Monoclonal antibodies that bind the juxta-membrane region of mesothelin and uses thereof

Abstract

Monoclonal antibodies, including humanized antibodies, that specifically bind the juxta-membrane region of the tumor antigen mesothelin are described. Antibody conjugates, including chimeric antigen receptors and bispecific antibodies, that include a mesothelin-specific monoclonal antibody are also described. The mesothelin-specific antibodies and conjugates can be used, for example, in the treatment and diagnosis of a mesothelin-positive cancer.

Description

[0001] MONOCLONAL ANTIBODIES THAT BIND THE JUXTA-MEMBRANE REGION OF MESOTHELIN AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 550,703, filed February 7, 2024, which is herein incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure concerns monoclonal antibodies that specifically bind the tumor antigen mesothelin and conjugates thereof, such as chimeric antigen receptors (CARs) and bispecific antibodies. The disclosure further concerns use of the monoclonal antibodies and conjugates, such as in the treatment of mesothelin- expressing tumors.

[0006] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0007] The electronic sequence listing, submitted herewith as an XML file named 4239-111381-02. xml (28,544 bytes), created on January 22, 2025, is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] The mesothelin (MSLN) gene encodes a ~70 kDa precursor protein that is processed to a ~30 kDa N-terminal protein and a ~ 40 kDa C-terminal membrane -bound mature mesothelin (Hassan and Ho, Eur J Cancer 44:46-53, 2008). Mesothelin is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in malignant mesotheliomas (Chang et al., Cancer Res 52:181-186, 1992; Chang and Pastan, Proc Natl Acad Sci USA 93: 136-140, 1996) and other solid tumors, such as stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer and ovarian cancer (Hassan et al., Clin. Cancer Res. 10:3937-3942, 2004; McGuire et al., N. Engl. J. Med. 334: 1-6, 1996; Argani et al., Clin. Cancer Res. 7:3862-3868, 2001; Hassan et al., Appl. Immunohistochem. Mol. Morphol. 13:243-247, 2005; Li et al., Mol. Cancer Ther. 7:286-296, 2008; Yu et al., J Cancer 1:141-1749, 2010; Tchou et al., Breast Cancer Res Treat 133(2):799-804, 2012; U.S. Patent No. 7,081,518).

[0010] Mesothelin is a well-established tumor target in multiple solid tumors. Thus, a needs exists for the identification and selection of mesothelin-specific antibodies for the development of mesothelin-targeted immunotherapies .

[0011] SUMMARY

[0012] Disclosed herein are monoclonal antibodies that specifically bind the juxta-membrane region of the tumor antigen mesothelin. Antibody RO4 was isolated from a rabbit immunized with a C-terminal fragment of the mesothelin protein. A humanized form of RO4, referred to as hRO4, was also generated by humanizing the framework regions of RO4.

[0013] Provided herein are monoclonal antibodies and antigen-binding fragments that specifically bind mesothelin. The mesothelin-specific antibodies include the complementarity determining region (CDR) sequences (or the complete variable domains) of antibody RO4 or hRO4 as disclosed herein.

[0014] Also provided herein are conjugates that include a disclosed monoclonal antibody or antigenbinding fragment (or the CDR sequences of a disclosed monoclonal antibody). In some aspects, provided are CARs, CAR-expressing cells (such as T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages and induced pluripotent stem cells (iPSCs)), fusion proteins (such as Fc fusion proteins), immunoconjugates (such as immunotoxins), multi-specific antibodies (such as bispecific or trispecific antibodies), antibody-drug conjugates (ADCs), antibody-nanoparticle conjugates, and antibody-photon absorber conjugates (such as 1R700 conjugates for immunoPET imaging) that include a monoclonal antibody or antigen-binding fragment disclosed herein.

[0015] Further provided are nucleic acid molecules encoding a disclosed monoclonal antibody or antigenbinding fragment, CAR, immunoconjugate, multi-specific antibody, or fusion protein. In some aspects, the nucleic acid molecule is part of a vector. Also provided are isolated cells that include a nucleic acid molecule or vector disclosed herein.

[0016] Also provided are compositions that include a pharmaceutically acceptable carrier and a mesothelin- specific monoclonal antibody or antigen-binding fragment, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, or fusion protein disclosed herein. In some examples, the composition is lyophilized.

[0017] Methods of detecting expression of mesothelin in a sample are further provided. In some aspects, the method includes contacting the sample with a mesothelin-specific monoclonal antibody or antigenbinding fragment disclosed herein and detecting binding of the monoclonal antibody or antigen-binding fragment to the sample. Methods of diagnosing a subject as having a mesothelin-positive tumor are further provided. In some aspects, the method includes contacting a sample obtained from the subject with a mesothelin-specific monoclonal antibody or antigen-binding fragment disclosed herein and detecting binding of the monoclonal antibody to the sample.

[0018] Also provided are methods of treating a mesothelin-positive cancer in a subject, and methods of inhibiting tumor growth or metastasis of a mesothelin-positive cancer in a subject. In some aspects, the methods include administering to the subject a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein. In some aspects, the mesothelin-positive cancer is a solid tumor, such as, but not limited to, mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, colon cancer, or thymus cancer. Further provided herein are kits that include a monoclonal antibody or antigen-binding fragment, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and / or instructional materials.

[0019] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1: Binding of antibody RO4 to mesothelin. ELISA plates were coated with full-length mesothelin with a methionine at position 593 (593M), full-length mesothelin with a valine at position 593 (593V), and mesothelin with a C-terminal truncation of positions 296-591 (A296-591) or positions 296-585 (A296-585). After blocking the plate, various concentrations of RO4 were added, followed by anti-rabbit IgG-HRP for detection. Antibody RO4 binds 593M and 593V mesothelin equally well, but does not bind the C-terminal truncated forms of mesothelin.

[0022] FIGS. 2A-2D: Binding curves showing affinity of RO4 and humanized RO4 (hRO4) for the 593M and 593V variants of mesothelin. Sample concentrations tested included 70, 35, 17.5, 8.75, 4.38, 2.19 and 1.09 nM. Shown are binding of RO4 to 593M mesothelin (FIG. 2A), hRO4 binding to 593M mesothelin (FIG. 2B), RO4 binding to 593V mesothelin (FIG. 2C), and hRO4 binding to 593V mesothelin (FIG. 2D).

[0023] FIG. 3: Humanized RO4 binds more strongly to OVCAR8 cells than monoclonal mouse antibody 15B6 (ml5B6). Antibodies were conjugated with ALEXA FLUOR™ 647 (AF647) and mixed with OVCAR8 cells, incubated for 30 minutes at room temperature, washed, and analyzed by flow cytometry. A monoclonal antibody (“MN”) that recognizes the N-terminal region (but not the C-terminal region) of cellsurface MSLN was also tested. A non-specific IgG antibody was used as a negative control.

[0024] FIG. 4: Inhibition of mesothelin shedding by rabbit antibody RO4. A total of 50 Ltg / ml of RO4 was incubated with RH16, RH29, OVCAR8, or KLM1 cells for 24 hours. Media were collected and shed mesothelin was detected by human mesothelin R-PLEX kit. Antibody RO4 showed greater inhibition of mesothelin shedding than antibody ml5B6 in RH29 human mesothelioma cells. Inhibition of mesothelin shedding in the other cell types was similar between the two antibodies.

[0025] FIG. 5: Specific binding of RO4 to normal human tissues by immunohistochemistry. Included in the tissue microarray were normal tissues from 32 human organs collected from three different individuals.

[0026] FIGS. 6A-6C: Binding of humanized 15B6 (hl5B6) and humanized RO4 (hRO4) to human cancer cell lines RH16 (FIG. 6A), RH29 (FIG. 6B), and KLM1 (FIG. 6C). hRO4 exhibited stronger binding than h!5B6 for all three cell types tested. FIGS. 7A-7E: Inhibition of mesothelin shedding by antibodies RO4, 15B6 and h!5B6, compared to a control antibody. Shown is mesothelin shedding by (FIG. 7A) T3M4, (FIG. 7B) HeLa, (FIG. 7C) H06, (FIG. 7D) KPC-hM-911, and (FIG. 7E) KPC-hM-994 cells.

[0027] FIG. 8: Inhibition of mesothelin shedding by humanized RO4 (hRO4) and humanized 15B6 (hl5B6). A total of 50 txg / ni I of hRO4, hl6B6 or a control antibody was incubated with RH16, RH29, OVCAR8, KLM1, H226, H06 or PDA cells for 24 hours. Media were collected and shed mesothelin was detected by human mesothelin R-PLEX kit. Antibody hRO4 showed greater inhibition of mesothelin shedding than antibody hl5B6 in RH16, RH29, OVCAR8, KLM1, and H226 cells.

[0028] FIG. 9: Schematic of a chimeric antigen receptor T cell (CART) vector. The CART construct includes a CD8 hinge region, a CD8 transmembrane (TM) domain, a 4- IBB co-stimulatory domain and a CD3^ signaling domain separated from a truncated form of human EGFR (hEGFRt) by a T2A sequence. The antibody is an scFv in the VL-linker-VH or VH-linker-VL format.

[0029] FIGS. 10A-10E: Cytotoxicity of hRO4 CART and hl5B6 CART against mesothelin-expressing cancer cells. (FIG. 10A) T3M4, (FIG. 10B) OVCAR-8, (FIG. 10C) SW48, (FIG. 10D) KLM1 or (FIG. 10E) RH29 cells were co-cultured with hRO4 or hl5B6 CAR-transduced T cells at the indicated effector to target (E / T) ratios for 20 hours. Specific lysis was measured using a luciferase-based assay. Mock-transfected T cells were used as a control (see FIGS. 10A-10B). In FIG. 10B, T cells expressing CARs containing mesothelin-specific antibody SSI were also included.

[0030] FIG. 11: Anti-tumor activity of hRO4 CAR T cells and hl5B6 CAR T cells. NSG mice were implanted subcutaneously with 5 million KLM1 cells and intravenously administered 2 million hRO4 or hl5B6 CAR T cells on day 6. Tumor volume was measured up to 25 days after implantation of KLM1 cells. hRO4 CAR T cells exhibited significantly greater anti-tumor activity than hl5B6 CAR T cells. On day 25 *, p = 0.00084. Control = untransfected T cells.

[0031] FIGS. 12A-12F: Cytotoxicity induced by bispecific antibody 13 (BsAb 13) and BsAb 7 in various cancer cell lines. (FIG. 12A) KB31, (FIG. 12B) OVCAR-8, (FIG. 12C) RH63, (FIG. 12D) KLM1, (FIG. 12E) A431 and (FIG. 12F) KLM1-E10 cells were seeded in 96-well plates and incubated with the indicated concentrations of bispecific antibody and donor T cells at a 10: 1 E / T ratio. After three days of incubation, viable cells were measured with the Luciferase Assay system, and cytotoxic activity was calculated.

[0032] FIGS. 13A-13D: BsAb 13-induced cytotoxicity is not inhibited by mesothelin (MSLN) 296-591 His, a recombinant protein version of truncated MSLN found in patient ascites. (FIG. 13A) KB31, (FIG. 13B) OVCAR-8, (FIG. 13C) RH63 and (FIG. 13D) KLM1 cells were seeded in 96-well plates at the following densities: 2000 cells / well (FIGS. 13B, 13D), 3000 cells / well (FIG. 13A), and 5000 cells / well (FIG. 13C). After overnight incubation, plates were treated with the following donor T cells: D15 at a 10:1 E / T ratio (FIGS. 13A, 13C) and D14 at a 20: 1 E / T ratio (FIGS. 13B, 13D). The co-cultures were then administered various doses of MSLN 296-591 His and either 0.1 pg / mL (FIGS. 13A, 13B) or 0.5 pg / mL (FIGS. 13C, 13D) bispecific antibody (BsAb 13 or BsAb 7). Cell viability was evaluated using the luciferase assay system (FIGS. 13B, 13D) or WST-8 assays (FIGS. 13A, 13C). Two technical replicates were performed with 3 replicates per MSLN 296-591 His dose.

[0033] FIG. 14: BsAb 13 inhibits growth of RH63 mesothelioma tumors. RH63 cells were implanted subcutaneously in NSG mice. On day 12, mice were injected I.P. with 10 million human PBMCs (upper arrow). Starting on day 13, mice were injected every other day with 3 mg / kg BsAb 13, for a total of 10 injections (lower arrows). Untreated (UT) mice and mice that received the PBMC injection but not BsAb 13 were included as control groups. Each group included 4 mice. Average tumor size is plotted for each group.

[0034] FIGS. 15A-15B: Comparison of the structures of RO4 and 15B6 complexed with mesothelin. (FIG. 15A) Structure of the RO4 variable fragment (Fv) in complex with the C-terminal peptide of mesothelin. (FIG. 15B) Structure of the 15B6 Fv in complex with the C-terminal peptide of mesothelin. The structures demonstrate that RO4 and 15B6 bind differently to mesothelin.

[0035] SEQUENCE LISTING

[0036] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0037] SEQ ID NO: 1 is the amino acid sequence the RO4 VH domain.

[0038] SEQ ID NO: 2 is the amino acid sequence the RO4 VL domain.

[0039] SEQ ID NO: 3 is the amino acid sequence the hRO4 VH domain.

[0040] SEQ ID NO: 4 is the amino acid sequence the hRO4 VL domain.

[0041] SEQ ID NOs: 5-10 are the amino acid sequences of the RO4 and hRO4 CDRs according to Kabat.

[0042] SEQ ID NOs: 11-16 are the amino acid sequences of the RO4 and hRO4 CDRs according to IMGT.

[0043] SEQ ID NOs: 17-22 are the amino acid sequences of the RO4 and hRO4 CDRs according to Paratome.

[0044] SEQ ID NO: 23 is a nucleic acid sequence encoding the RO4 VH domain.

[0045] SEQ ID NO: 24 is a nucleic acid sequence encoding the RO4 VL domain.

[0046] SEQ ID NO: 25 is the amino acid sequence of the hRO4 heavy chain of the BsAbl3 bispecific antibody.

[0047] SEQ ID NO: 26 is the amino acid sequence of the hRO4 light chain of the BsAbl3 bispecific antibody.

[0048] SEQ ID NO: 27 is the amino acid sequence of the anti-CD3 antibody heavy chain of the BsAb 13 bispecific antibody.

[0049] SEQ ID NO: 28 is the amino acid sequence of the anti-CD3 antibody light chain of the BsAbl3 bispecific antibody.

[0050] SEQ ID NO: 29 is the amino acid sequence of a C-terminal fragment of human mesothelin. DETAILED DESCRIPTION

[0051] I. Introduction

[0052] A previously described mouse monoclonal antibody (15B6) recognizing the C-terminal region of mesothelin (MSLN) was generated by immunizing mice with a peptide containing the C-terminal residues of mesothelin (IPNGYLVLDLSMQEALS; SEQ ID NO: 29). The present disclosure describes the immunization of rabbits with the same C-terminal region of MSLN to identify antibodies with superior properties. Rabbits have a robust immune system capable of producing high-affinity antibodies against a wide range of antigens. The rabbit immune system can recognize a broader range of epitopes compared to smaller rodents, such as mice. This diversity can be beneficial in generating antibodies against a specific epitope.

[0053] As disclosed herein, rabbits were immunized with the C-terminal peptide of MSLN (SEQ ID NO: 29) attached to keyhole limpet hemocyanin (KLH). B cell cloning was performed from PBMC collected from one of the four rabbits. Seventeen clones that reacted with mesothelin were isolated and sequenced. The RO4 antibody was selected for pre-clinical development because it recognizes both the MSLN(M) and MSLN(V) forms of mesothelin (which have a methionine and valine residue, respectively, at position 593 of mesothelin). In addition, RO4 exhibited very high affinity and specificity for mesothelin. The RO4 antibody was also humanized, but retains the same CDR sequences as the rabbit antibody.

[0054] II. Abbreviations

[0055] ADC antibody-drug conjugate

[0056] BsAb bispecific antibody

[0057] CAR chimeric antigen receptor

[0058] CDR complementarity determining region

[0059] CTL cytotoxic T lymphocyte

[0060] E / T effector to target

[0061] ELISA enzyme-linked immunosorbent assay

[0062] FACS fluorescence activated cells sorting

[0063] GMCSFRss granulocyte-macrophage colony stimulating factor receptor signal Sequence

[0064] HRP horseradish peroxidase huEGFRt human truncated epidermal growth factor receptor

[0065] KLH keyhole limpet hemocyanin

[0066] MSLN mesothelin

[0067] NK natural killer

[0068] PE Pseudomonas exotoxin

[0069] PET positron emission tomography scFv single-chain variable fragment TM transmembrane

[0070] UT untreated

[0071] VH variable heavy

[0072] VL variable light

[0073] III. Summary of Terms

[0074] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin 's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0075] 4- IBB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4- IBB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response.

[0076] Administration: To provide or give a subject an agent, such as a monoclonal antibody, CAR, CAR-expressing cell, bispecific antibody or other composition provided herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprost atic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0077] Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen. Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians. Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three- dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of numbering schemes, including those described by Kabat el al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php).

[0078] A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

[0079] A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies can be produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0080] A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.

[0081] A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85- 90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Antibody-drug conjugate (ADC): A molecule that includes an antibody (or antigen-binding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with ADCs include anti-microtubule agents (such as maytansinoids, auristatin E and auristatin F) and interstrand crosslinking agents (for example, pyrrolobenzodiazepines; PBDs). In some cases, the ADC is a bi-specific ADC, which is comprised of two monoclonal antibodies or antigen-fragments thereof, each directed to a different antigen or epitope, conjugated to a drug. In one example, the agent attached to the antibody is IRDye® 700 DX (IR700, Li-cor, Lincoln, NE), which can then be used with near infrared light NIR light to kill cancer cells to which the antibody binds (photoimmunotherapy; see for example US 8,524,239 and 10,538,590). For example, amino-reactive IR700 can be covalently conjugated to an antibody using the NHS ester of IR700.

[0082] Anti-microtubule agent: A type of drug that blocks cell growth by stopping mitosis. Antimicrotubule agents, also referred to as “anti-mitotic agents,” are used to treat cancer.

[0083] Binding affinity: Affinity of an antibody for an antigen. In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another aspect, binding affinity is measured by an antigen / antibody dissociation rate. In another aspect, a high binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry or by surface plasmon reference. An antibody that “specifically binds” an antigen (such as mesothelin) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0084] In some examples, a monoclonal antibody (such as an anti-mesothelin antibody provided herein) specifically binds to a target (such as a mesothelin) with a binding constant that is at least 103M1greater, 104M-1greater or 105M1greater than a binding constant for other molecules in a sample or subject. In some examples, an antibody (e.g., monoclonal antibody) has an equilibrium constant (KD) of 5 llM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a monoclonal antibody binds to a target, such as mesothelin with a binding affinity of at least about 1 x 10'6M, at least about 0.5 x 1045M, at least about 1 x IO'7M, at least about 0.5 x 10'7M, at least about 1 x 10'8M, at least about 0.5 x 10'8M, at least about 1 x 10'9M, at least about 0.5 x 109M, or at least about 0.1 x 109. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of <1000 nM, <750 nM, 500 nM, <250 nM, <100 nM, <50 nM, <25 nM, <10 nM, <5 nM, <2.5 nM, <1 nM, <0.5 nM, <0.25 nM, <0.01 nM, or <0.001 nM (e.g., 10'6M or less, e.g., from 10‘6M to 1010M, e.g., from 10‘10M to 10‘12M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on bio-layer interferometry (BLI) technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.).

[0085] Bispecific antibody: A recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies (such as two single -domain antibodies), and is thereby capable of binding two different antigens. In some aspects, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3), macrophages or effector natural killer (NK) cells, and a tumor antigen (such as mesothelin). Similarly, a multi-specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies (such as scFv forms of the monoclonal antibodies).

[0086] Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk). Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2 / neu protein. Triple negative breast cancer is also called ER-negative PR-negative HER2 / neu-negative breast cancer.

[0087] Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a mesothelin-positive tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nded., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.f. Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds mesothelin used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as an anti-mesothelin antibody provided herein, as well as other anti-cancer antibodies, such as anti-PDl or anti-PDLl (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA- 4).

[0088] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv or single-domain antibody) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3Q. Typically, CARs are comprised of an antigen-binding moiety, a transmembrane domain and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3^ or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and / or DAP10. In some examples, the CAR is bispecific or bicistronic. A bispecific CAR is a single CAR molecule comprised of two antigen-binding domains (such as two scFv or two single-domain antibodies) that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337).

[0089] Cholangiocarcinoma: A type of cancer that develops in cells that line the bile ducts in the liver.

[0090] Complementarity determining region (CDR): A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of a mammalian immunoglobulin each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively. A single-domain antibody contains three CDRs, referred to as CDR1, CDR2 and CDR3.

[0091] Conjugate: In the context of the present disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, photon absorber, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an “immunoconjugate.” When the conjugate comprises an antibody linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an “antibodydrug conjugate” or “ADC.” Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0092] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to mesothelin. For example, a monoclonal antibody that specifically binds mesothelin can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the mesothelin polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds mesothelin. Nonconservative substitutions are those that reduce an activity or binding to mesothelin.

[0093] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0094] 1) Alanine (A), Serine (S), Threonine (T);

[0095] 2) Aspartic acid (D), Glutamic acid (E);

[0096] 3) Asparagine (N), Glutamine (Q);

[0097] 4) Arginine (R), Lysine (K);

[0098] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0099] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0100] Contacting: Placement in direct physical association; includes both in solid and liquid form.

[0101] Cytotoxic agent: Any drug or compound that kills cells. Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to the cells intended to be targeted, as opposed to the cells of the rest of an organism. In contrast, the term “toxicity” refers to toxicity of an immunotoxin to cells other than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term “animal toxicity” refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.

[0102] Diagnostic: Identifying the presence or nature of a pathologic condition, such as a mesothelin- positive cancer. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Prognostic" is the probability of development (such as severity) of a pathologic condition, such as mesothelioma.

[0103] Drug: Any compound used to treat, ameliorate or prevent a disease or condition in a subject. In some aspects herein, the drug is an anti-cancer agent, for example a cytotoxic agent, such as an anti-mitotic or anti-microtubule agent.

[0104] Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. Effector molecule is also known as an effector moiety (EM), therapeutic agent, diagnostic agent, or similar terms. Therapeutic agents (or drugs) include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety, such as an anti-mesothelin antibody, may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as35S, "C,13N,15O,18F,19F,99mTc,1311,3H,14C,1SN,90Y,99TC,11'In and125I, fluorophores, chemiluminescent agents, and enzymes.

[0105] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as mesothelin.

[0106] Framework region: Amino acid sequences interposed between CDRs. Framework regions of an immunoglobulin molecule include variable light and variable heavy framework regions. Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins. In some examples, a fusion protein includes an antigen-binding fragment of an antibody fused to an Fc domain, such as a human Fc domain.

[0107] Heterologous: Originating from a separate genetic source or species.

[0108] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigen-specific response”). In one aspect, an immune response is a T cell response, such as a CD4+response or a CD8+response. In another aspect, the response is a B cell response, and results in the production of specific antibodies.

[0109] Immunoconjugate: A covalent linkage of an effector molecule to an antibody or functional fragment thereof. The effector molecule can be, for example, a detectable label, a photon absorber (such as IR700), or a toxin (to form an immunotoxin, such as an immunotoxin comprising Pseudomonas exotoxin or a variant thereof). Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. In one aspect, an antibody is joined to an effector molecule. In another aspect, an antibody joined to an effector molecule is further joined to a lipid or other molecule, such as to increase its half-life in the body. The linkage can be either by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because immunoconjugates were originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.” The term “chimeric molecule,” as used herein, therefore refers to a targeting moiety, such as a ligand or an antibody, conjugated (coupled) to an effector molecule. The term “conjugated” or “linked” refers to making two polypeptides into one contiguous polypeptide molecule.

[0110] Immunoliposome: A liposome with antibodies or antibody fragments conjugated to its surface. Immunoliposomes can carry cytotoxic agents or other drugs to antibody-targeted cells, such as tumor cells.

[0111] Interstrand crosslinking agent: A type of cytotoxic drug capable of binding covalently between two strands of DNA, thereby preventing DNA replication and / or transcription.

[0112] Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, for example other chromosomal and extra- chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0113] Label (or detectable label): A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a “labeled antibody” refers to incorporation of another molecule in the antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as35S, “C,13N,15O,18F,19F,99mTc,1311,3H,14C,15N,90Y, "Tc,H 1In and125I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some aspects, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0114] Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a cytotoxin or a detectable label. The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an antibody. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0115] Lung cancer: Cancer that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer (NSCLC). These types are diagnosed based on how the cells look under a microscope.

[0116] Mesothelin: A 40 kDa cell-surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. The human mesothelin protein is synthesized as a 70 kD precursor which is then proteolytically processed. The 30 kD amino terminus of mesothelin is secreted and is referred to as megakaryocyte potentiating factor (Yamaguchi et al., J. Biol. Chem. 269:805 808, 1994). The 40 kD carboxyl terminus remains bound to the membrane as mature mesothelin (Chang et al., Natl. Acad. Sci. USA 93: 136 140, 1996). Exemplary nucleic acid and amino acid sequences of mesothelin are as described in PCT Publication No. WO 97 / 25,068; U.S. Patent No. 6,083,502; Chang and Pastan, Int. J. Cancer 57:90, 1994; Chang and Pastan, Proc. Natl. Acad. Sci USA 93: 136, 1996; Brinkmann et al. , Int. J. Cancer 71 :638, 1997; and Chowdhury et al., Mol. Immunol. 34:9, 1997. Mesothelin also refers to mesothelin proteins or polypeptides which remain intracellular as well as secreted and / or isolated extracellular mesothelin protein.

[0117] Mesothelioma: A type of neoplasm derived from the cells lining the pleura and peritoneum, which grow as a thick sheet covering the viscera. The lining is composed of spindle cells or fibrous tissue which may enclose gland-like spaces lined by cuboidal cells. Mesotheliomas often originate in the tissue lining the lung, heart or abdomen. In some cases, mesotheliomas are caused by exposure to asbestos.

[0118] Mesothelin-positive cancer: A cancer that expresses or overexpresses mesothelin. Examples of mesothelin-positive cancers include, but are not limited to, mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), ovarian cancer, endometrial cancer, cervical cancer, colon cancer, and thymus cancer.

[0119] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0120] Ovarian cancer: Cancer that forms in tissues of the ovary (one of a pair of female reproductive glands in which the ova, or eggs, are formed). Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which originates in cells that release hormones and connect the different structures of the ovaries.

[0121] Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The vast majority (-94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (IPMN), and mucinous cystadenocarcinoma. In some examples, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.

[0122] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the antibodies and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0123] Photoimmunotherapy: A targeted cancer therapy that utilizes an antigen- specific antibodyphotoabsorber conjugate that can be activated by near-infrared light to kill targeted cells. The photon absorber is typically based on phthalocyanine dye, such as a near infrared (NIR) phthalocyanine dye (for example, IRDye® 700DX, also know knows as IR700). The antibody (for example, a mesothelin-specific antibody) binds to the appropriate cell surface antigen (e.g. mesothelin) and the photo-activatable dye induces lethal damage to cell membranes after NIR-light exposure. NIR-light exposure (690 nm) induces highly selective, necrotic cancer cell death within minutes without damage to adjoining cells (see, for example, U.S. Application No. 2018 / 0236076).

[0124] Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0125] Prostate cancer: Cancer that forms in tissues of the prostate (a gland in the male reproductive system found below the bladder and in front of the rectum). Prostate cancer usually occurs in older men.

[0126] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, nonlimiting example, a substantially purified protein is 90% free of other proteins or cellular components.

[0127] Pyrrolobenzodiazepine (PBD): A class of sequence-selective DNA minor-groove binding crosslinking agents originally discovered in Streptomyces species. PDBs are significantly more potent than systemic chemotherapeutic drugs. The mechanism of action of PBDs is associated with their ability to form an adduct in the minor groove of DNA, thereby interfering with DNA processing. In the context of the present disclosure, PBDs include naturally produced and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric, dimeric and hybrid PBDs (for a review see Gerratana, Med Res Rev 32(2):254-293, 2012). Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0128] Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, a sample includes a tumor biopsy.

[0129] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.

[0130] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981: Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sei. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5: 151 , 1989; Corpet et al., Nucleic Acids Research 16: 10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0131] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0132] Homologs and variants of a VH domain of an antibody that specifically binds a mesothelin polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0133] Small molecule: A molecule, typically with a molecular weight less than about 1000 Daltons, or in some aspects, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.

[0134] Squamous cell carcinoma: A malignant neoplasm derived from stratified squamous epithelium, but which may also occur in sites such as bronchial mucosa where glandular or columnar epithelium is normally present. Squamous cell carcinoma is the most common type of skin cancer.

[0135] Stomach cancer: Cancer that forms in tissues lining the stomach. Also called gastric cancer. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0136] Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein (for example, an antibody) can be chemically synthesized in a laboratory.

[0137] Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor, such as reduce a tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size / volume / number prior to treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase survival time of a subject with a mesothelin-positive cancer, for example by at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months (such as 2-72 months, 6-12 months, or 3-24 months), for example as compared to a survival time without a disclosed treatment. When administered to a subject, a dosage can be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

[0138] Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus, adeno- associated virus, or adenovirus vector.

[0139] IV. Monoclonal Antibodies Specific for Mesothelin

[0140] Monoclonal antibodies that specifically bind mesothelin with high affinity are described. The mesothelin-specific RO4 monoclonal antibody was isolated from a rabbit immunized with a C-terminal fragment of the mesothelin protein. The rabbit RO4 antibody was humanized to generate the hRO4 antibody. The amino acid sequences of the VH domain and VL domain of the disclosed antibodies (RO4 and hRO4) are provided below. The CDR sequences according to Kabat, IMGT and Paratome are listed in Table 1 below. In the VH and VL domain sequences below, the Kabat CDRs are underlined, the IMGT CDRs are in italics and the Paratome CDRs are in bold font. Although the CDR locations were identified using Kabat, IMGT and Paratome, a skilled person understands that other numbering schemes, such as Chothia, can also be used to determine the boundaries of each CDR.

[0141] RO4 VH domain (

[0142] OSLEESGGGLVT SKTSSTTVDLKMTSLTTEDTATYFCVRGVGGGLWGPGTLVTVSS

[0143] RO4 VL domain

[0144] ADVVMTOTPSS

[0145] GSGTQFTLTISDLECADAATYYCOSAAZJGGVVFGAFGGGTEVVV hRO4 VH domain

[0146] EVOLVESGGGLV

[0147] FTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVGGGEWGOGTLVTVSS hRO4 VL domain (SE

[0148] DIOMTOSPSSLSASV

[0149] TDFTLTISSLQPEDVATYYCOSAALZGGVVFGAFGGGTKVEIK

[0150] Table 1. CDR sequences of RO4 and hRO4

[0151] Provided herein are monoclonal antibodies and antigen-binding fragments thereof that bind, such as specifically bind, mesothelin. The monoclonal antibody or antigen-binding fragment includes a variable heavy (VH) domain and a variable light (VL) domain, and the VH domain includes a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3, and the VL domain includes a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3. In some aspects, and the VH domain includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 1 or SEQ ID NO: 3, and the VL domain includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 2 or SEQ ID NO: 4. In some examples, the monoclonal antibody or antigen-binding fragment includes one or more (such as all three) HCDR sequences from SEQ ID NO: 1 and / or one or more (such as all three) LCDR sequences from SEQ ID NO: 2, as determined using any numbering scheme, such as IMGT, Kabat, Paratome or Chothia, or any combination thereof. In some examples, the monoclonal antibody or antigen-binding fragment includes one or more (such as all three) HCDR sequences from SEQ ID NO: 3 and / or one or more (such as all three) LCDR sequences from SEQ ID NO: 4, as determined using any numbering scheme, such as IMGT, Kabat, Paratome or Chothia, or any combination thereof.

[0152] In some aspects, the VH domain of the monoclonal antibody or antigen-binding fragment includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1 or SEQ ID NO: 3; and the VL domain comprises the LCDR1, LCDR1 and LCDR3 sequences of SEQ ID NO: 2 or SEQ ID NO: 4. In some examples, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively includes SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and / or the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively include SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In other examples, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and / or the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively include SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In yet other examples, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:

[0153] 19, and / or the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively include SEQ ID NO:

[0154] 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0155] In some examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.

[0156] In some examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3 and includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 3; and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 3 and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 4.

[0157] In some aspects, the monoclonal antibody is an IgG, IgA, or IgM molecule. In some examples, the monoclonal antibody is an IgGl, IgG2, IgG3 or IgG4.

[0158] In some aspects, the antigen-binding fragment is selected from a Fab fragment, an Fab’ fragment, an F(ab)’2 fragment, a single chain variable fragment (scFv) and a disulfide stabilized variable fragment (dsFv).

[0159] In some aspects, the monoclonal antibody or antigen-binding fragment is a humanized monoclonal antibody or antigen-binding fragment.

[0160] In some aspects, the monoclonal antibody further includes a constant region, such as an IgG constant region, for example, an IgGl constant region, such as a human IgGl constant region. In some examples, the constant region includes at least one amino acid modification to increase the half-life, stability and / or function of the monoclonal antibody.

[0161] Also provided herein are chimeric antigen receptors (CARs) that include a mesothelin-specific monoclonal antibody or antigen-binding fragment disclosed herein. In some aspects, the antigen-binding fragment of the CAR is an scFv. In some aspects, the CAR further includes a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof. In some examples, the hinge region includes a CD8 hinge region; the transmembrane domain includes a CD8 transmembrane domain; the costimulatory signaling moiety includes a 4-1BB signaling moiety; and / or the signaling domain includes a CD3^ signaling domain. Isolated cells that expressed a CAR disclosed herein are also provided. In some aspects, the cell is an immune cell, such as but not limited to a T cell, a B cell, a natural killer (NK) cell, a macrophage, or a dendritic cell (DC). In other aspects, the cell is an induced pluripotent stem cell (iPSC). CARs and CAR-expressing cells are further described in section V.

[0162] Further provided herein are multi-specific antibodies that include an antibody or antigen-binding fragment disclosed herein and at least one additional monoclonal antibody or antigen-binding fragment. In some aspects, the multi-specific antibody is a bispecific antibody. In other aspects, the multi-specific antibody is a trispecific antibody. In some examples, the at least one additional monoclonal antibody or antigen-binding fragment includes a monoclonal antibody or antigen-binding fragment that specifically hinds T cells (such as CD3 on T cells), NK cells (such as CD16, NKp46 or NKG2D on NK cells), or macrophages (such as CD89 / FcocRI). In particular non-limiting examples, the multi-specific antibody is a bispecific antibody and includes amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and / or SEQ ID NO: 28, or includes or consists of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and / or SEQ ID NO: 28. Multi-specific antibodies arc further described in section VI.

[0163] Further provided herein are immunoconjugates that include a mesothelin-specific monoclonal antibody disclosed herein and an effector molecule. In some aspects, the effector molecule is a toxin, such as a Pseudomonas exotoxin or a variant thereof, for example PE38. In other aspects, the effector molecule is a detectable label, such as a fluorophore, an enzyme or a radioisotope. In other aspects, the effector molecule is a photon absorber, such as IR700. Immunoconjugates that include a photon absorber can be used, for example, for photoimmunotherapy or in vivo diagnostic imaging. Immunoconjugates are further described in section VII.

[0164] Further provided herein are antibody-drug conjugates (ADCs) that include a drug conjugated to a mesothelin-specific monoclonal antibody disclosed herein. In some aspects, the drug is a small molecule, for example an anti-cancer agent, anti-microtubule agent, an anti-mitotic agent and / or a cytotoxic agent. ADCs are further described in section VIII.

[0165] Also provided herein are antibody-nanoparticle conjugates that include a nanoparticle conjugated to a mesothelin-specific monoclonal antibody disclosed herein. In some aspects, the nanoparticle includes a polymeric nanoparticle, nanosphere, nanocapsule, liposome, dendrimer, polymeric micelle, or niosome. In some aspects, the nanoparticle includes a cytotoxic agent. Antibody-nanoparticle conjugates are further described in section VIII.

[0166] Further provided herein are fusion proteins that include a mesothelin-specific monoclonal antibody disclosed herein and a heterologous protein or peptide. In some aspects, the heterologous protein is an Fc protein, such as a human Fc protein (e.g., human IgGl Fc). In some examples, the fusion protein further includes a linker, such as protein linker.

[0167] Also provided herein are nucleic acid molecules that encode a monoclonal antibody or antigenbinding fragment, CAR, multi-specific antibody, immunoconjugate, or fusion protein, disclosed herein. In some aspects, the nucleic acid molecule is operably linked to a promoter. In some aspects in which the nucleic acid molecule encodes a monoclonal antibody or antigen-binding fragment, the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 23, or a degenerate variant thereof, and / or SEQ ID NO: 24, or a degenerate variant thereof. In some examples, the nucleotide sequence includes or consists of SEQ ID NO: 23 and / or SEQ ID NO: 24. In some aspects, the nucleic acid molecule is operably linked to a promoter.

[0168] Vectors that include a disclosed nucleic acid are also provided. In some aspects, the vector is an expression vector. In other aspects, the vector is a viral vector. Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided. In some examples, the isolated cell is a prokaryotic cell, such as an E. coli cell. In other examples, the isolated cell is a mammalian cell, such as a human cell. Nucleic acid molecules, vectors and host cells are further described in section X.

[0169] Compositions that include a pharmaceutically acceptable carrier and a mesothelin-specific monoclonal antibody or antigen-binding fragment, fusion protein, CAR, isolated cell (such as a CAR expressing cell, for example a CAR T cell, a CAR NK cell or a CAR macrophage), immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, isolated nucleic acid molecule or vector disclosed herein are further provided by the present disclosure. Compositions are further described in section XI.

[0170] Also provided are methods of detecting expression of mesothelin in a sample. In some aspects, the method includes contacting the sample with a mesothelin-specific monoclonal antibody or antigen-binding fragment disclosed herein and detecting binding of the monoclonal antibody or antigen-binding fragment to the sample, thereby detecting expression of mesothelin in the sample.

[0171] Further provided are methods of diagnosing a subject as having a mesothelin-positive tumor. In some aspects, the method includes contacting the sample with a mesothelin-specific monoclonal antibody or antigen-binding fragment disclosed herein and detecting binding of the monoclonal antibody or antigenbinding fragment to the sample, thereby diagnosing the subject as having a mesothelin-positive tumor. In some examples of the disclosed methods, the monoclonal antibody or antigen-binding fragment is directly labeled. In other examples, the method includes contacting the monoclonal antibody or antigen-binding fragment with a detection antibody, and detecting the binding of the detection antibody to the monoclonal antibody or antigen-binding fragment, thereby detecting expression of mesothelin in the sample or diagnosing the subject as having a mesothelin-positive cancer. In some examples, the sample is obtained from a subject suspected of having a mesothelin-positive cancer. In particular examples, the sample is a tumor biopsy. Methods for diagnosis and detection are described in section XIII.

[0172] Further provided are methods of treating a mesothelin-positive cancer in a subject and / or methods of inhibiting tumor growth or metastasis of a mesothelin-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a mesothelin-specific monoclonal antibody or antigen-binding fragment, CAR, isolated cell (such as a CAR expressing immune cell, for example a CAR T cell, a CAR B cell, a CAR NK cell, a CAR DC, or a CAR macrophage; or an iPSC), immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein. In some examples of the disclosed methods, the mesothelin-positive cancer is a solid tumor. In particular examples, the solid tumor is a mesothelioma, a prostate cancer, a lung cancer, a stomach cancer, a squamous cell carcinoma, a pancreatic cancer, a cholangiocarcinoma, a breast cancer, an ovarian cancer, an endometrial cancer, a cervical cancer, a colon cancer, or a thymus cancer. Therapeutic methods are further described in section XII.

[0173] Also provided herein are kits that include a monoclonal antibody or antigen-binding fragment, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and one or more of a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and instructional materials. Kits are further described in section XIII.

[0174] V. Chimeric Antigen Receptors (CARs)

[0175] The disclosed anti-mesothelin monoclonal antibodies (including antigen-binding fragments) can be used to produce CARs (also known as chimeric T cell receptors, artificial T cell receptors or chimeric immunoreceptors) and / or immune cells, such as T lymphocytes (such as CTLs), B cells, natural killer (NK) cells, dendritic cells or macrophages, engineered to express CARs. Induced pluripotent stem cells (iPSCs) can also be used to express CARs. Generally, CARs include a binding moiety, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many instances, the binding moiety is an antigen binding fragment of a monoclonal antibody, such as a scFv, or a single-domain antibody. The spacer / hinge region typically includes sequences from IgG subclasses, such as IgGl, IgG4, IgD, CD8, or CD28. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3^, CD4, CD8 or CD28. Several different endodomains have been used to generate CARs. For example, the endodomain can consist of a signaling chain having an IT AM, such as CD3C or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27 and / or DAP 10.

[0176] Immune cells, such as T cells, B cells, NK cells, dendritic cells, or macrophages, or iPSCs expressing CARs can be used to target a specific cell type, such as a mesothelin-positive tumor cell. Thus, the antibodies disclosed herein can be used to engineer immune cells or iPSCs that express a CAR containing the mesothelin-specific monoclonal antibody or antigen-binding fragment thereof, thereby targeting the engineered cells to mesothelin-positive tumor cells.

[0177] Multispecific (such as bispecific) or bicistronic CARs are also contemplated by the present disclosure. In some aspects, the multispecific or bispecific CAR includes a monoclonal antibody (such as an scFv) specific for mesothelin and a monoclonal antibody specific for a different antigen (or a different epitope of mesothelin). Similarly, a bicistronic CAR includes two CAR molecules expressed from the same construct where one CAR molecule is a mesothelin-targeted CAR and the second CAR targets a second antigen, such as a second tumor antigen (see, for example, Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337).

[0178] Accordingly, provided herein are CARs that include a mesothelin-specific antibody, such as RO4 or humanized RO4, or any antibody having the CDR sequences of RO4 or hRO4. Also provided are isolated nucleic acid molecules and vectors encoding the CARs (including bispecific and bicistronic CARs), and host cells, such as T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing the CARs, bispecific CARs or bicistronic CARs. T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing CARs comprised of a mesothelin-specific monoclonal antibody or antigen-binding fragment can be used for the treatment of a mesothelin-positive cancer. In some aspects herein, the CAR is a monospecific CAR. In other aspects, the CAR is a bispecific CAR. In other aspects herein, the CAR is a bicistronic CAR.

[0179] In some aspects, the CAR includes a signal peptide sequence, for example, N-terminal to the antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2. While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence may be cleaved off of the CAR. Accordingly, in some aspects, the CAR lacks a signal peptide sequence.

[0180] In some aspects, the mesothelin-targeted CAR includes the following features in an N-terminal to C- terminal direction: signal peptide, an antigen-binding fragment (such as a scFv in a VH domain-linker- VL domain orientation or in a VL domain-linker- VH domain orientation), a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain. In some examples, the signal peptide is a GMCSFR signal peptide. In some examples, the linker sequence is a glycine- and serine -rich linker sequence. In some examples, the hinge region is a CD8 hinge region. In some examples, the transmembrane domain is a CD8 transmembrane domain. In some examples, the co-stimulatory domain is a 4- IBB co-stimulatory domain. In some examples, the signaling domain is a CD3 signaling domain.

[0181] Also provided herein are mesothelin-specific monoclonal antibodies modified to enable their use with a universal CAR system. Universal CAR systems have been developed to increase CAR flexibility and expand their use to additional antigens. Currently, for each patient who receives CAR immune cell therapy, autologous immune cells (such as T cells) must be cultured, expanded, and modified to express an antigen- specific CAR. This process is lengthy and expensive, limiting its use. Universal CARs are based on a system in which the signaling components of the CAR are split from the antigen-binding portion of the molecule but come together using a “lock-key” system. For example, biotin-binding immune receptor (BBIR) CARs are comprised of an intracellular T cell signaling domain fused to an extracellular domain comprising avidin. Biotinylated antigen-specific (such as mesothelin-specific) monoclonal antibodies can then bind the BBIR to direct immune cells to antigen-expressing cells. Another example is the split, universal and programmable (SUPRA) CAR system. In the SUPRA system, the CAR includes the intracellular signaling domains fused to an extracellular leucine zipper, which is paired with an antigenspecific monoclonal antibody fused to a cognate leucine zipper. For a review of universal CAR systems, see, for example, Zhao et al., J Hematol Oncol 11(1): 132, 2018; and Cho et al., Cell 173:1426-1438, 2018. In some aspects herein, the mesothelin-specific monoclonal antibody is fused to one component of a specific binding pair. In some examples, the monoclonal antibody is fused to a leucine zipper or biotin.

[0182] Another type of universal CAR can be generated using a sortase enzyme. A sortase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. Sortase catalyzes transpeptidation between a sortase recognition motif and a sortase acceptor motif. Thus, antigen-specific CARs can be generated by contacting an antigen-specific antibody fused to a sortase recognition motif with a portion of a CAR molecule that includes the intracellular signaling domain(s), a transmembrane region and an extracellular portion that includes a sortase acceptor motif. In the presence of the sortase enzyme, the two components become covalently attached to form a complete antigen-specific CAR. Accordingly, in some aspects herein, a mesothelin-specific monoclonal antibody is modified to include a sortase recognition motif (see, for example, PCT Publication No. WO 2016 / 014553).

[0183] In some aspects, the mesothelin-targeted CAR is expressed in allogeneic immune cells, such as allogeneic T cells, B cells, NK cells, DCs or macrophages from a healthy donor(s). In some examples, the allogeneic immune cells are genetically engineered to express the mesothelin-targeted CAR, for example by disrupting expression of the endogenous T cell receptor by insertion of the CAR (see, for example, MacLeod et al., Mol Ther 25(4): 949-961, 2017). Gene editing can be performed using any appropriate gene editing system, such as CRISPR / Cas9, zinc finger nucleases or transcription activator-like effector nucleases (TALEN).

[0184] VI. Multi-Specific Antibodies

[0185] Multi-specific antibodies are recombinant proteins comprised of two or more monoclonal antibodies or antigen-binding fragments of two or more different monoclonal antibodies. For example, bispecific antibodies are comprised of two different monoclonal antibodies or antigen-binding fragments thereof. Thus, bispecific antibodies bind two different antigens (or two different epitopes of an antigen) and trispecific antibodies bind three different antigens (or three different epitopes of an antigen).

[0186] Provided herein are multi-specific, such as trispecific or bispecific, monoclonal antibodies that include a first mesothelin-specific monoclonal antibody. In some aspects, the multi-specific monoclonal antibody further includes a second antibody that specifically binds a different epitope of mesothelin or a different cell-surface antigen. In some aspects, the multi-specific antibody further includes a second antibody that specifically binds CD3. In some aspects, the multi-specific monoclonal antibody further includes a second antibody that specifically binds PD-1 (such as nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP-514). In some aspects, the multispecific monoclonal antibody further includes a second antibody that specifically binds CTLA-4 (such as ipilimumab or tremelimumab).

[0187] In some aspects disclosed herein, the multi-specific monoclonal antibody includes a first monoclonal antibody specific for mesothelin (such as a mesothelin-specilic scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a component of the T cell receptor, such as CD3. In other aspects, the multi-specific monoclonal antibody includes a first monoclonal antibody specific for mesothelin (such as a mesothelin-specific scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a NK cell activating receptor, such as CD16, Ly49, or CD94.

[0188] In some aspects, the multi -specific antibody is a bispecific antibody that includes the VH and VL domains of antibody RO4 or hRO4 and the VH and VL domains of a monoclonal antibody that specifically binds CD3. In specific examples, the bispecific antibody has the amino acid sequences of the heavy and light chains of bispecific antibody BsAbl3, which includes the hRO4 heavy chain, the hRO4 light chain, and the heavy and lights chains of a CD3-specific monoclonal antibody (CD3-specific antibody described in Smith et al., Sci Rep 5: 17943, 2015).

[0189] In the sequences below, the signal sequence is underlined and the variable domain is in bold and the constant regions are in normal font. hRO4 heavy chain sequence (SEQ ID NO: 25) MGWSCIILFLVATATGVHSEVOLVESGGGLVQPGGSLRLSCAASGFDISRHYMTWVROAPGKG LEWIGSIYGGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVGGGLWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK hRO4 light chain sequence (SEQ ID NO: 26)

[0190] MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCQASQSISTALVWYQQKPGKVPKL LIRSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQSAALIGGVVFGAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0191] Anti-CD3 antibody heavy chain sequence (SEQ ID NO: 27)

[0192] MGWSCIILFLVATATGVHSEVOLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVROAPGK GLEWVSGISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGH YYYGMDVWGQGTTVTVASRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

[0193] Anti-CD3 antibody light chain sequence (SEQ ID NO: 28)

[0194] MGWSCIILFLVATATGVHSEIVMTOSPATLSVSPGERATLSCRASOSVSSNLAWYOQKPGOAPR LLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIKAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0195] Also provided are isolated nucleic acid molecules and vectors encoding the multi-specific antibodies, and host cells comprising the nucleic acid molecules or vectors. Multi-specific antibodies that include a mesothelin-specific antibody can be used for the treatment of a mesothelin-positive cancer. Thus, provided herein are methods of treating a subject with a mesothelin-positive cancer by administering to the subject a therapeutically effective amount of the mesothelin-targeting multi-specific antibody.

[0196] VII. Immunoconjugates

[0197] The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody. A therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule. One of skill will appreciate that therapeutic agents can include various drugs, such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as125I,32P,14C,3H and3’S, photon absorbers such as IR700, and other labels, target moieties and ligands. The choice of a particular therapeutic agent depends on the target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a mesothelin-expressing cell). Conversely, where it is desired to invoke a non-lethal biological response, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0198] With the therapeutic agents and antibodies described herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence, but which encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0199] Effector molecules can be linked to an antibody of interest using any number of known means. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (- SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0200] In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.

[0201] In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, a skilled person will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0202] The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), an Fc protein, a detection agent, a photon absorber, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a poly histidine tag).

[0203] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.

[0204] The antibody can be conjugated with a detectable marker, for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP). An antibody can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, 0-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

[0205] An antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles, such as superparamagnetic iron oxide, are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some aspects, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0206] An antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides:3H,14C, ’N, An antibody disclosed herein can also be conjugated to a photon absorber. In some aspects, the photon absorber is a phthalocyanine dye, such as, but not limited to, IRDye® 700DX (also known as “IR700”). Antibody-photoabsorber conjugates can be used for photoimmunotherapy (for example to kill mesothelin-positive tumor cells).

[0207] An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.

[0208] Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401). In one aspect, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264:14256-14261 , 1989).

[0209] PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; PCT Publication Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; and Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0210] Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to PE-LR, PE-6X, PE-8X, PE-LR / 6X and PE-LR / 8X (see, for example, Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32): 11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954 and WO 2011 / 032022).

[0211] In some examples, the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other examples, the PE is a variant designated PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other examples, the PE variant is PE with reducing immunogenicity. In yet other examples, the PE is a variant designated PE-LR / 8M (PCT Publication No. WO 2011 / 032022). Modification of PE may occur in any previously described variant, including cytotoxic fragments of PE (for example, PE38, PE-LR and PE-LR / 8M). Modified PEs may include any substitution(s), such as for one or more amino acid residues within one or more T-cell epitopes and / or B cell epitopes of PE, or deletion of one or more T-cell and / or B-cell epitopes (see, for example, U.S. Patent Application Publication No. 2015 / 0099707).

[0212] Contemplated forms of PE also include deimmunized forms of PE, for example versions with domain II deleted (for example, PE24). Deimmunized forms of PE are described in, for example, PCT Publication Nos. WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0213] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing mesothelin on their surface (e.g., mesothelin-positive tumor cells). Thus, an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing mesothelin. This can be done for therapeutic, diagnostic or research purposes. Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, and recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.

[0214] Alternatively, the molecule linked to an antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are known (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0215] Antibodies described herein can also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example,3H,12,I,35S,14C, or32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.

[0216] Means of detecting such labels are known. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. VIII. Antibody-Drug Conjugates (ADCs)

[0217] ADCs are compounds comprised of an antigen-specific antibody (such as a monoclonal antibody or antigen-binding fragment of an immunoglobulin provided herein that binds mesothelin) and a drug, for example a cytotoxic agent (such as an anti-microtubule agent or cross-linking agent). Because ADCs are capable of specifically targeting cells expressing a particular antigen, the drug can be much more potent than agents used for standard systemic therapy. For example, the most common cytotoxic drugs currently used with ADCs have an IC50 that is 100- to 1000-fold more potent than conventional chemotherapeutic agents. Common cytotoxic drugs include anti-microtubule agents, such as maytansinoids and auristatins (such as auristatin E and auristatin F). Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PBDs), which covalently bind the minor groove of DNA to form interstrand crosslinks. In many instances, ADCs comprise a 1:2 to 1:4 ratio of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0218] The antibody and drug can be linked by a cleavable or non-cleavable linker. However, in some instances, a linker is stable in the circulation to prevent systemic release of the cytotoxic drug that could result in significant off-target toxicity. Non-cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, digestion of the antibody by lysosomal proteases results in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0219] One method for site-specific and stable conjugation of a drug to a monoclonal antibody (or antigenbiding fragment fused to Fc) is via glycan engineering. Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant (31,4-galactosyltransferase enzyme (Y289L-Gal-Tl; U.S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162), 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical handle for conjugation.

[0220] The oligosaccharide chain attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-Gl) or completely degalactosylated (IgG-GO). Treatment of a monoclonal antibody with |31 ,4-galactosidasc converts the antibody to the IgG-GO glycoform. The mutant (31 ,4- galactosyltransferase enzyme is capable of transferring 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-Gl and IgG-GO glycoforms. The chemical handle on the transferred sugar enables conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0221] Provided herein are ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) mesothelin. In some aspects, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent and / or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, a PBD, an auristatin, a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0222] In some aspects, the ADC includes a pyrrolobenzodiazepine (PBD). The natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al., J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs, both naturally- occurring and synthetic analogues, have been reported (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As one example, PBD dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010 / 0203007). Exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US 2011 / 0256157; and WO 2011 / 130598).

[0223] In some aspects, the ADC includes an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0224] In some aspects, the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin- 10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5- benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, for example, U.S. Publication No. 2013 / 0129753).

[0225] In some aspects, the ADC includes an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and analogues thereof, can produce double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0226] In some aspects, the ADC includes an anthracy cline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA- dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and / or interactions of the drug molecules with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4): 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grandi et al., Cancer Treat Rev 17: 133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0227] In some aspects, the ADC can further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC. In some aspects, ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.

[0228] In some examples, a linker has a functionality that can react with a free cysteine present on an antibody to form a covalent bond. Exemplary linkers with such reactive functionalities include maleimide, haloacetamides, oc-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0229] In some examples, a linker has a functionality that can react with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

[0230] In some examples, the linker is a cleavable linker, which facilitates release of the drug. Examples of cleavable linkers include acid-labile linkers (for example, comprising hydrazone), protease-sensitive linkers (for example, peptidase- sensitive), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020).

[0231] The ADCs disclosed herein can be used for the treatment of a mesothelin-positive tumor alone or in combination with another therapeutic agent and / or in combination with any standard therapy for the treatment of a mesothelin-positive cancer. IX. Antibody-Nanoparticle Conjugates

[0232] The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic agents directly to mesothelin-expressing cells via binding of the antibody to mesothelin expressed on the surface of cells. The use of nanoparticles reduces off-target side effects and can also improve drug bioavailability and reduce the dose of a drug required to achieve a therapeutic effect. Nanoparticle formulations can be tailored to suit the drug that is to be carried or encapsulated within the nanoparticle. For example, hydrophobic molecules can be incorporated inside the core of a nanoparticle, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell. Examples of nanoparticles include, but at not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, niosomes, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3):381 -394, 2011).

[0233] Liposomes are common types of nanoparticles used for drug delivery. An antibody conjugated to a liposome is often referred to as an “immunoliposome.” The liposomal component of an immunoliposome is typically a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, the phospholipids are composed of a hydrophilic head group and two hydrophobic chains to enable encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from the circulation via macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size and charge of the liposome can be modulated. The surface of the liposome may also be modified, such as with a glycolipid or sialic acid. For example, the inclusion of polyethylene glycol (PEG) significantly increases circulation half-life. Liposomes for use as drug delivery agents, including for preparation of immunoliposomes, have been described (see, for example, Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012; Immordino et al., hit J Nanomedicine l(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655; 2010 / 00329981).

[0234] Niosomes are non-ionic surfactant-based vesicles having a structure similar to liposomes. The membranes of niosomes are composed only of nonionic surfactants, such as polyglyceryl-alkyl ethers or N- palmitoylglucosamine. Niosomes range from small, unilamellar to large, multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and increase bioavailability of encapsulated drugs.

[0235] Dendrimers include a range of branched polymer complexes. These nanoparticles are water-soluble, biocompatible and are sufficiently non-immunogenic for human use. Generally, dendrimers consist of an initiator core, surrounded by a layer of a selected polymer that is grafted to the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly (amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, chemotherapeutic agents and other drugs.

[0236] Polymeric micelles are composed of aggregates of amphiphilic co-polymers (consisting of both hydrophilic and hydrophobic monomer units) assembled into hydrophobic cores, surrounded by a corona of hydrophilic polymeric chains exposed to the aqueous environment. In many cases, the polymers used to prepare polymeric micelles are heterobifunctional copolymers composed of a hydrophilic block of PEG, polytvinyl pyrrolidone) and hydrophobic poly(L-lactide) or poly(L-lysine) that forms the particle core. Polymeric micelles can be used to carry drugs that have poor solubility. These nanoparticles have been used to encapsulate a number of drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0237] Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The formulation selected typically depends on the solubility of the therapeutic agent to be carried / encapsulated; poorly water-soluble drugs are more readily encapsulated within nanospheres, while water-soluble and labile drugs, such as DNA and proteins, are more readily encapsulated within nanocapsules. The polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkylcyanoacrylates), poly(lactic acid) (PLA), polytglycolic acids) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0238] Antibodies can be conjugated to a suitable nanoparticle according to standard known methods. For example, conjugation can be either covalent or non-covalent. In some aspects in which the nanoparticle is a liposome, the antibody is attached to a sterically stabilized, long circulation liposome via a PEG chain. Coupling of antibodies or antibody fragments to a liposome can also involve thioester bonds, for example by reaction of thiols and maleimide groups. Cross-linking agents can be used to create sulfhydryl groups for attachment of antibodies to nanoparticles (Paszko and Senge, CurrMed Chem 19(31)5239-5277, 2012).

[0239] X. Nucleic Acid Molecules

[0240] Nucleic acid molecules (for example, DNA, cDNA, mRNA, or RNA molecules) encoding the amino acid sequences of the disclosed antibodies, fusion proteins, CARs and other conjugates that specifically bind to mesothelin, are provided. Nucleic acid molecules encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and the variable domain sequences), sequences available (such as framework or constant region sequences), and the genetic code. In some aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell or a bacterial cell) to produce a disclosed antibody, fusion protein or antibody conjugate (e.g., CAR, immunotoxin, multi-specific antibody).

[0241] Exemplary nucleic acid sequences encoding the VH and VL domains of antibody RO4 are provided below.

[0242] RO4 VH domain DNA sequence (SEQ ID NO: 23)

[0243] CAGTCGCTGGAGGAGTCCGGAGGAGGCCTGGTAACGCCTGGAGGAATCCTGACACTCACCTGC ACAGCCTCTGGATTCGACATCAGTAGGCATTACATGACCTGGGTCCGCCAGGCTCCAGGGGAG GGGCTGGAATGGATCGGAAGCATTTATGGTGGTAGCACATACTACGCGAGCTGGGCGAAAGGC CGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACGAGTCTGACAACC GAGGACACGGCCACCTATTTCTGTGTCAGAGGTGTTGGTGGTGGCTTGTGGGGCCCAGGCACC CTGGTCACCGTCTCCTCA

[0244] RO4 VL domain DNA sequence (SEQ ID NO: 24)

[0245] GCCGACGTCGTGATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACC ATGAAGTGCCAGGCCAGTCAGAGCATTAGCACTGCATTAGTCTGGTATCAGCAGAAACCAGGG CAGCCTCCCAAGCTCCTGATCCGTTCTACATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCA AAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTG CCACTTATTATTGTCAATCTGCTGCTTTGATTGGTGGTGTTGTTTTTGGTGCTTTCGGCGGAGGG ACCGAGGTGGTGGTCAGA

[0246] In some aspects, the nucleotide sequence of a nucleic acid molecule encoding a mesothelin-specific monoclonal antibody (or portion thereof) disclosed herein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23, SEQ ID NO: 24, or both SEQ ID NO: 23 and SEQ ID NO: 24, or degenerate variants thereof. In some examples, the nucleotide sequence of the nucleic acid molecule encoding a disclosed antibody (or portion thereof) includes or consists of SEQ ID NO: 23, SEQ ID NO: 24, or both SEQ ID NO: 23 and SEQ ID NO: 24, or degenerate variants thereof.

[0247] The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that differ in their sequence, but which encode the same antibody sequence, or encode a conjugate or fusion protein including the antibody sequence.

[0248] Nucleic acid molecules encoding the antibodies, fusion proteins, bispecific antibodies, CARs and other conjugates that specifically bind to mesothelin can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0249] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements).

[0250] Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).

[0251] The nucleic acid molecules can be expressed in a recombinantly engineered cell such as in bacterial, plant, yeast, insect and mammalian cells. The antibodies and conjugates can be expressed as individual proteins including the antibody (linked to an effector molecule or detectable marker as needed) or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011).

[0252] One or more DNA sequences encoding the antibodies, CARs, bispecific antibodies, fusion proteins, or other conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, for example mammalian cells, such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, may be used.

[0253] The expression of nucleic acids encoding the antibodies, CARs and other conjugates described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (z.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

[0254] To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription / translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and / or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and / or acceptor sequences (for example, CMV and / or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

[0255] Modifications can be made to a nucleic acid encoding an antibody described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the antibody into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.

[0256] Once expressed, the antibodies, CARs, bispecific antibodies, fusion proteins, and other conjugates can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, CARs, bispecific antibodies, fusion proteins, and other conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylactically, the antibodies should be substantially free of endotoxin.

[0257] Methods for expression of antibodies, CARs, fusion proteins, and conjugates, and / or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544-546, 1989.

[0258] XI. Compositions and Administration

[0259] Compositions are provided that include one or more of the disclosed monoclonal antibodies or antigen-binding fragments that bind (for example specifically bind) mesothelin in a carrier. Compositions comprising fusion proteins (such as scFv-Fc fusion proteins), ADCs, CARs (and immune cells expressing CARs), multi-specific (such as bispecific or trispecific) antibodies, antibody- nanoparticle conjugates, immunoliposomes and immunoconjugates are also provided, as are nucleic acid molecule and vectors encoding the antibodies or antibody conjugates. The compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The antibody, fusion protein, ADC, CAR, CAR-expressing cell, multi-specific antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate can be formulated for systemic or local administration.

[0260] The compositions for administration can include a solution of the antibody, fusion protein, ADC, CAR, CAR-expressing cell (such as a T cell, B cell, DC, NK cell, macrophage or iPSC), multi-specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

[0261] An exemplary pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of an antibody (or fusion protein, ADC, CAR, multi-specific antibody, antibody-nanoparticle conjugate, or immunoconjugate), per subject per day. Dosages from 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. In some aspects, the composition can be a liquid formulation including one or more antibodies in a concentration range from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 1 mg / ml to about 10 mg / ml. Actual methods for preparing administrable compositions will be known or apparent to a skilled person and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005).

[0262] Monoclonal antibodies (or antibody conjugates, or nucleic acid molecules encoding such molecules) may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg / kg of body weight. Considerable experience is available in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN™ in 1997. Antibodies, Fc fusion proteins, ADCs, CARs (or CAR-expressing cells), multi-specific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes or immunoconjugates can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg / kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg / kg infused over a 30-minute period if the previous dose was well tolerated.

[0263] Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 Ltm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0264] Polymers can be used for ion-controlled release of the antibody -based compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, poloxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It is an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al.. Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al. , Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al.. Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271 ,961 ; 5,254,342 and 5,534,496).

[0265] XII. Therapeutic Methods

[0266] The antibodies, compositions, CARs (and CAR-expressing immune cells or iPSCs), ADCs, multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes and immunoconjugates disclosed herein can be administered to slow or inhibit the growth of tumor cells or inhibit the metastasis of tumor cells, such as a mesothelin-positive solid tumor. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a solid tumor that expresses mesothelin, such as, but not limited to, mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), ovarian cancer, endometrial cancer, cervical cancer, colon cancer, or thymus cancer.

[0267] Provided herein is a method of treating a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of a mesothelin-specific monoclonal antibody, immunoconjugate, CAR (or an immune cell or iPSC expressing a CAR), ADC, multi-specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of a mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of a mesothelin-specific monoclonal antibody, immunoconjugate, CAR (such as an immune cell or iPSC expressing a CAR), ADC, multi-specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. In some aspects, the mesothelin-positive cancer is a solid tumor, such as mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), ovarian cancer, endometrial cancer, cervical cancer, colon cancer, or thymus cancer.

[0268] The tumor does not need to be completely eliminated or inhibited for the method to be effective. For example, the method can decrease tumor size (e.g., volume) or metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to the absence of the treatment.

[0269] In one aspect, the method increases survival time of a subject with a mesothelin-positive cancer, for example by at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months (such as 2-72 months, 6-12 months, or 3-24 months), for example as compared to a survival time without a disclosed treatment.

[0270] A therapeutically effective amount of a mesothelin-specific monoclonal antibody, ADC, CAR (for example an immune cell or iPSC expressing a CAR), multi-specific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the antibody-based composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. Antibodies and conjugates thereof can be administered, for example, by intravenous infusion. Doses of the antibody or conjugate thereof can vary, but generally range between about 0.5 mg / kg to about 50 mg / kg, such as a dose of about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg. In some aspects, the dose of the antibody or conjugate can be from about 0.5 mg / kg to about 5 mg / kg, such as a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg or about 5 mg / kg. The antibody or conjugate is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or conjugate is administered weekly, every two weeks, every three weeks, or every four weeks.

[0271] In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.

[0272] In several aspects, a subject (such as a human subject with a mesothelin-positive tumor) is administered an effective amount of a viral vector that includes one or more nucleic acid molecules encoding a disclosed antibody. The viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody, and administration of the effective amount of the viral vector to the subject leads to expression of an effective amount of the antibody in the subject. Non-limiting examples of viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al., Nat. Med., 15(8) :901 -906, 2009 and Gardner et al., Nature, 519(7541) :87- 91, 2015.

[0273] In one aspect, a nucleic acid encoding a disclosed antibody, or conjugate thereof, is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

[0274] Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 JJ-g / kg to about 50 mg / kg, and typically are about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0275] Single or multiple administrations of a composition including a disclosed antibody or antibody conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit growth or metastasis of a mesothelin-positive cancer without producing unacceptable toxicity to the patient.

[0276] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0277] The mesothelin-specific antibody, antibody conjugate, nucleic acid molecule encoding such molecules, or a composition including such molecules, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intraarterially, intraperitoneally, intramuscularly, intradermally, intratumorally, intraprostatically, or intrathecally. In some aspects, the composition is administered by inhalation, such as by using an inhaler. In one aspect, the antibody, antigen binding fragment, or nucleic acid molecule encoding such molecules, or a composition including such molecules, is administered by a single subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, intratumoral or intrathecal injection once a day. The antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, can also be administered by direct injection at or near the site of disease. A further method of administration is by osmotic pump (e.g., an ALZET pump) or minipump (e.g., an ALZET mini-osmotic pump), which allows for controlled, continuous and / or slow-release delivery of the antibody, antibody conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre-determined period. The osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.

[0278] In one example, a mesothelin-specific antibody provided herein is conjugated to IR700, and photoimmunotherapy is used to treat a mesothelin-positive cancer. For example, such a method can include administering to the subject with a mesothelin-positive cancer a therapeutically effective amount of one or more mesothelin-specific antibody-IR700 conjugates, wherein the mesothelin-specific antibody specifically binds to mesothelin-expressing cells. Following administration of the conjugate, irradiation is performed at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) and at a dose of at least 1 J cm'2(such as at least 1 to about 50 J cm'2) thereby treating the mesothelin-positive cancer in the subject. In some examples, the mesothelin-positive tumor is irradiated at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) at a dose of at least 1 J cm'2(such as at least 1 J cm'2, at least 4 J cm'2,at least 10 J cm'2, at least 50 J cm'2, or at least 100 J cm'2) thereby treating the cancer in the subject. In some examples, multiple rounds of treatment are performed, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 treatment cycles. In particular examples, a therapeutically effective dose of a mesothelin-specific antibody-IR700 conjugates is at least 0.5 milligram per 60 kilogram (mg / kg), at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg, for example 0.5 to 50 mg / 60 kg, such as a dose of 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg, for example when administered intravenously. In another example, a therapeutically effective dose of a mesothelin-specific antibody-IR700 conjugates is at least 10 pg / kg, such as at least 100 pg / kg, at least 500 pg / kg, or at least 500 pg / kg, for example 10 pg / kg to 1000 pg / kg, such as a dose of 100 pg / kg, 250 pg / kg, about 500 pg / kg, 750 pg / kg, or 1000 pg / kg, for example when administered i.p. In one example, a therapeutically effective dose of an mesothelin-specific antibody-IR700 conjugates is at least 1 pg / ml, such as at least 500 pg / ml, such as between 20 pg / ml to 100 pg / ml, such as 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml administered in a topical solution.

[0279] In some aspects, the treatment methods further include administration of other anti-cancer agents or therapeutic treatments. Any suitable anti-cancer agent can be administered in combination with the compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and antiangiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells. Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0280] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0281] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0282] Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide) .

[0283] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Eeustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0284] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Eabs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IU-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0285] Exemplary biologies that can be used in combination with the disclosed methods include one or more monoclonal antibodies (mAbs) used to treat cancer, such as mAbs specific for EGFR (e.g., cetuximab), VEGF (e.g., bevacizumab), PD-1 (e.g., nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP-514), mesothelin (e.g., atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (envafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737), CD25 (e.g., daclizumab or basiliximab), CD20 (e.g., Tositumomab (Bexxar®); Rituximab (Rituxan, Mabthera); Ibritumomab tiuxetan (Zevalin, for example in combination with yttrium-90 or indium-ill therapy); Ofatumumab (Arzerra®), veltuzumab, obinutuzumab, ublituximab, ocaratuzumab), CD22 (e.g., narnatumab, inotuzumab ozogamicin, moxetumomab pasudotox) or CTLA4 (e.g., ipilimumab, tremelimumab). In some examples, the additional therapeutic agent administered is an anti-cancer monoclonal antibody, for example one or more of: 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox, Qregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, Zalutumumab, or combinations thereof In a specific example, the disclosed methods are used in combination with a therapeutic PD-1 mAb, such as one or more of nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI3O8), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514.

[0286] In some examples, the methods further include surgical treatment, for example surgical resection of the cancer or a portion of it. In some examples, the methods further include administration of radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

[0287] XIII. Methods for Diagnosis and Detection

[0288] Methods are also provided for the detection of the presence of mesothelin in vitro or in vivo. For example, the disclosed monoclonal antibodies can be used for in vivo imaging to detect a mesothelin- positive cancer. To use the disclosed antibodies as diagnostic reagents in vivo, the antibodies are labelled with a detectable moiety, such as a radioisotope, fluorescent label, or positron emitting radionuclides. As one example, the monoclonal antibodies (or antigen-binding fragments thereof) disclosed herein can be conjugated to a positron emitting radionuclide for use in positron emission tomography (PET); this diagnostic process is often referred to as immunoPET. To use the disclosed antibodies as diagnostic reagents in vitro, the antibodies can be directly or indirectly labelled with a detectable moiety (e.g., by using a labeled secondary antibody that binds to the mesothelin antibody), such as a radioisotope, enzyme, or fluorescent label. In some examples, the presence of mesothelin is detected in a biological sample from a subject and can be used to identify a subject with a mesothelin-positive cancer. The sample can be any sample, including, but not limited to, blood, serum, urine, semen, sputum, saliva, mucus, nasal wash, nasopharyngeal samples, oropharyngeal samples, tissue, cells, tissue biopsy, fine needle aspirate, surgical specimen, feces, cerebral spinal fluid (CSF), and bronchoalveolar lavage (BAL) fluid. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. The method of detection can include contacting a cell or sample, with an antibody or antibody conjugate e.g., a conjugate including a detectable marker) that specifically binds to mesothelin under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment).

[0289] Provided herein is a method of determining if a subject has a mesothelin-positive cancer by contacting a sample from the subject with a mesothelin-specific monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample. An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample identifies the subject as having a mesothelin-positive cancer.

[0290] In another aspect, provided is a method of confirming a diagnosis of a mesothelin-positive cancer in a subject by contacting a sample from a subject diagnosed with a mesothelin-positive cancer with a mesothelin monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample. An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample confirms the diagnosis of a mesothelin-positive cancer in the subject.

[0291] In one aspect, the antibody or antigen binding fragment is directly labeled with a detectable marker. In another aspect, the antibody that binds mesothelin (the primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection. The secondary antibody that is chosen is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

[0292] Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include12’I,131I, "S or 'H.

[0293] In an alternative aspect, mesothelin can be assayed in a biological sample by a competition immunoassay utilizing mesothelin standards labeled with a detectable substance and an unlabeled antibody that specifically binds mesothelin. In this assay, the biological sample, the labeled mesothelin standards and the antibody that specifically binds mesothelin are combined and the amount of labeled mesothelin standard bound to the unlabeled antibody is determined. The amount of mesothelin in the biological sample is inversely proportional to the amount of labeled mesothelin standard bound to the antibody that specifically binds mesothelin.

[0294] The immunoassays and methods disclosed herein can be used for a number of purposes. In one aspect, the antibody that specifically binds mesothelin may be used to detect the production of mesothelin in cells in cell culture. In another aspect, the antibody can be used to detect the amount of mesothelin in a biological sample, such as a sample obtained from a subject having or suspected or having a mesothelin- positive cancer.

[0295] In one aspect, a kit is provided for detecting mesothelin in a biological sample, such as a tissue biopsy, fine needle aspirate, core biopsy, blood, serum, urine, semen, CSF, nasopharyngeal, oropharyngeal, sputum, or saliva sample. Kits for detecting mesothelin-positive cells can include a monoclonal antibody that specifically binds mesothelin, such as monoclonal antibody disclosed herein. In a further aspect, the monoclonal antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label). In some examples, the antibody is present on a solid support, such as a bead or multi-well plate. In some examples, the kit further includes a detectably labeled secondary antibody that permits detection of the antibody that specifically binds mesothelin.

[0296] In one aspect, a kit includes instructional materials disclosing means of use of an antibody that binds mesothelin. The instructional materials may be written, in an electronic form or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. The kits may additionally include materials to obtain a sample, such as a swab, syringe, needle, and the like. Such kits and appropriate contents are well known.

[0297] In one aspect, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting mesothelin in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to mesothelin. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0298] The monoclonal antibodies disclosed herein can also be utilized in immunoassays, such as, but not limited to radioimmunoassays (RIAs), ELISA, lateral flow assay (LFA), or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS), such as for identifying / detecting mesothelin-positive cells. FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620). Any of the monoclonal antibodies that bind mesothelin, as disclosed herein, can be used in these assays. Thus, the monoclonal antibodies can be used in a conventional immunoassay, including, without limitation, ELISA, RIA, LFA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. The disclosed antibodies can also be used in nanotechnology methods, such as microfluidic immunoassays, which can be used to capture mesothelin, or exosomes containing mesothelin. Suitable samples for use with a microfluidic immunoassay or other nanotechnology method, include but are not limited to, saliva, blood, and fecal samples. Microfluidic immunoassays are described in U.S. Patent Application No. 2017 / 0370921, 2018 / 0036727, 2018 / 0149647, 2018 / 0031549, 2015 / 0158026 and 2015 / 0198593; and in Lin et al., JALA June 2010, pages 254-274; Lin et al., Anal Chem 92: 9454-9458, 2020; and Herr et l., Proc Natl Acad Sei USA 104(13): 5268-5273, 2007).

[0299] EXAMPLES

[0300] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0301] Example 1: Isolation and characterization of mesothelin-specific rabbit antibody RO4

[0302] A previously described mouse monoclonal antibody, 15B6, recognizes the C-terminal region of mesothelin (MSLN). The 15B6 antibody was made by immunizing mice with a peptide containing the C- terminal residues of mesothelin (IPNGYLVLDLSMQEALS; SEQ ID NO: 29). To generate additional antibodies recognizing this C-terminal region of mesothelin, rabbits were selected because they frequently produce antibodies with different properties than antibodies produced by mice.

[0303] Four rabbits were immunized with the C-terminal peptide of MSLN (SEQ ID NO: 29) attached to KLH (MSLN-KLH). Each rabbit was injected four times with 200 pg MSLN-KLH and B cell cloning was performed from PBMC using 1 of the 4 rabbits. Seventeen clones that reacted with mesothelin were isolated and sequenced.

[0304] Two forms of human MSLN naturally exist - one form has a methionine (M) at position 593 while the other has a valine (V) at position 593. These two forms of MSLN are referred to as MSLN(M) and MSLN(V), respectively. An ELISA assay using MSLN(M)-His and MSLN(V)-His was performed to select an antibody that reacted equally well with both forms of mesothelin. ELISA plates were coated with full- length mesothelin with a methionine at position 593 (593M), full-length mesothelin with a valine at position 593 (593V), and mesothelin with a C-terminal truncation of positions 296-591 (A296-591) or positions 296- 585 (A296-585). After blocking the plate, various concentrations of RO4 were added, followed by antirabbit IgG-HRP for detection. Antibody RO4 bound 593M and 593V mesothelin with equal affinity, but as expected, did not bind the C-terminal truncated forms of mesothelin (FIG. 1). Based on this data, antibody RO4, which exhibited a very high affinity and specificity for mesothelin, was selected for pre-clinical development.

[0305] The ability of rabbit antibody RO4 to detect mesothelin protein in normal human tissues was also evaluated by tissue microarray. Normal tissues from 32 human organs collected from three different individuals were included in the microarray. Tissues were sectioned at 5 pm. Automated immunohistochemistry (IHC) staining was performed using Bond-RX (Leica Biosystems, Buffalo Grove, IL) with the following conditions: Epitope Retrieval 2 (EDTA) for 20 minutes, RO40.02 pg / ml for 30 minutes, and Bond Polymer Refine Detection Kit (Leica Biosystems #DS9800) with omission of the Post Primary Reagent. DAB (3,3’ -diaminobenzidine) chromogen was used to stain the sections, which were subsequently counterstained with hematoxylin. Rabbit monoclonal IgG XP® Isotype Control (Cell Signaling Technology) used in place of the primary antibody for the negative control. Images were captured using the Aperio ScanScope FL (Leica Biosystems) whole slide scanner. One independent research pathologist evaluated the expression of mesothelin using the RO4 antibody.

[0306] The IHC results are shown in FIG. 5. Mesothelin was considered positive when localized along the cytoplasmic membrane. Mesothelial cells limning mesentery and pericardium were positive (KI and K2). The mesothelial sample (K3) had an artifact and could not be evaluated. Rare epithelial cells lining the tonsil (El and E3) were also positive. Mesothelin expression was negative in the rest of the evaluated organs: brain, adrenal gland, ovary, pancreas, lymph node, trachea, testis, breast, spleen, thymus, bone marrow, lung, heart, esophagus, stomach, small and large intestine, liver, salivary gland, kidney, prostate, uterus, skeletal muscle, skin, and peripheral nerve tissue. Rare false positive cytoplasmic staining was present in several organs including stomach, kidney, and uterus but no membranous staining was in any normal organs that were evaluated. Weak blood vessel staining was also present in several organs including the submucosal tissue of the larynx. Thyroid gland, cervix, and cancer tissue surrounding eye could not be evaluated due to artifact. A subsequent independent section from normal human thyroid tissue was evaluated and revealed negative expression of mesothelin in the follicular epithelial cells liming the thyroid gland.

[0307] In summary, mesothelin expression detected by the RO4 antibody was present in the mesothelial cells comprising the serosa of the abdominal and pleural organs with rare positivity in the epithelial cells lining the tonsil. All other normal organs showed no expression of mesothelin. Example 2: Comparison of RO4 and humanized RO4 (hRO4)

[0308] Rabbit antibody RO4 was humanized to produce to hRO4. The amino acid sequences of the VH domain and VL domain of each antibody are set forth herein as SEQ ID NOs: 1-4. RO4 and hRO4 have identical CDR sequences, but have sequence variation in the framework regions.

[0309] An ELISA was performed to compare the affinity of RO4 and hRO4 to both the 593M and 593V forms of mesothelin. Sample concentrations included in the assay were 70, 35, 17.5, 8.75, 4.38, 2.19 and 1.09 nM. FIGS. 2A-2D show binding of RO4 to 593M mesothelin (FIG. 2A), hRO4 binding to 593M mesothelin (FIG. 2B), RO4 binding to 593V mesothelin (FIG. 2C), and hRO4 binding to 593V mesothelin (FIG. 2D). Affinity measurements are listed in Table 2.

[0310] Table 2. Affinity data of mesothelin binding to antibodies RO4 and hRO4

[0311] Example 3: Comparison of hRO4, mouse 15B6 and humanized 15B6 (hl5B6)

[0312] Affinities of humanized RO4 and mouse monoclonal antibody 15B6 were compared by ELISA. Antibodies were conjugated with ALEXA FLUOR™ 647 (AF647) and mixed with mesothelin-expressing OVCAR8 cells, incubated for 30 minutes at room temperature, washed, and analyzed by flow cytometry. A monoclonal antibody (“MN”) that recognizes the N-terminal region (but not the C-terminal region) of cellsurface MSLN was also tested. A non-specific IgG antibody was used as a negative control. As shown in FIG. 3, hRO4 bound more strongly to OVCAR8 cells than ml5B6.

[0313] Next, binding of hRO4 and the humanized form of 15B6 (h!5B6) to the M and V forms of mesothelin was measured by BIACORE (KD was calculated from individual concentrations). Data was processed using BIACORE 8K Evaluation Software version 4.0. Flow cell and blank injections of running buffer in each cycle were used as double reference for resonance units (RU) subtraction. Calculated affinities are show in Table 3 below.

[0314] Table 3. Affinity of hRO4 and h!5B6 for mesothelin These results demonstrate that the affinity of hRO4 for MSLN-M is 16-fold greater than affinity of hl5B6 for MSLN-M. Similarly, the affinity of hRO4 for MSLN-V is 6-fold greater than the affinity of h!5B6 for MSLN-V.

[0315] Binding of hl5B6 and hRO4 to the human cancer cell lines RH16, RH29 and KLM1 was tested by flow cytometry. The results are shown in FIGS. 6A-6C. hRO4 exhibited stronger binding than hl5B6 for all three cell types tested.

[0316] As shown in FIGS. 15A-15B, RO4 binds differently to the C-terminal fragment of mesothelin than 15B6.

[0317] Example 4: Inhibition of mesothelin shedding experiments by RO4 and hRO4

[0318] Several studies were performed to evaluate whether rabbit antibody RO4 or the humanized hRO4 could inhibit shedding of mesothelin from cancer cell lines.

[0319] In a first experiment, inhibition of mesothelin shedding by rabbit antibody RO4 and mouse antibody 15B6 (15B6) was tested. A total of 50 (tg / ml of RO4 or 15B6 was incubated with RH16, RH29, OVCAR8, or KLM1 cells for 24 hours. A control IgG antibody and no antibody (NON) served as controls. Media were collected and shed mesothelin was detected by human mesothelin R-PLEX kit. As shown in FIG. 4, antibody RO4 showed greater inhibition of mesothelin shedding than antibody ml5B6 in RH29 human mesothelioma cells. Inhibition of mesothelin shedding in the other cell types was similar between the two antibodies.

[0320] A second experiment evaluated inhibition of mesothelin shedding by T3M4, HeLa, H06, KPC-hM- 911, and KPC-hM-994 cells using antibodies RO4, 15B6, hl5B6, or a control antibody. Cells were incubated with 15B6, hl5B6, RO4 or control antibody for 24 hours. Media were collected and shed mesothelin was detected by human mesothelin R-PLEX kit. RO4 inhibited mesothelin shedding by T3M4 (FIG. 7 A), HeLa (FIG. 7B) and H06 (FIG. 7C) to a significantly greater extent than either 15B6 or hl5B6. Inhibition of mesothelin shedding was similar amongst the three antibodies for KPC-hM-911 (FIG. 7D) and KPC-hM-994 (FIG. 7E) cells.

[0321] An additional study compared the ability of hRO4 and hl5B6 to inhibit mesothelin shedding by several different cancer cells lines. A total of 50 (ig / rnl of hRO4, hl6B6 or a control antibody was incubated with RH16, RH29, OVCAR8, KLM1, H226, H06 or PDA cells for 24 hours. Media were collected and shed mesothelin was detected by human mesothelin R-PLEX kit. As shown in FIG. 8, antibody hRO4 showed greater inhibition of mesothelin shedding than antibody hl5B6 in RH16, RH29, OVCAR8, KLM1, and H226 cells.

[0322] These studies demonstrated that antibodies RO4 and hRO4 were effective in inhibiting shedding of mesothelin from a variety of cancer cell lines and were significantly more effective in several cells lines than antibodies 15B6 and hl5B6, which bind the same C-terminal region of mesothelin. Example 5: Characterization of hRO4 chimeric antigen receptor T (CART) cells

[0323] A CAR containing hRO4 in scFv format was constructed (FIG. 9). The CART vector included a CD8 hinge region, a CD8 transmembrane (TM) domain, a 4- IBB co-stimulatory domain and a CD3^ signaling domain separated from a truncated form of human EGFR (hEGFRt) by a T2A sequence. For the studies described below, the hRO4 scFv was in the VL-linker-VH format.

[0324] Cytotoxicity of hRO4 CART and hl5B6 CART cells against mesothelin-expressing cancer cells was tested. T3M4, OVCAR-8, SW48, KLM1 or RH29 cells were co-cultured with hRO4 CAR- or h!5B6 CAR- transduced T cells at various effector to target (E / T) ratios for 20 hours. Specific lysis was measured using a luciferase-based assay. As shown in FIGS. 10A-10E, hRO4 CART cells were more cytotoxic against mesothelin-expressing cancer cells than hl5B6 CART cells.

[0325] Anti-tumor activity of hRO4 CAR T cells and h!5B6 CAR T cells in vivo was next tested. NOD scid gamma (NSG) mice were implanted subcutaneously with 5 million KLM1 cells and intravenously administered 2 million hRO4 CAR T or hl5B6 CAR T cells on day 6. As shown in FIG. 11, hRO4 CAR T cells exhibited significantly greater anti-tumor activity than h!5B6 CAR T cells.

[0326] Example 6: Bispecific antibody specific for mesothelin and CD3

[0327] A bispecific antibody (BsAb 13) containing the variable fragments of hRO4 and the variable fragments of an anti-CD3 antibody was constructed using the CrossMab format. Also tested was a bispecific antibody containing the variable fragments of anti-mesothelin antibody SSI and the variable fragments of the anti-CD3 antibody (referred to as BsAb 7). Cytotoxicity induced by BsAb 13 and BsAb 7 in various cancer cell lines was evaluated. Mesothelin-positive KB31, OVCAR-8, RH63, and KLM1 cells, and mesothelin-negative A431 and KLM1-E10 cells were seeded in 96-well plates and incubated with the 0.01, 0.1, 1, 10, or 100 nM BsAb 13 or BsAb 7 and donor T cells at a 10:1 E / T ratio. After three days of incubation, viable cells were measured with the Luciferase Assay system, and cytotoxic effect was calculated. As shown in FIGS. 12A-12D, BsAb 13 induced cytotoxicity in all mesothelin-expressing cancer lines in a dose-dependent manner. BsAb 7 also induced cytotoxicity of KB31, OVCAR-8 and RH63 cells. As expected, neither bispecific antibody induced cytotoxicity of the mesothelin-negative cell lines (FIGS. 12E-12F).

[0328] A further study evaluated whether BsAb 13-induced cytotoxicity is inhibited by mesothelin (MSLN) 296-591 His, a recombinant protein version of truncated MSLN found in patient ascites. KB31 (FIG. 13A), OVCAR-8 (FIG. 13B), RH63 (FIG. 13C) and KLM1 (FIG. 13D) cells were seeded in 96-well plates at the following densities: 2000 cells / well (FIGS. 13B, 13D), 3000 cells / well (FIG. 13A), and 5000 cells / well (FIG. 130). After overnight incubation, plates were treated with the following donor T cells: D15 at a 10:1 E / T ratio (FIGS. 13A, 13C) and D14 at a 20: 1 E / T ratio (FIGS. 13B, 13D). The co-cultures were then administered various doses of MSLN 296-591 His and either 0.1 pg / mL (FIGS. 13A, 13B) or 0.5 pg / mL (FIGS. 13C, 13D) bispecific antibody (BsAb 13 or BsAb 7). Cell viability was evaluated using the luciferase assay system (FIGS. 13B, 13D) or WST-8 assays (FIGS. 13A, 13C). Two technical replicates were performed with 3 replicates per MSLN 296-591 His dose. The results showed that BsAb 13-induced cytotoxicity was not inhibited by MSLN 296-591 His. In contrast, MSLN 296-591 His significantly inhibited cytotoxicity of BsAb 7.

[0329] A further study was conducted to determine the effect of BsAb 13 in an in vim tumor model. RH63 cells were implanted subcutaneously in NSG mice. On day 12, mice were injected I.P. with 10 million human PBMCs. Starting on day 13, mice were injected every other day with 3 mg / kg BsAb 13, for a total of 10 injections. Untreated (UT) mice and mice that received the PBMC injection but not BsAb 13 were included as control groups. Each group included 4 mice. In FIG. 14, average tumor size is plotted for each group. The results showed that BsAb 13 inhibited tumor growth in treated mice.

[0330] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

CLAIMS1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds mesothelin, comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: the VH domain comprises a heavy chain complementarity determining region (HCDR) 1, a HCDR2 and a HCDR3, and the VL domain comprises a light chain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3; and the VH domain comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1; and the VL domain comprises the LCDR1, LCDR1 and LCDR3 sequences of SEQ ID NO: 2.

2. The monoclonal antibody of antigen-binding fragment of claim 1, wherein:(i) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively comprise SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10;(ii) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and(iii) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and the amino acid sequences of the LCDR1, LCDR1 and LCDR3 respectively comprise SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

3. The monoclonal antibody or antigen-binding fragment of claim 1 or claim 2, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3; and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

4. The monoclonal antibody or antigen-binding fragment of any one of claims 1-3, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2; or the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 3 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 4.

5. The monoclonal antibody or antigen-binding fragment of any one of claims 1-4, wherein the monoclonal antibody is an IgG, IgA, or IgM molecule.

6. The monoclonal antibody or antigen-binding fragment of any one of claims 1-4, wherein the antigen-binding fragment is a Fab fragment, a Fab’ fragment, a F(ab)’2 fragment, a single chain variable fragment (scFv) or a disulfide stabilized variable fragment (dsFv).

7. The monoclonal antibody or antigen-binding fragment of any one of claims 1-6, which is a humanized monoclonal antibody or antigen-binding fragment.

8. A chimeric antigen receptor (CAR) comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-7.

9. The CAR of claim 8, wherein the antigen-binding fragment is an scFv.

10. The CAR of claim 8 or claim 9, further comprising a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof.

11. The CAR of claim 10, wherein: the hinge region comprises a CD8 hinge region; the transmembrane domain comprises a CD8 transmembrane domain; the costimulatory signaling moiety comprises a 4- IBB signaling moiety; and / or the signaling domain comprises a CD3^ signaling domain.

12. An isolated cell expressing the CAR of any one of claims 8-11.

13. The isolated cell of claim 12, which is a T cell, B cell, natural killer (NK) cell, macrophage, dendritic cell (DC), or induced pluripotent stem cell (iPSC).

14. A multi-specific antibody comprising the antibody or antigen-binding fragment of any one of claims 1-7 and at least one additional monoclonal antibody or antigen-binding fragment.

15. The multi-specific antibody of claim 14, which is a bispecific antibody or a trispecific antibody.

16. The multi-specific antibody of claim 14 or claim 15, wherein the at least one additional monoclonal antibody or antigen-binding fragment comprises a monoclonal antibody or antigen-binding fragment that specifically binds:CD3 on T cells;CD16, NKp46 or NKG2D on NK cells; orCD89 on macrophages.

17. The multi-specific antibody of claim 16, which is a bispecific antibody that specifically binds mesothelin and CD3, and comprises the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

18. An immunoconjugate comprising the antibody or antigen-binding fragment of any one of claims 1-7 and an effector molecule.

19. The immunoconjugate of claim 18, wherein the effector molecule is a toxin, a photon absorber, or a detectable label.

20. The immunoconjugate of claim 19, wherein the toxin comprises a Pseudomonas exotoxin or a variant thereof.

21. The immunoconjugate of claim 19, wherein the detectable label comprises a fluorophore, an enzyme or a radioisotope.

22. An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody or antigen -binding fragment of any one of claims 1-7.

23. The ADC of claim 22, wherein the drug is a small molecule.

24. The ADC of claim 22 or claim 23, wherein the drug is an anti-microtubule agent, an antimitotic agent and / or a cytotoxic agent.

25. An antibody-nanoparticle conjugate, comprising a nanoparticle conjugated to the antibody of any one of claims 1-7.

26. The antibody-nanoparticle conjugate of claim 25, wherein the nanoparticle comprises a polymeric nanoparticle, nanosphere, nanocapsule, liposome, dendrimer, polymeric micelle, or niosome.

27. The antibody-nanoparticle conjugate of claim 25 or claim 26, wherein the nanoparticle comprises a cytotoxic agent.

28. A fusion protein comprising the antibody of any one of claims 1-7 and a heterologous protein or peptide.

29. The fusion protein of claim 28, wherein the heterologous protein is an Fc protein.

30. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment of any one of claims 1-7, the CAR of any one of claims 8-11, the multi-specific antibody of any one of claims 14-17, the immunoconjugate of any one of claims 18-21, or the fusion protein of any one of claims 28-29.

31. The isolated nucleic acid molecule of claim 30, wherein the nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment is: at least 90% identical to SEQ ID NO: 23, or a degenerate variant thereof; and / or at least 90% identical to SEQ ID NO: 24, or a degenerate variant thereof.

32. The isolated nucleic acid molecule of claim 31, wherein the nucleotide sequence comprises or consists of:SEQ ID NO: 23, or a degenerate variant thereof; and / orSEQ ID NO: 24, or a degenerate variant thereof.

33. The isolated nucleic acid molecule of any one of claims 30-32, operably linked to a promoter.

34. A vector comprising the isolated nucleic acid molecule of claim 33.

35. An isolated cell comprising the isolated nucleic acid molecule of any one of claims 30-33 or the vector of claim 34.

36. A composition comprising a pharmaceutically acceptable carrier and the monoclonal antibody of any one of claims 1-7, the CAR of any one of claims 8-11, the isolated cell of any one of claims 12, 13 and 35, the multi-specific antibody of any one of claims 14-17, the immunoconjugate of any one of claims 18-21, the ADC of any one of claims 22-24, the antibody-nanoparticle conjugate of any one of claims 25-27, the fusion protein of any one of claims 28-29, the isolated nucleic acid molecule of any one of claims 30-33, or the vector of claim 34.

37. A method of detecting expression of mesothelin in a sample, comprising contacting the sample with the monoclonal antibody or antigen-binding fragment of any of claims 1-7; and detecting binding of the monoclonal antibody or antigen-binding fragment to the sample, thereby detecting expression of mesothelin in the sample.

38. A method of diagnosing a subject as having a mesothelin-positive cancer, comprising: contacting a sample obtained from the subject with the monoclonal antibody or antigen-binding fragment of any of claims 1-7; and detecting binding of the monoclonal antibody or antigen-binding fragment to the sample, thereby diagnosing the subject as having a mesothelin-positive cancer.

39. A method of treating a mesothelin-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody of any one of claims 1-7, the CAR of any one of claims 8-11, the isolated cell of any one of claims 12-13, the multi-specific antibody of any one of claims 14-17, the immunoconjugate of any one of claims 18-21, the ADC of any one of claims 22-24, the antibody-nanoparticle conjugate of any one of claims 25-27, the fusion protein of claim 28 or claim 29, or the composition of claim 36.

40. The method of claim 39, wherein the mesothelin-positive cancer is a solid tumor.

41. The method of claim 39 or claim 40, wherein the mesothelin-positive cancer is a mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, colon cancer, or thymus cancer.

42. A kit, comprising: the monoclonal antibody of any one of claims 1-7, the CAR of any one of claims 8-11, the isolated cell of any one of claims 12, 13 and 35, the multi -specific antibody of any one of claims 14-17, the immunoconjugate of any one of claims 18-21, the ADC of any one of claims 22-24, the antibody- nanoparticle conjugate of any one of claims 25-27, the fusion protein of any one of claims 28-29, the isolated nucleic acid molecule of any one of claims 30-33, the vector of claim 34, or the composition of claim 36; and one or more of a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and instructional materials.

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