundings.

[0070] An “isolated antibody” is one which has been separated from a component of its natural environment. In certain embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr (2007); B 848:79-87.

[0071] An “isolated nucleic acid” refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0072] An “isolated nucleic acid encoding an antibody” (including references to a specific antibody, e.g., an anti-mesothelin antibody) refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0073] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been 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."

[0074] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0075] An “effective amount” (or, “therapeutically effective amount”) is an amount sufficient to effect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.

[0076] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters; guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0077] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In certain embodiments, antibodies of the presently disclosed subject matter are used to delay development of a disease or to slow the progression of a disease, e.g., a tumor, e.g., a tumor associated with mesothelin.

[0078] The terms “comprises”, “comprising”, and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.

[0079] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.

[0080] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0081] Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the ambit of the presently disclosed subject matter.

[0082] 2. Mesothelin

[0083] Mesothelin is an immunogenic cell surface antigen that is highly expressed in solid cancers. Mesothelin is involved in cell proliferation, adhesion, invasion, cell signaling, and metastasis. Studies have demonstrated that serum soluble mesothelin-related peptide secreted by mesothelin-expressing tumors can be measured in both humans and mice, and has been shown to correlate with therapy response and prognosis. In normal tissues, mesothelin is expressed only in the pleura, pericardium, and peritoneum, at low levels. The anti-mesothelin recombinant immunotoxin SS1P has shown in vivo specificity and significant antitumor activity in patients. In a pancreatic cancer vaccine trial, patients with survival advantage had consistent CD8+ T cell responses to mesothelin associated with vaccine-induced delay ed-type hypersensitivity response. Specific T cell epitopes derived from mesothelin were shown to activate human T cells to efficiently lyse human tumors expressing mesothelin. Thus, there is strong supportive evidence that adoptive immunotherapy targeting mesothelin can target mesothelin-expressing tumors.

[0084] In certain embodiments, the antigen-recognizing receptor binds to human mesothelin. In certain embodiments, the human mesothelin comprises or consists of the amino acid sequence with a NCBI Reference No: AAV87530.1 (SEQ ID NO: 5) or a fragment thereof. SEQ ID NO: 5 is provided below: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLS PRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLL FLNPDAFSGPQACTHFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALG GLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDAL RGLLPVLGQPI IRS IPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKARE IDESLI FYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHL GYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSS IWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKI QSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRP VRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTVLALLLAST

[0085] LA [ SEQ ID NO : 5 ]

[0086] In certain embodiments, the human mesothelin comprises or consists of 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% identical to the amino acid sequence set forth in SEQ ID NO: 5 or a portion thereof.

[0087] 3. Anti-mesothelin Antibodies

[0088] The antibodies of the presently disclosed subject matter are characterized by particular functional features or properties of the antibodies. For example, the antibodies bind specifically to mesothelin (e.g., bind to human mesothelin).

[0089] In certain embodiments, a presently disclosed antibody or antigen-binding fragment binds to mesothelin (e.g., human mesothelin) with a binding affinity, for example with a dissociation constant (KD) of 1 x 10'8M or less, e.g., about 1 x 10'8M or less, about 5 x 1 O’9M or less, about 1 x 10'9M or less, about 5 x 1 O’10M or less, about 1 x IO'10M or less, or about 1 x io-11M or less.

[0090] In certain embodiments, a presently disclosed antibody or antigen-binding fragment binds to a cell expressing mesothelin with a Half maximal effective concentration (ECso) value of from about 1 nM to about 50 nM, from about 5 nM to about 50 nM, from about 10 nM to about 50 nM, from about 20 nM to about 50 nM, from about 30 nM to about 50 nM, from about 40 nM to about 50 nM, or greater than about 50 nM.

[0091] The heavy and light chains of a presently disclosed antibody or antigen-binding fragment can be full-length (e.g., an antibody can include at least one (e.g., one or two) complete heavy chains, and at least one (e.g., one or two) complete light chains) or can include an antigen-binding fragment (a Fab, F(ab’)2, Fv or a single chain Fv fragment (“scFv”)). In certain embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. In certain embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, and IgG4. In certain embodiments, the immunoglobulin isotype is IgGl (e.g., human IgGl). The choice of antibody isotype can depend on the immune effector function that the antibody is designed to elicit. In certain embodiments, the antibody light chain constant region is chosen from, e.g., kappa or lambda. In certain embodiments, the antibody light chain constant region is kappa.

[0092] In constructing a recombinant immunoglobulin, appropriate amino acid sequences for constant regions of various immunoglobulin isotypes and methods for the production of a wide array of antibodies are known to those of skill in the art.

[0093] 3.1. Single-Chain Variable Fragments (scFvs)

[0094] In certain embodiments, the presently disclosed subject matter includes antibodies or antigen-binding fragments thereof that have the scFv sequence fused to one or more constant domains to form an antibody with an Fc region of a human immunoglobulin to yield a bivalent protein, increasing the overall avidity and stability of the antibody. In addition, the Fc portion allows the direct conjugation of other molecules, including but not limited to fluorescent dyes, cytotoxins, radioisotopes etc. to the antibody for example, for use in antigen quantitation studies, to immobilize the antibody for affinity measurements, for targeted delivery of a therapeutic agent, to test for Fc-mediated cytotoxicity using immune effector cells and many other applications. In certain embodiments, the Fc portion is conjugated to a cytotoxin.

[0095] The results presented here highlight the specificity, sensitivity and utility of the presently disclosed antibodies or antigen-binding fragments in targeting mesothelin (e.g., human mesothelin).

[0096] In certain embodiments, the anti-mesothelin antibody is an scFv, an scFv-Fc fusion protein or a full-length human IgG (or antibody fragment thereof) with VH and VL regions or CDRs selected from Table 1. In certain embodiments, the anti-mesothelin scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the anti-mesothelin scFv comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO: 12 and SEQ ID NO: 13 are provided in Table 1. In certain embodiments, the scFv is designated as “R6P1-B1.” In certain embodiments, the anti- mesothelin antibody comprises the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NO: 16 is provided in Table 1,

[0097] In certain embodiments, the anti-mesothelin scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-mesothelin scFv comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof. SEQ ID NOs: 6-8 are provided in Table 1.

[0098] In certain embodiments, the anti-mesothelin scFv comprises a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof. SEQ ID NOs: 9-11 are provided in Table 1.

[0099] In certain embodiments, the anti-mesothelin scFv comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

[0100] In certain embodiments, the anti-mesothelin scFv comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0101] In certain embodiments, the anti-mesothelin scFv comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0102] In certain embodiments, the variable regions are linked one after another such that a heavy chain variable region (VH) is positioned at the N-terminus. In certain embodiments, the variable regions are positioned from the N- to the C-terminus: VH-VL. In certain embodiments, a light chain variable region (VL) is positioned at the N-terminus. In certain embodiments, the variable regions are positioned from the N- to the C-terminus: VL-VH.

[0103] Table 1

[0104] 3.2. Monoclonal Antibodies

[0105] The presently disclosed subject matter provides antibodies (e.g., human antibodies, e.g., human monoclonal antibodies) that specifically bind to mesothelin (e.g., human mesothelin). The VH amino acid sequences of the presently disclosed anti-mesothelin antibody is set forth in SEQ ID NO: 12. The VL amino acid sequences of the presently disclosed anti-mesothelin antibody is set forth in SEQ ID NO: 13.

[0106] In certain embodiments, the presently disclosed subject matter provides an antibody or an antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 12; and (b) a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 13; wherein the antibody or antigen-binding fragment specifically binds to mesothelin, e.g., human mesothelin.

[0107] In certain embodiments, the presently disclosed subject matter provides antibodies or antigen-binding fragments thereof that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s of the presently disclosed scFv (e.g., one disclosed in Section 3.1).

[0108] The amino acid sequences of the VH CDRls is set forth in SEQ ID NO: 6. The amino acid sequences of the VH CDR2S is set forth in SEQ ID NO: 7. The amino acid sequences of the VH CDR3S is set forth in SEQ ID NO: 8. The amino acid sequences of the VL CDRls is set forth in SEQ ID NO: 9. The amino acid sequences of the VL CDR2S is set forth in SEQ ID NO: 10. The amino acid sequences of the VL CDR3S is set forth in SEQ ID NO: 11. The CDR regions are delineated using the Kabat system.

[0109] In certain embodiments, the anti-mesothelin antibody or antigen-binding fragment thereof comprises:

[0110] (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6;

[0111] (b) a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7;

[0112] (c) a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8;

[0113] (d) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9;

[0114] (e) a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10; and

[0115] (f) a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0116] In certain embodiments, the anti-mesothelin antibody or antigen-binding fragment thereof comprises a heavy chain constant region and / or a light chain constant region.

[0117] In certain embodiments, the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below.

[0118] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK [ SEQ ID NO : 18 ] In certain embodiments, the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 19. SEQ ID NO:

[0119] 19 is provided below.

[0120] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK [ SEQ ID NO : 19 ]

[0121] In certain embodiments, the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NO:

[0122] 20 is provided below.

[0123] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPP PCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHEALHNRFTQKSLS LSPGK [ SEQ ID NO : 20 ]

[0124] In certain embodiments, the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO:

[0125] 21 is provided below. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVL TVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK [ SEQ ID NO : 21 ]

[0126] In certain embodiments, the light chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below.

[0127] RTVAAPSVEI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [ SEQ ID NO : 22 ]

[0128] In certain embodiments, the anti-mesothelin antibody or an antigen-binding fragment thereof comprises: (a) a heavy chain constant region comprising 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and (b) a light chain constant region comprising 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% at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the anti-mesothelin antibody or an antigen-binding fragment thereof comprises: (a) a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and (b) a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0129] The constant regions / framework regions of the anti-mesothelin antibodies disclosed herein can be altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation etc., the number of cysteine residues, effector cell function, effector cell function, complement function or introduction of a conjugation site). In certain embodiments, a presently disclosed anti-mesothelin antibody is a fully- human antibody. Fully-human mAbs, when administered to humans, causing serious side effects, including anaphylaxis and hypersensitivity reactions.

[0130] The use of phage display libraries has made it possible to select large numbers of antibody repertoires for unique and rare Abs against very defined epitopes (for more details on phage display see McCafferty et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348: 552-554.) The rapid identification of human Fab or single chain Fv (scFv) fragments highly specific for tumor antigen-derived peptide-MHC complex molecules has thus become possible. In addition, by engineering full-length monoclonal antibody (mAb) using the Fab fragments, it is possible to directly generate a therapeutic human mAb, bypassing months of time-consuming work, normally needed for developing therapeutic mAbs. The presently disclosed subject matter involves the development of a fully human mAb that recognizes, for example, a human mesothelin polypeptide (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 5) for cancer therapy.

[0131] 3.3. Homologous Antibodies

[0132] In certain embodiments, a presently disclosed anti-mesothelin antibody or antigenbinding fragment thereof comprises heavy and light chain variable regions comprising amino acid sequences that are homologous or identical to the amino acid sequences of the antibodies described herein, and wherein the antibodies or antigen-binding fragments thereof retain the desired functional properties of the anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter.

[0133] For example, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0134] (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12; and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the VH and / or VL amino acid sequences can be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the sequences set forth above. An antibody having VH and VL regions having high (i.e., 80% or greater) homology or identity to the VH and VL regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis), followed by testing of the encoded altered antibody for retained function (i.e., the binding affinity) using the binding assays described herein.

[0135] In certain embodiments, a presently disclosed anti-mesothelin antibody or antigenbinding fragment thereof comprises heavy and light chain constant regions comprising amino acid sequences that are homologous or identical to the amino acid sequences of the antibodies described herein, and wherein the antibodies or antigen-binding fragments thereof retain the desired functional properties of the anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter.

[0136] For example, the presently disclosed subject matter provides an anti-mesothelin antibody or an antigen-binding fragment thereof, comprising a heavy chain constant region and a light chain constant region, wherein:

[0137] (a) the heavy chain constant region (CH) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and

[0138] (b) the light chain constant region (CL) comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0139] In certain embodiments, the CH and / or CL amino acid sequences can be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the sequences set forth above. An antibody having CH and CL regions having high (i.e., 80% or greater) homology or identity to the CH and CL regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis), followed by testing of the encoded altered antibody for retained function (i.e., the binding affinity) using the binding assays described herein.

[0140] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity or homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0141] The percent homology or identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci (1988);14 : 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol (1970);48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0142] Additionally or alternatively, the protein sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al., J Mol Biol (1990);215 :403- 10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res (I997);25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0143] 3.4. Antibodies with Conservative Modi fications In certain embodiments, a presently disclosed anti-mesothelin antibody or an antigenbinding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specified amino acid sequences based on the preferred antibodies described herein, or a conservative modification thereof, and wherein the antibodies retain the desired functional properties of the anti- mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter. The presently disclosed subject matter provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

[0144] (a) the heavy chain variable region CDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 8, and conservative modifications thereof;

[0145] (b) the light chain variable region CDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 11, and conservative modifications thereof.

[0146] In certain embodiments, the heavy chain variable region CDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 8, and conservative modifications thereof; and the light chain variable region CDR3 sequence comprises the amino acid sequence set forth in SEQ ID NO: 11, and conservative modifications thereof.

[0147] In certain embodiments, the heavy chain variable region CDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 7, and conservative modifications thereof; and the light chain variable region CDR2 sequence comprises the amino acid sequence set forth in SEQ ID NO: 10, and conservative modifications thereof.

[0148] In certain embodiments, the heavy chain variable region CDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 6, and conservative modifications thereof; and the light chain variable region CDR1 sequence comprises the amino acid sequence set forth in SEQ ID NO: 9, and conservative modifications thereof.

[0149] As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Exemplary conservative amino acid substitutions are shown in Table 2. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. In certain embodiments, a sequence disclosed herein, e.g., a CDR sequence, a VH sequence or a VL sequence, can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about six, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and / or substituted.

[0150] Table 2 Amino acids may be grouped according to common side-chain properties:

[0151] • hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0152] • neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0153] • acidic: Asp, Glu;

[0154] • basic: His, Lys, Arg;

[0155] • residues that influence chain orientation: Gly, Pro;

[0156] • aromatic: Trp, Tyr, Phe.

[0157] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0158] 3.5. Anti-mesothelin Antibodies that Cross-compete for Binding to mesothelin with Anti-mesothelin Antibodies of the Present Disclosure

[0159] The presently disclosed subject matter provides antibodies or antigen-binding fragments thereof that cross-compete with any of the disclosed anti-mesothelin antibodies for binding to mesothelin (e.g., human mesothelin). For example, and not by way of limitation, the cross-competing antibodies can bind to the same epitope region, e.g., same epitope, adjacent epitope, or overlapping as any of the anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter. In certain embodiments, the reference antibody or reference antigen-binding fragments thereof for cross-competition studies can be the anti-mesothelin antibodies or antigen-binding fragments thereof disclosed herein.

[0160] Such cross-competing antibodies can be identified based on their ability to cross- compete with any one of the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof in standard mesothelin binding assays. For example, Biacore analysis, ELISA assays or flow cytometry can be used to demonstrate cross-competition with the antibodies of the presently disclosed subject matter. The ability of a test antibody to inhibit the binding of, for example, any one of the presently disclosed anti-mesothelin antibodies to mesothelin (e.g., human mesothelin) demonstrates that the test antibody can compete with any one of the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof for binding to mesothelin (e.g., human mesothelin) and thus binds to the same epitope region on mesothelin (e.g., human mesothelin) as any one of the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof. In certain embodiments, the cross-competing antibody or antigen-binding fragment thereof binds to the same epitope on mesothelin (e.g., human mesothelin) as any one of the presently disclosed anti-mesothelin antibodies or antigenbinding fragments thereof.

[0161] 3.6. Characterization of Antibody Binding to Antigen

[0162] Antibodies or antigen-binding fragments thereof of the presently disclosed subject can be tested for binding to mesothelin by, for example, standard ELISA. To determine if the selected anti-mesothelin antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using mesothelin coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a streptavidin-alkaline phosphatase probe.

[0163] To determine the isotype of purified antibodies, isotype ELISAs can be performed using reagents specific for antibodies of a particular isotype. Anti-mesothelin human IgGs can be further tested for reactivity with mesothelin antigen by Western blotting.

[0164] In certain embodiments, the KD is measured by a radiolabeled antigen binding assay (RIA). In certain embodiments, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody- coated plate (see, e.g., Chen et al., J Mol Biol (1999);293 :865-881).

[0165] In certain embodiments, the KD is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE® -2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ).

[0166] 3. 7. Multi-specific Molecules

[0167] The presently disclosed subject matter provides multi-specific molecules comprising an anti-mesothelin antibody, or a fragment thereof, disclosed herein. A presently disclosed or an antigen-binding fragment thereof can be derivatized or linked to one more functional molecules, e.g., one or more peptides or proteins (e.g., one or more antibodies or ligands for a receptor) to generate a multi-specific molecule that binds to two or more different binding sites or target molecules. The presently disclosed anti-mesothelin antibody or antigen-binding fragment thereof can in fact be derivatized or linked to more than one other functional molecules to generate multi-specific molecules that bind to more than two different binding sites and / or target molecules. To create a multi-specific molecule, a presently disclosed anti- mesothelin antibody or an antigen-binding fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule.

[0168] In certain embodiments, the multi-specific molecule is a bispecific molecule. In certain embodiments, the bispecific molecules comprise at least a first binding specificity for mesothelin and a second binding specificity for a second target epitope region. The second target epitope region can be a mesothelin epitope, or a non-mesothelin epitope, e.g., a different antigen. In certain embodiments, the multi-specific molecule comprises a first binding specificity for mesothelin, a second binding specificity for a second target, and a third binding specificity for a third target. In certain embodiments, the second target is an antigen expressed on the surface of an immune cell (e.g., a T cell, or a human immune effector cell). In certain embodiments, the multi-specific molecule is capable of recruiting the activity of that immune effector cell by specifically binding to the effector antigen on the human immune effector cell, thereby enhancing effector function. In certain embodiments, the third target is an antigen expressed on a senescent cell.

[0169] The multi-specific molecules of the presently disclosed subject matter can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the multi-specific molecule can be generated 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), 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidom ethyl) cyclohaxane-1 -carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Conjugating agents can be SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0170] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In certain embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the multi-specific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab’)2 or ligand x Fab fusion protein.

[0171] Binding of the multi-specific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography.

[0172] 3.8. Antibody Fragments

[0173] The presently disclosed subject matter provides antibody fragments of an anti- mesothelin antibody disclosed herein. In certain embodiments, the antibody fragment comprises an scFv as disclosed herein in Section 3.1. In certain embodiments, the antibody fragment is an scFv as disclosed herein in Section 3.1. In certain embodiments, the antibody fragment is a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, or a F(ab’)2 fragment.

[0174] In certain embodiments, the antibody fragment is a “Fab” fragments. “Fab” fragments can be produced by papain digestion of full length antibodies. Traditionally, Fab fragments contain the heavy-chain variable domain (VH) and light-chain variable domain (VH) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI).

[0175] In certain embodiments, the antibody fragment is a “Fab”’ fragments. Fab’ fragments can be distinguished from Fab fragments because including additional residues at the carboxy terminus of the CHI domain. In certain embodiments, the Fab’ fragments include one or more cysteines from the antibody hinge region.

[0176] In certain embodiments, the antibody fragment is a “Fab’-SH” fragments. Fab’-SH are Fab’ fragments where at least one cysteine residue of the constant domains includes a free thiol group. In certain embodiments, the antibody fragment is a “F(ab’)2” fragment. F(ab’)2 fragments can be obtained by pepsin digestion of full length antibodies. F(ab’)2 fragments have two antigen-binding sites (e.g., two Fab fragments) and a portion of the Fc region.

[0177] In certain embodiments, the antibody fragment is a single-domain antibody. Singledomain antibody are antibody fragments including the heavy chain variable domain or a portion thereof of an antibody or the light chain variable domain or a portion thereof of an antibody.

[0178] 3.9. Humanized or Chimeric Antibodies

[0179] The presently disclosed subject matter further provides chimeric and / or humanized versions of an anti-mesothelin antibody, or a fragment thereof, disclosed herein.

[0180] In certain embodiments, the anti-mesothelin antibody is a chimeric antibody. In certain embodiments, the chimeric antibody comprises a variable region derived from a non-human species (e.g., a variable region derived from a mouse, a rat, a hamster, a rabbit, or a non-human primate) and a human constant region. Alternatively or additionally, a chimeric antibody can be a “class-switched” antibody. In certain embodiments, the cl ass- switched antibody is an antibody wherein the class or subclass has been modified from that of the parent antibody.

[0181] In certain embodiments, the anti-mesothelin antibody provided herein is a humanized antibody. In certain embodiments, the humanized antibody comprises at least one variable domain. In certain embodiments, the variable domain comprises CDRs derived from a non- human antibody, e.g., a mouse antibody.

[0182] In certain embodiments, the variable domain comprises framework regions (FR) derived from human antibody sequences. In certain embodiments, the FR include substitutions and / or modification. In certain embodiments, residues in a humanized antibody can be substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), to restore or improve antibody specificity or affinity.

[0183] In certain embodiments, the humanized antibody can also include a human constant region. In certain embodiments, humanization of a non-human antibody, e.g. a mouse antibody, reduces immunogenicity of the antibody in humans. In certain embodiments, humanization of a non-human antibody, e.g. a mouse antibody, does not impair the specificity and affinity of the parental non-human antibody.

[0184] 4. I mmu noconjugates

[0185] The presently disclosed subject provides an anti-mesothelin antibody or an antigenbinding fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Non-limiting examples of cytotoxins include taxol (such as ricin, diphtheria and gelonin), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin di one, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, calecheamicin, aureastatin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), hypomethylating agents (azacytidine and decitabine), and anti-mitotic agents (e.g., vincristine and vinblastine). Other non-limiting examples of therapeutic cytotoxins that can be conjugated to an anti-mesothelin antibody disclosed herein include duocarmycins, calicheamicins, maytansines, auristatins, calicheamicin, tubulysin, and pyrrolobenzodiazepines (PBD), and derivatives thereof.

[0186] Cytotoxins can be conjugated to an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein using linker technology available in the art. In certain embodiments, a linker is a molecule with two reactive termini, one for conjugation to an antibody and the other for conjugation to a cytotoxin. The antibody conjugation reactive terminus of the linker is typically a site that is capable of conjugation to the antibody through a cysteine thiol or lysine amine group on the antibody, and so is typically a thiol -reactive group such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo, or iodo, or an R-sulfanyl group, or an amine-reactive group such as a carboxyl group; while the antibody conjugation reactive terminus of the linker is typically a site that is capable of conjugation to the cytotoxin through formation of an amide bond with a basic amine or carboxyl group on the cytotoxin, and so is typically a carboxyl or basic amine group. Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). In certain embodiments, the linker can be conjugated to an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein using a cysteine rebridging approach called ThioBridge®. Additional information on ThioBridge® can be found in Bird et al., Methods Mol Biol. 2020;2078: 113- 129, the content of which is incorporated in its entirety.

[0187] For further discussion of types of cytotoxins, linkers, and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199- 215; Trail, P.A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3 :207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3 : 1089-1091; Senter, P.D. and Springer, C.J. (2001) Adv. Drug Deliv. Rev. 53 :247-264.

[0188] Anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Non-limiting examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include47Sc,67Cu,90Y,131I,149Tb,161Tb,177Lu,225Ac,213Bi,223Ra, and227Th. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the presently disclosed anti-mesothelin antibodies.

[0189] In certain embodiments, anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter can be conjugated to non-radioactive isotopes that can be irradiated, e.g., with neutrons, to undergo a decay reaction and generate alpha particles. For example, but not by way of limitation, an anti-mesothelin antibodies or antigenbinding fragments thereof of the presently disclosed subject matter also can be conjugated to a molecule comprising boron (e.g.,10B), a stable non-radioactive isotope of boron. The presently disclosed conjugates comprising boron can be used in boron neutron capture therapy (BNCT). As used herein, “boron neutron capture therapy” or “BNCT” refers to a targeted radiotherapy, wherein boron of the presently disclosed immunoconjugates comprising boron are irradiated with low energy thermal neutrons to yield biologically destructive alpha particles and lithium- 7 nuclei. Additional information concerning immunoconjugates comprising boron and BNCT can be found in Guan et \., Proceedings of the National Academy of Sciences 95, no. 22 (1998): 13206-13210; Barth et al., Cancer Communications 38, no. 1 (2018): 1-15; Nedunchezhian et al., Journal of clinical and diagnostic research: JCDR 10, no. 12 (2016): ZE01; Malouff et al., Frontiers in oncology 11 (2021): 601820; and U.S. Pat. No. 4,624,846; the content of each of which is incorporated by reference in its entirety.

[0190] Anti-mesothelin antibodies or antigen-binding fragments thereof of the presently disclosed subject matter also can be conjugated to a self-assembly disassembly (SAD A) polypeptide. A SADA domain is composed of multimerization domains which are each composed of helical bundles that associate in a parallel or anti-parallel orientation. Nonlimiting examples of SADA domain containing human polypeptides include p53, p63, p73, heterogeneous nuclear Ribonucleoprotein C (hnRNPC), C or N-terminal domain of Synaptosomal-associated protein 23 (SNAP-23), Cyclin-D-related protein (CBFA2T1), variants thereof, or fragments thereof. In certain embodiments, these conjugates can multimerize to form a complex of a desired size under relevant conditions (e.g., in a solution in which the conjugate is present above a threshold concentration or pH and / or when present at a target site characterized by a relevant level or density of receptors for the payload), and disassemble to a smaller form under other conditions (e.g., absent the relevant environmental multimerization trigger). Additional information concerning the SADA polypeptides and immunoconjugates comprising the same can be found in International Patent Publication No. WO 2018 / 204873, the content of which is incorporated by reference in its entirety.

[0191] The antibody conjugates of the presently disclosed subject matter can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor (TNF) or interferon-y; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.

[0192] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

[0193] 5. Nucleic Acids encoding the Antibodies or Antigen-binding Fragments and / or VH and / or VL thereof

[0194] The presently disclosed subject matter provides nucleic acids encoding the anti- mesothelin antibodies or antigen-binding fragments thereof disclosed herein. The presently disclosed subject matter provides nucleic acids encoding the heavy chain variable region sequence of any one of the presently disclosed anti-mesothelin antibodies.

[0195] The presently disclosed subject matter provides nucleic acids encoding the light chain variable region sequence of any one of the presently disclosed anti-mesothelin antibodies.

[0196] In certain embodiments, the nucleic acids encoding the anti-mesothelin antibodies or antigen-binding fragments thereof disclosed herein can include codons that are optimized for expression in a particular cell type or organism. These codon-optimized sequences are synthetic sequences that encode an identical polypeptide encoded by the non-codon-optimized parent polynucleotide. In certain embodiments, the coding region of the nucleic acid encoding the antibodies or antigen-binding fragments thereof disclosed herein can include an altered sequence to optimize codon usage for a particular cell type (e.g., a eukaryotic or prokaryotic cell). For example, the coding sequence for a human or humanized heavy (or light) chain variable region as described herein can be optimized for expression in bacterial cells. Additionally or alternatively, the coding sequence can be optimized for expression in a mammalian cell (e.g., a CHO, an NSO cell, and a Sp2 / 0 cell). Further provided are vectors comprising the presently disclosed nucleic acids. In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is a low copy vector. In certain embodiments, a low copy vector is a vector that is present in an amount of at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 copy per host cell. Non-limiting examples of low copy vectors are yeast replicating plasmids or yeast centromeric plasmids.

[0197] The presently disclosed subject matter further provides host cells comprising the vectors disclosed herein. In certain non-limiting embodiments, the host cells can be T cells, Chinese Hamster Ovary (CHO) cells, E. coli cells, NSO cells, and Sp2 / 0 cells. In certain embodiments, the host cells transformed with an expression vector can be grown under conditions allowing expression and production of a presently disclosed antibody or antigenbinding fragment thereof.

[0198] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein can be synthesized and purified by any technique that allows for the formation of a stable antibody or antigen-binding fragments thereof. For example, but without any limitation, the antibodies or antigen-binding fragments thereof disclosed herein can be recovered from cells either as soluble polypeptides or as inclusion bodies. Additional exemplary methods for the purification of the antibodies or antigen-binding fragments thereof disclosed herein include ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, and gel filtration.

[0199] 6. Pharmaceutical Compositions and Methods of Treatment

[0200] The presently disclosed subject matter provides compositions comprising a presently disclosed anti-mesothelin antibody or an antigen-binding fragment thereof, a presently disclosed immunoconjugate, a presently disclosed multi-specific molecule. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0201] Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.

[0202] The presently disclosed subject matter provides various methods of using the anti- mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multispecific molecule, and the composition disclosed herein. For example, the presently disclosed subject matter provides methods for treating or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering one or more of the anti- mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multispecific molecule, or the composition disclosed herein to the subject. In certain embodiments, the disease or disorder is associated with mesothelin. In certain embodiments, the disease or disorder is associated with overexpression of mesothelin. In certain embodiments, the disease or disorder is tumor. The presently disclosed subject matter provides methods of reducing tumor burden in a subject. In certain embodiments, the method comprises administering one or more of the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein to the subject. The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject.

[0203] The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject having a tumor. In certain embodiments, the method comprises administering one or more of the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein to the subj ect. The method can reduce or eradicate tumor burden in the subj ect.

[0204] The presently disclosed subject matter further provides methods for treating and / or preventing a tumor in a subject. In certain embodiments, the method comprises administering one or more of the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein to the subject.

[0205] Such methods comprise administering the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof in an amount effective, a presently disclosed composition (e.g., a pharmaceutical composition) to achieve the desired effect, be it palliation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0206] In certain embodiments, the tumor or neoplasia can be treated by the presently disclosed cells and compositions include a solid tumor (e.g., mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, and / or cholangiocarcinoma).

[0207] In certain embodiments, the tumor or neoplasia is associated with mesothelin. In certain embodiments, the tumor or neoplasia expresses mesothelin. In certain embodiments, the tumor is cancer.

[0208] In certain embodiments, the tumor or neoplasia is a solid tumor. Non-limiting examples of solid tumors include mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, and cholangiocarcinoma.

[0209] In certain embodiments, the neoplasm is a solid tumor. The neoplasm can a primary tumor or primary cancer. In addition, the neoplasm can be in metastatic status.

[0210] Cancers whose growth may be inhibited using the immunoresponsive cells of the presently disclosed subject matter comprise cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include mesothelioma, lung cancer (e.g., nonsmall cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, cervical cancer, and salivary gland cancer. Additionally, the presently disclosed subject matter comprises refractory or recurrent malignancies whose growth may be inhibited using the immunoresponsive cells of the presently disclosed subject matter.

[0211] Examples of other neoplasms or cancers that may be treated using the methods of the presently disclosed subject matter include bone cancer, intestinal cancer, liver cancer, skin cancer, cancer of the head or neck, melanoma (cutaneous or intraocular malignant melanoma), renal cancer (e.g. clear cell carcinoma), throat cancer, prostate cancer (e.g. hormone refractory prostate adenocarcinoma), blood cancers (e.g. leukemias, lymphomas, and myelomas), uterine cancer, rectal cancer, cancer of the anal region, bladder cancer, brain cancer, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin’s disease, non-Hodgkin’s disease), cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, include Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0212] The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.

[0213] Any suitable method or route can be used to administer a presently disclosed anti- mesothelin antibody, and optionally, to co-administer antineoplastic agents. Routes of administration include, but are not limited to, oral, intravenous, intraperitoneal, subcutaneous, intramuscular, intranodal, intratumoral, intraosseous, intrathecal, pleural, intrapleural, topical, and direct administration. It should be emphasized, however, that the presently disclosed subject matter is not limited to any particular method or route of administration.

[0214] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can be administered as a conjugate, which binds specifically to the receptor and delivers a toxic, lethal payload following ligand-toxin internalization.

[0215] In certain embodiments, the presently disclosed methods include administering the anti- mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multispecific molecule, or the composition disclosed herein in combination with a second therapeutic agent. As used here, the term “in combination with” means that the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein, and one or more agents, e.g., an second therapeutic agent, are administered to a subject as part of a treatment regimen or plan.

[0216] In certain embodiments, the second therapeutic agent targets components of the immune system to fight cancer can be used with the presently disclosed methods. Non-limiting examples of second therapeutic agents include immune checkpoint inhibitors, dendritic cells, therapeutic antibodies (e.g., anti-CD33 antibodies, anti-CDl lb antibodies), cancer vaccines, cytokines (e.g., IL-12, GM-CSF, IL-2, IFNP, IFNy, MIP-1, MCP-1, IL-8), Bacillus Calmette- Guerin (BCG), and any combinations thereof. In certain embodiments, the second therapeutic agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is selected from anti-PDl antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, anti-BTLA antibodies, anti-TIM3 antibodies, anti-LAG-3 antibodies, and any combinations thereof. For example, but without any limitation, the immune checkpoint inhibitor can be pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cemiplimab (LIBTAYO®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), durvalumab (IMFINZI®), or ipilimumab (YERVOY®).

[0217] In certain embodiments, the second therapeutic agent is a chemotherapy. For example, but without any limitation, chemotherapy includes CHOP (cyclophosphamide, doxorubicin, vincristie, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or any other multidrug regimens.

[0218] In certain embodiments, the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent can be administered to the subject as part of a treatment regimen. In certain embodiments, the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent can be administered concurrently to the subject. In certain embodiments, the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent can be administered at the same time. In certain embodiments, the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent can be administered sequentially in any order (e.g., the anti-mesothelin antibodies or antigenbinding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein are administered to the subject after the second therapeutic agent is administered) or at different points in time (e.g., the anti-mesothelin antibodies or antigenbinding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent are administered to the subject on the same day but different hours; or the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein and the second therapeutic agent are administered to the subject in the same week but on different days).

[0219] 7. Diagnostic and Prognostic Methods

[0220] The presently disclosed anti-mesothelin antibodies, antigen-binding fragments thereof, multi-specific molecules, and nucleic acids encode thereof can be used for diagnostic and prognostic applications as well as use as research tools for detection of mesothelin in a biological sample, in a cell, a tissue, or a blood sample. The presently disclosed subject matter provides methods for detecting mesothelin in a cell, a tissue, or a blood sample. In certain embodiments, the method comprises: contacting a cell, a tissue, or a blood sample with the antibody, antigen-binding fragment thereof, or multi-specific molecule disclosed herein, wherein the antibody, antigen-binding fragment thereof or multi-specific molecule comprises a detectable label; and determining the amount of the labeled antibody, antigen-binding fragment thereof, or multi-specific molecule bound to the cell, tissue, or blood sample by measuring the amount of detectable label associated with the cell or tissue, wherein the amount of bound antibody, antigen-binding fragment thereof, or multi-specific molecule indicates the amount of mesothelin in the cell, tissue, or a blood sample. The cell or tissue can be any cell or tissue, including any normal, healthy, or cancerous cells and tissues. In certain embodiments, the blood sample is a peripheral blood sample.

[0221] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can be used in methods known in the art relating to the localization and / or quantitation of mesothelin polypeptides (e.g., for use in measuring levels of the mesothelin protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the polypeptide, and the like). The presently disclosed anti-mesothelin antibodies or antigenbinding fragments thereof can be used to isolate a mesothelin polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can facilitate the purification of natural immunoreactive.

[0222] Mesothelin proteins from biological samples, e.g., mammalian sera or cells as well as recombinantly-produced immunoreactive mesothelin proteins expressed in a host system. Moreover, anti-mesothelin antibodies of the present technology can be used to detect an immunoreactive mesothelin protein (e.g., in plasma, a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the immunoreactive polypeptide. The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can be used diagnostically to monitor immunoreactive mesothelin protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As noted above, the detection can be facilitated by coupling (i.e., physically linking) the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof to a detectable substance.

[0223] An exemplary method for detecting the presence or absence of an immunoreactive mesothelin protein in a biological sample comprises contacting a biological sample from a subject with a presently disclosed anti-mesothelin antibody or an antigen-binding fragment thereof, wherein the presence of an immunoreactive mesothelin protein is detected in the biological sample. Detection may be accomplished by means of a detectable label attached to the antibody.

[0224] The term “labeled” with regard to the anti-mesothelin antibody or antigen-binding fragment thereof is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.

[0225] In certain embodiments, the presently disclosed anti-mesothelin antibodies or antigenbinding fragments thereof are conjugated to one or more detectable labels. For such uses, the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof may be detectably labeled by covalent or non-covalent attachment of a chromogenic, enzymatic, radioisotopic, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance contrast agent or other label.

[0226] The presently disclosed detection methods can be used to detect an immunoreactive mesothelin protein in a biological sample in vitro as well as in vivo. Non-limiting examples of in vitro techniques for detection of an immunoreactive mesothelin protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, radioimmunoassay, and immunofluorescence. Furthermore, in vivo techniques for detection of an immunoreactive mesothelin protein include introducing into a subject a labeled anti- mesothelin antibody or an antigen-binding fragment thereof. For example, the anti-mesothelin antibody or antigen-binding fragment thereof can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In certain embodiments, the biological sample comprises mesothelin protein molecules from the test subject.

[0227] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can be used to assay immunoreactive mesothelin protein levels in a biological sample (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes or other radioactive agent, such as iodine (1251,1211,131I), carbon (14C), sulfur (35S), tritium (3H), indium (H 1In), and technetium (99mTc), and fluorescent labels, such as fluorescein, rhodamine, and green fluorescent protein (GFP), as well as biotin.

[0228] In addition to assaying immunoreactive mesothelin protein levels in a biological sample, the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof may be used for in vivo imaging of mesothelin. Antibodies useful for this method include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the anti- mesothelin antibodies by labeling of nutrients for the relevant scFv clone.

[0229] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof, which are labeled with an appropriate detectable imaging moiety (such as a radioisotope (e.g.,1311,1 UIN "mTc,18F,89Zr), a radio-opaque substance, or a material detectable by nuclear magnetic resonance) are introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc. The labeled anti-mesothelin antibody or antigen-binding fragment thereof then accumulates at the location of cells which contain the specific target polypeptide. For example, the labeled anti-mesothelin antibodies or antigen-binding fragments thereof accumulate within the subject in cells and tissues in which the mesothelin protein has localized.

[0230] Thus, the presently disclosed subject matter provides diagnostic methods of a medical condition. In certain embodiments, the method comprises: (a) assaying the expression of immunoreactive mesothelin protein by measuring binding of a presently disclosed anti- mesothelin antibody or an antigen-binding fragment thereof in cells or body fluid of an individual; and (b) comparing the amount of immunoreactive mesothelin protein present in the sample with a standard reference, wherein an increase or decrease in immunoreactive mesothelin protein levels compared to the standard is indicative of a medical condition.

[0231] Furthermore, the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof may be used to purify immunoreactive mesothelin protein from a sample. In certain embodiments, the antibodies are immobilized on a solid support. Non-limiting examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art.

[0232] The simplest method to bind the antigen to the antibody-support matrix is to collect the beads in a column and pass the antigen solution down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using low flow rates. The immobilized antibody captures the antigen as it flows past. Alternatively, an antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating the slurry, allowing maximum contact between the antigen and the immobilized antibody. After the binding reaction has been completed, the slurry is passed into a column for collection of the beads. The beads are washed using a suitable washing buffer and then the pure or substantially pure antigen is eluted.

[0233] An antibody or polypeptide of interest can be conjugated to a solid support, such as a bead. In addition, a first solid support such as a bead can also be conjugated, if desired, to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugation of a polypeptide to a support. Accordingly, any of the conjugation methods and means disclosed herein with reference to conjugation of a polypeptide to a solid support can also be applied for conjugation of a first support to a second support, where the first and second solid support can be the same or different.

[0234] Appropriate linkers, which can be cross-linking agents, for use for conjugating a polypeptide to a solid support include a variety of agents that can react with a functional group present on a surface of the support, or with the polypeptide, or both. Reagents useful as crosslinking agents include homo-bi-functional and, in particular, hetero-bi-functional reagents. Useful bi-functional cross-linking agents include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected to provide a selectively cleavable bond between a polypeptide and the solid support. For example, a photolabile cross-linker, such as 3-amino-(2-nitrophenyl)propionic acid can be employed as a means for cleaving a polypeptide from a solid support. (Brown et al., Mol. Divers, pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and US. Pat. No. 5,643,722). Other cross-linking reagents are well-known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

[0235] An antibody or polypeptide can be immobilized on a solid support, such as a bead, through a covalent amide bond formed between a carboxyl group functionalized bead and the amino terminus of the polypeptide or, conversely, through a covalent amide bond formed between an amino group functionalized bead and the carboxyl terminus of the polypeptide. In addition, a bi-functional trityl linker can be attached to the support, e.g., to the 4-nitrophenyl active ester on a resin, such as a Wang resin, through an amino group or a carboxyl group on the resin via an amino resin. Using a bi-functional trityl approach, the solid support can require treatment with a volatile acid, such as formic acid or trifluoroacetic acid to ensure that the polypeptide is cleaved and can be removed. In such a case, the polypeptide can be deposited as a beadless patch at the bottom of a well of a solid support or on the flat surface of a solid support. After addition of a matrix solution, the polypeptide can be desorbed into a MS.

[0236] Hydrophobic trityl linkers can also be exploited as acid-labile linkers by using a volatile acid or an appropriate matrix solution, e.g., a matrix solution containing 3-HPA, to cleave an amino linked trityl group from the polypeptide. Acid lability can also be changed. For example, trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can be changed to the appropriate p-substituted, or more acid-labile tritylamine derivatives, of the polypeptide, i.e., trityl ether and tritylamine bonds can be made to the polypeptide. Accordingly, a polypeptide can be removed from a hydrophobic linker, e.g., by disrupting the hydrophobic attraction or by cleaving tritylether or tritylamine bonds under acidic conditions, including, if desired, under typical MS conditions, where a matrix, such as 3-HPA acts as an acid.

[0237] Orthogonally cleavable linkers can also be useful for binding a first solid support, e.g., a bead to a second solid support, or for binding a polypeptide of interest to a solid support. Using such linkers, a first solid support, e.g., a bead, can be selectively cleaved from a second solid support, without cleaving the polypeptide from the support; the polypeptide then can be cleaved from the bead at a later time. For example, a disulfide linker, which can be cleaved using a reducing agent, such as DTT, can be employed to bind a bead to a second solid support, and an acid cleavable bi-functional trityl group could be used to immobilize a polypeptide to the support. As desired, the linkage of the polypeptide to the solid support can be cleaved first, e.g., leaving the linkage between the first and second support intact. Trityl linkers can provide a covalent or hydrophobic conjugation and, regardless of the nature of the conjugation, the trityl group is readily cleaved in acidic conditions.

[0238] For example, a bead can be bound to a second support through a linking group which can be selected to have a length and a chemical nature such that high density binding of the beads to the solid support, or high density binding of the polypeptides to the beads, is promoted. Such a linking group can have, e.g., "tree-like" structure, thereby providing a multiplicity of functional groups per attachment site on a solid support. Examples of such linking group; include polylysine, polyglutamic acid, penta-erythrole and tris-hydroxy-aminomethane.

[0239] Noncovalent Binding Association. An antibody or polypeptide can be conjugated to a solid support, or a first solid support can also be conjugated to a second solid support, through a noncovalent interaction. For example, a magnetic bead made of a ferromagnetic material, which is capable of being magnetized, can be attracted to a magnetic solid support, and can be released from the support by removal of the magnetic field. Alternatively, the solid support can be provided with an ionic or hydrophobic moiety, which can allow the interaction of an ionic or hydrophobic moiety, respectively, with a polypeptide, e.g., a polypeptide containing an attached trityl group or with a second solid support having hydrophobic character.

[0240] A solid support can also be provided with a member of a specific binding pair and, therefore, can be conjugated to a polypeptide or a second solid support containing a complementary binding moiety. For example, a bead coated with avidin or with streptavidin can be bound to a polypeptide having a biotin moiety incorporated therein, or to a second solid support coated with biotin or derivative of biotin, such as iminobiotin.

[0241] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, biotin, e.g., can be incorporated into either a polypeptide or a solid support and, conversely, avidin or other biotin binding moiety would be incorporated into the support or the polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzyme, and their substrates, a nucleotide sequence and its complementary sequence, an antibody and the antigen to which it interacts specifically, and other such pairs knows to those skilled in the art.

[0242] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof are useful in diagnostic methods. As such, the presently disclosed subject matter provides methods using the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof in diagnosis of mesothelin activity in a subject. The presently disclosed anti- mesothelin antibodies or antigen-binding fragments thereof may be selected such that they have any level of epitope binding specificity and high binding affinity to a mesothelin polypeptide.

[0243] The presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof can be used to detect an immunoreactive mesothelin protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject is at an early stage of cancer. In certain embodiments, the early stage of cancer is determined by the level or expression pattern of mesothelin protein in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsi ed body tissue.

[0244] Immunometric or sandwich assays are one format for the diagnostic methods of the present technology. Such assays use one antibody, e.g., the anti-mesothelin antibody or a population of anti-mesothelin antibodies immobilized to a solid phase, and another anti- mesothelin antibody or a population of anti-mesothelin antibodies in solution. Typically, the solution anti-mesothelin antibody or population of anti-mesothelin antibodies is labeled. If an antibody population is used, the population can contain antibodies binding to different epitope specificities within the target polypeptide. Accordingly, the same population can be used for both solid phase and solution antibody. If anti-mesothelin monoclonal antibodies are used, first and second mesothelin monoclonal antibodies having different binding specificities are used for the solid and solution phase. Solid phase (also referred to as “capture”) and solution (also referred to as “detection”) antibodies can be contacted with target antigen in either order or simultaneously. If the solid phase antibody is contacted first, the assay is referred to as being a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as being a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the mesothelin protein with the anti- mesothelin antibody, a sample is incubated for a period that usually varies from about 10 min to about 24 hr and is usually about 1 hr. A wash step is then performed to remove components of the sample not specifically bound to the anti-mesothelin antibody being used as a diagnostic reagent. When solid phase and solution antibodies are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, typically by detecting a label linked to the solid phase through binding of labeled solution antibody. Usually for a given pair of antibodies or populations of antibodies and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of the immunoreactive mesothelin protein in samples being tested are then read by interpolation from the calibration curve (i.e., standard curve). Analyte can be measured either from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of the mesothelin protein in a sample.

[0245] Suitable supports for use in the above methods include, e.g., nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, and also particles, such as agarose, a dextran-based gel, dipsticks, particulates, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEXTM (Amersham Pharmacia Biotech, Piscataway N.J.), and the like. Immobilization can be by absorption or by covalent attachment. Optionally, anti- mesothelin antibodies can be joined to a linker molecule, such as biotin for attachment to a surface bound linker, such as avidin.

[0246] In certain embodiments, the presently disclosed anti-mesothelin antibody or antigenbinding fragment thereof is conjugated to a diagnostic agent. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography or ultrasound), and the radioactive label can be a gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope. A diagnostic agent is a molecule which is administered conjugated to an antibody moiety, i.e., antibody or antibody fragment, or subfragment, and is useful in diagnosing or detecting a disease by locating the cells comprising the antigen.

[0247] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). In certain embodiments, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. Chelates may be coupled to the presently disclosed anti-mesothelin antibodies or antigen-binding fragments thereof using standard chemistries. The chelate is normally linked to the antibody by a group which enables formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking.

[0248] 8. Kits

[0249] The presently disclosed subject matter provides kits for treatment or ameliorating a disease or disorder associated with mesothelin (e.g., a metastatic cancer cell), and / or detecting mesothelin. In certain embodiments, the kit comprises the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein. In certain embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0250] In certain embodiments, the kit further comprises instructions for administering the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein to a subject in need the treatment. The instructions can generally include information about the use of the anti-mesothelin antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, and the composition disclosed herein for the treatment or ameliorating a disease or disorder. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment and / or prevention of a tumor or neoplasm or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0251] 9. Exemplary Embodiments

[0252] Al. An anti-mesothelin antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

[0253] A2. The anti-mesothelin antibody or an antigen-binding fragment thereof of Al, wherein the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0254] A3. The anti-mesothelin antibody or an antigen-binding fragment thereof of Al or A2, comprising (a) a heavy chain variable region comprising 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12; or (b) a light chain variable region comprising 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% at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0255] A4. The anti-mesothelin antibody or an antigen-binding fragment thereof of any one of A1-A3, comprising a heavy chain variable region comprising 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%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising 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%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0256] A5. The antibody or antigen-binding fragment thereof of any one of A1-A4, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0257] A6. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A5, wherein the antibody comprises a heavy chain constant region and / or a light chain constant region.

[0258] A7. The anti-mesothelin antibody or antigen-binding fragment thereof of A6, wherein: (a) the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or (b) the light chain constant region comprises 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% at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0259] A8. The anti-mesothelin antibody or antigen-binding fragment thereof of A6 or A7, wherein: (a) the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or (b) the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0260] A9. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A8, wherein the antibody comprises a human variable region framework region.

[0261] A10. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A9, which is a fully human or an antigen-binding fragment thereof.

[0262] Al l. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A8, which is a chimeric antibody or an antigen-binding fragment thereof.

[0263] A12. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A8, which is a humanized antibody or an antigen-binding fragment thereof.

[0264] Al 3. The anti-mesothelin antibody or antigen-binding fragment thereof of any one of A1-A12, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, variable fragment (Fv), or single chain variable region (scFv).

[0265] A14. The anti-mesothelin antibody or antigen-binding fragment thereof of A13, wherein the antigen-binding fragment is an scFv.

[0266] Al 5. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0267] Al 6. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a single chain variable region (scFv) comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. Al 7. The anti-mesothelin antibody or an antigen-binding fragment thereof of Al 6, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0268] Al 8. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0269] Al 9. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0270] A20. An antibody or an antigen-binding fragment thereof, which cross-competes for binding to mesothelin with an anti-mesothelin antibody or an antigen-binding fragment thereof of any one of Al -Al 9.

[0271] A21. An antibody or an antigen-binding fragment thereof, which binds to the same epitope region on mesothelin as an anti-mesothelin antibody or an antigen-binding fragment thereof of any one of Al -Al 9.

[0272] A22. An immunoconjugate comprising the anti-mesothelin antibody or antigen-binding fragment thereof of any one of Al A-21, linked to a therapeutic agent.

[0273] A23. The immunoconjugate of A22, wherein the therapeutic agent is a drug, a cytotoxin, or a radioactive isotope.

[0274] A24. A multi-specific molecule comprising the antibody or antigen-binding fragment thereof of any one of A1-A21, linked to one or more functional moi eties.

[0275] A25. The multi-specific molecule of A23, wherein the one or more functional moi eties have a different binding specificity than the antibody or antigen binding fragment thereof.

[0276] A26. A composition comprising the antibody of any one of A1-A21, the immunoconjugate of A22 or A23, or the multi-specific molecule of A24 or A25.

[0277] A27. The composition of A26, which is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0278] A28. A nucleic acid that encodes an antibody or antigen-binding fragment thereof of any one of A1-A21.

[0279] A29. A nucleic acid that encodes a heavy chain variable region of an antibody or antigen-binding fragment thereof of any one of A1-A21.

[0280] A30. A nucleic acid that encodes a light chain variable region of an antibody or antigenbinding fragment thereof of any one of A1-A21.

[0281] A31. A vector comprising the nucleic acid of any one of A28-A30.

[0282] A32. The vector of A31, wherein the vector is a low copy number vector. A33. A host cell comprising the vector of A31 or A32.

[0283] A34. A method for detecting mesothelin in a whole cell, a tissue, or a blood sample, comprising: (a) contacting a cell, tissue or blood sample with the antibody or antigen-binding fragment thereof of any one of A1-A21, wherein the antibody or antigen-binding fragment thereof comprises a detectable label; and (b) determining the amount of the labeled antibody or antigen-binding fragment thereof bound to the cell, tissue or blood sample by measuring the amount of detectable label associated with said cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of mesothelin in the cell, tissue or blood sample.

[0284] A35. A method of treating or ameliorating a disease or disorder associated with mesothelin in a subject, comprising administering to the subject the antibody or antigenbinding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27.

[0285] A36. The method of A35, wherein the disease or disorder is a tumor.

[0286] A37. A method of reducing tumor burden in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27.

[0287] A38. The method of A37, wherein the method reduces the number of the tumor cells, reduces the tumor size, and / or eradicates the tumor in the subject.

[0288] A39. A method of treating and / or preventing a tumor in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of Al-21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27.

[0289] A40. A method of increasing or lengthening survival of a subject having a tumor, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27.

[0290] A41. The method of A40, wherein the method reduces or eradicates tumor burden in the subject.

[0291] A42. The method of any one of A35-A41, wherein the tumor is a solid tumor.

[0292] A43. The method of A42, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

[0293] A44. The method of any one of A35-A43, wherein the subject is a human.

[0294] A45. A kit comprising the antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or 25, the composition of A26 or K 1 the nucleic acid of any one of A28-A30, the vector of A31 or A32, or the host cell of A32.

[0295] A46. The kit of A45, wherein the kit further comprises written instructions for using the antibody or antigen-binding fragment thereof, immunoconjugate, multi-specific molecule, composition, nucleic acid, vector, or host cell.

[0296] A47. The kit of A45 or A46 for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor.

[0297] Bl. The antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27 for use in treating or ameliorating a disease or disorder associated with mesothelin in a subject.

[0298] B2. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multispecific molecule, or the composition for use of B 1, wherein the disease or disorder is a tumor.

[0299] B3. The antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27 for use in reducing tumor burden in a subject.

[0300] B4. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multispecific molecule, or the composition for use of B3, wherein the method reduces the number of the tumor cells, reduces the tumor size, and / or eradicates the tumor in the subject.

[0301] B5. The antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27 for use in treating and / or preventing a tumor in a subject.

[0302] B6. The antibody or antigen-binding fragment thereof of any one of A1-A21, the immunoconjugate of A22 or A23, the multi-specific molecule of A24 or A25, or the composition of A26 or A27 for use in increasing or lengthening survival of a subject having a tumor. B7. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multispecific molecule, or the composition for use of B4, wherein the method reduces or eradicates tumor burden in the subject.

[0303] B8. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multispecific molecule, or the composition for use of B1-B7, wherein the tumor is a solid tumor.

[0304] B9. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multispecific molecule, or the composition for use of B8, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

[0305] BIO. The antibody or antigen-binding fragment thereof, the immunoconjugate, the multi-specific molecule, or the composition for use of B1-B9, wherein the subject is a human.

[0306] EXAMPLES

[0307] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the antibodies, make and use immunoconjugates of such antibodies, compositions comprising thereof, screening, and therapeutic methods of the presently disclosed subject matter, and are not intended to limit the scope of what the inventors regard as their presently disclosed subject matter. It is understood that various other embodiments can be practiced, given the general description provided above.

[0308] Example 1

[0309] The present example illustrates scFv with cross-reactivity to human and mouse mesothelin developed using a phage based biopanning process. A phage display library with a diversity of 5.37xlO10different clones was subjected to six rounds of panning against human and mouse mesothelin. The identified binders were further analyzed by ELISA. Phage DNA was extracted for antibody sequencing and the binding specificity of the identified clone was validated in mesothelin expressing cell lines by flow cytometry analysis.

[0310] Biopanning

[0311] Six rounds of solid phase panning were carried out with peptide antigens as shown to Table 3. Briefly, the peptide antigens were coated onto tubes followed by standard washing and blocking steps. The tubes were incubated with phage library and washed to remove the unbound phages. The bound phages were eluted with glycine-HCl and the concentration of the eluted phages were determined by calculating plaque-forming units (pfu) in an E.coli TGI based assay. Eluted phages were amplified with helper phage and used in the subsequent rounds of biopanning or ELISA assays. As seen in Table 4, the biopanning data shows significant and progressive enrichment (bold) over the rounds of biopanning. Since significant enrichment was observed after six rounds, the assay was stopped in order to prevent a decrease in phage diversity.

[0312] Table 3. Selection conditions for each round

[0313] Table 4. Biopanning results

[0314] Polyclonal phase ELISA (phase pools screenins)

[0315] Pooled phage screening was carried out with the eluted phages from each round of biopanning. Briefly, an ELISA plate was coated with antigen (5 pg / mL) or control followed by standard washing and blocking steps. The prepared ELISA plate was incubated with amplified phages followed by standard washing and incubation with a phage specific HRP antibody. The plate was then incubated with TMB solution followed by HC1 and the corresponding ELISA signal was read at 450 nm. As seen in Tables 5A and 5B, enrichment for antigen specific binders started slightly from Round 3, as indicated by the higher ELISA signal compared to the control (NC) at low dilutions of phage. Enrichment strengthened in Round 4 with very high specific ELISA signals from Round 5, with all concentrations nearing saturation at Round 6. Rounds 4, 5 and 6 were selected for monoclonal ELISA in which 384 single phage-binders were tested in order to maximize the chances of identifying all relevant clones.

[0316] Table 5 A. Results of polyclonal phage ELISA

[0317] Table 5B. Results of polyclonal phage ELISA

[0318] Agl : 4 pg / ml Human Mesothelin; Ag2: 4 pg / ml Mouse Mesothelin; NC: PBS.

[0319] Monoclonal phase ELISA (single phage binders screenins) Single phage binders were generated by randomly picking and amplifying 288 single

[0320] TGI clones from the pfu assay of Rounds 4, 5, and 6. ELISA plates were coated with antigen (5 pg / mL) or control followed by the washing, blocking steps, and incubation steps described above.

[0321] The highest proportion of positive clones were observed from Round 6 (Tables 15-17), followed by Round 5 (Tables 12-14), and Round 4 (Table 6-11). Since a high proportion of positive clones were observed on the ELISA plates from Rounds 5 and 6, all clones highlighted in bold (Tables 12-17) were sent for sequencing. One unique sequence was obtained from the positive binders, indicating that the cross-panning was successful at eliminating the non-cross reactive binders. Clone R6P1-B1 was selected for further analysis.

[0322] Table 6. Results of R4P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0323] Table 7. Results of R4P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0324] Table 8. Results of R4P1 output phage ELISA for NC (PBS)

[0325] Table 9. Results of R4P2 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0326] Table 10. Results of R4P2 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0327] Table 11. Results of R4P2 output phage ELISA for NC (PBS)

[0328] Table 12. Results of R5P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0329] Table 13. Results of R5P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0330] Table 14. Results of R5P1 output phage ELISA for NC (PBS)

[0331] Table 15. Results of R6P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0332] Table 16. Results of R6P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0333] Table 17. Results of R6P1 output phage ELISA for NC (PBS)

[0334] Confirmation ELISA after sequencing

[0335] A second verification ELISA was carried out on clone R6P1-B1. As seen in Table 18, the single identified unique clone shows human-mouse cross-reactivity against both recombinant protein antigens.

[0336] Table 18. Result of ELISA verification of 21055-R6P1-B1

[0337] AG1 : 4 pg / mL Human Mesothelin; AG2: 4 pg / mL Mouse Mesothelin; NC: PBS.

[0338] Validation by FACS

[0339] The binding specificity of clone R6P1-B1 was validated by FACS analysis in cultured human mesothelin expressing MGM cells, mouse mesothelin expressing AB 12 cells and non- mesothelin expressing MSTO-211H cells. As seen in Figure 1 and Table 19, the 21055-R6P1- B1 clone showed a strong positive signal in flow cytometry against the human mesothelin expressing MGM cells, confirming the activity of the clone against the native form of the protein. A weak positive signal was detected on the mouse mesothelin expressing AB 12 cells, which likely indicates that there is low expression of mouse mesothelin in AB 12 cells rather than a lack of activity of the phage clone, since an equivalent ELISA signal was observed against the recombinant human and mouse mesothelin proteins in Table 18.

[0340] Table 19. Result of FACS verification

[0341] % True positive Cells = (% Positive Cells Stained - % Positive Cells Unstained) - (% Positive Stained MSTO Cells - % Positive Unstained MSTO Cells)

[0342] Structure prediction analysis

[0343] The sequence identified from the biopanning process was compared to the previously characterized mesothelin (m912) scFv. The presently disclosed scFv was compared to m912 by sequence alignment analysis (Table 20). Further structural analysis highlights differences in the electrostatic forces, lipophilicity, and the predicted binding pocket between m912 and the presently disclosed scFv (Figures 2A-2E).

[0344] Table 20. Sequence alignment: m912 scFv vs. R6P1-B1 scFv

[0345] Embodiments of the presently disclosed subject matter

[0346] From the foregoing description, it will be apparent that variations and modifications may be made to the presently disclosed subject matter to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An anti-mesothelin antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

2. The anti-mesothelin antibody or an antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

3. The anti-mesothelin antibody or an antigen-binding fragment thereof of claim 1, comprising(a) a heavy chain variable region comprising 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12; or(b) a light chain variable region comprising 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% at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13.

4. The anti-mesothelin antibody or an antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising 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%, at least about 100% identical to theamino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising 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%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 13.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

6. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a heavy chain constant region and / or a light chain constant region.

7. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 6, wherein:(a) the heavy chain constant region comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or(b) the light chain constant region comprises 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% at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 22.

8. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 6, wherein:(a) the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and / or(b) the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 22.

9. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a human variable region framework region.

10. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, which is a fully human or an antigen-binding fragment thereof.

11. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, which is a chimeric antibody or an antigen-binding fragment thereof.

12. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, which is a humanized antibody or an antigen-binding fragment thereof.

13. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, variable fragment (Fv), or single chain variable region (scFv).

14. The anti-mesothelin antibody or antigen-binding fragment thereof of claim 13, wherein the antigen-binding fragment is an scFv.

15. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

16. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a single chain variable region (scFv) comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16.

17. The anti-mesothelin antibody or an antigen-binding fragment thereof of claim 16, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

18. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

19. An anti-mesothelin antibody or an antigen-binding fragment thereof comprising the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16.

20. An antibody or an antigen-binding fragment thereof, which cross-competes for binding to mesothelin with an anti-mesothelin antibody or an antigen-binding fragment thereof of any one of claims 1-19.

21. An antibody or an antigen-binding fragment thereof, which binds to the same epitope region on mesothelin as an anti-mesothelin antibody or an antigen-binding fragment thereof of any one of claims 1-19.

22. An immunoconjugate comprising the anti-mesothelin antibody or antigen-binding fragment thereof of any one of claims 1-19, linked to a therapeutic agent.

23. The immunoconjugate of claim 22, wherein the therapeutic agent is a drug, a cytotoxin, or a radioactive isotope.

24. A multi-specific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1-19, linked to one or more functional moi eties.

25. The multi-specific molecule of claim 23, wherein the one or more functional moieties have a different binding specificity than the antibody or antigen binding fragment thereof.

26. A composition comprising the antibody of any one of claims 1-21, the immunoconjugate of claim 22 or 23, or the multi-specific molecule of claim 24 or 25.

27. The composition of claim 26, which is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

28. A nucleic acid that encodes an antibody or antigen-binding fragment thereof of any one of claims 1-21.

29. A nucleic acid that encodes a heavy chain variable region of an antibody or antigen-binding fragment thereof of any one of claims 1-21.

30. A nucleic acid that encodes a light chain variable region of an antibody or antigenbinding fragment thereof of any one of claims 1-21.

31. A vector comprising the nucleic acid of any one of claims 28-30.

32. The vector of claim 31, wherein the vector is a low copy number vector.

33. A host cell comprising the vector of claim 31 or 32.

34. A method for detecting mesothelin in a whole cell, a tissue, or a blood sample, comprising:(a) contacting a cell, tissue or blood sample with the antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof comprises a detectable label; and(b) determining the amount of the labeled antibody or antigen-binding fragment thereof bound to the cell, tissue or blood sample by measuring the amount of detectable label associated with said cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of mesothelin in the cell, tissue or blood sample.

35. A method of treating or ameliorating a disease or disorder associated with mesothelin in a subject, comprising administering to the subject the antibody or antigenbinding fragment thereof of any one of claims 1-21, the immunoconjugate of claim 22 or 23, the multi-specific molecule of claim 24 or 25, or the composition of claim 26 or 27.

36. The method of claim 35, wherein the disease or disorder is a tumor.

37. A method of reducing tumor burden in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-21, the immunoconjugate of claim 22 or 23, the multi-specific molecule of claim 24 or 25, or the composition of claim 26 or 27.

38. The method of claim 37, wherein the method reduces the number of the tumor cells, reduces the tumor size, and / or eradicates the tumor in the subject.

39. A method of treating and / or preventing a tumor in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-21, the immunoconjugate of claim 22 or 23, the multi-specific molecule of claim 24 or 25, or the composition of claim 26 or 27.

40. A method of increasing or lengthening survival of a subject having a tumor, comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1-21, the immunoconjugate of claim 22 or 23, the multi-specific molecule of claim 24 or 25, or the composition of claim 26 or 27.

41. The method of claim 40, wherein the method reduces or eradicates tumor burden in the subject.

42. The method of any one of claims 35-41, wherein the tumor is a solid tumor.

43. The method of claim 42, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

44. The method of any one of claims 35-43, wherein the subject is a human.

45. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-21, the immunoconjugate of claim 22 or 23, the multi-specific molecule of claim 24 or 25, the composition of claim 26 or 27, the nucleic acid of any one of claims 28-30, the vector of claim 31 or 32, or the host cell of claim 32.

46. The kit of claim 45, wherein the kit further comprises written instructions for using the antibody or antigen-binding fragment thereof, immunoconjugate, multi-specific molecule, composition, nucleic acid, vector, or host cell.

47. The kit of claim 45 or 46 for treating or ameliorating a disease or disorder in a subject, reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor.

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