Cross-species single-domain antibodies targeting mesothelin for the treatment of solid tumors

Abstract

The present invention discloses camel single domain monoclonal antibodies that specifically bind to human and mouse mesothelin. The present invention also discloses chimeric antigen receptor (CAR) T cells and antibody conjugates based on mesothelin-specific antibodies. For example, the disclosed CAR T cells, mesothelin-specific antibodies and conjugates thereof can be used in the diagnosis or treatment of mesothelin-positive cancers.

Description

[0001] Cross-application in related fields

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 789,650, filed January 8, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to camelid single domain monoclonal antibodies that specifically bind to human and mouse mesothelin. The present invention also relates to the use of mesothelin-specific antibodies, such as for immunotherapy for treating tumors expressing mesothelin.

[0004] Recognition of government support

[0005] This invention was made with government support under Grant No. Z01 BC010891 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0006] The mesothelin (MSLN) gene encodes a -70 kDa precursor protein that is processed into 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). Mesothelial cells in the pleura, peritoneum, and pericardium of healthy individuals have relatively low levels of mesothelial cells, but they can be expressed in malignant mesothelioma (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 gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, bile duct cancer, 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. 11:394-395, 2008; McGuire et al., N. Engl. J. Med. 335:1-6, 1996; Argani et al., Clin. Cancer Res. 11:395-396, 2008; McGuire et al., N. Engl. J. Med. 336:1-6, 1996; Argani et al., Clin. Cancer Res. 11:394-396, 2008; 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 7,081,518).

[0007] Mesothelin is a recognized tumor target in multiple solid tumors. Although many mesothelin-specific antibodies and CAR-expressing T cells have been developed, and many clinical trials evaluating these antibodies and CAR T cells targeting mesothelin are ongoing, no antibodies have been reported to be cross-species, especially with high binding affinity to human and mouse mesothelin-positive cells. Moreover, CAR T cells targeting mesothelin have not shown effective antitumor activity in both mice and humans. Therefore, there is still a need for mesothelin-specific antibodies that bind to mouse and human mesothelin to evaluate the safety and efficacy of therapeutic antibodies in animal models. Summary of the invention

[0008] The present invention provides two cross-species mesothelin-specific camelid single domain monoclonal antibodies isolated by phage display. The mesothelin-specific antibodies, also known as A101 and G8, specifically bind human and mouse mesothelin with high affinity. Chimeric antigen receptor (CAR) T cells composed of the disclosed antibodies can effectively kill mesothelin-positive tumor cells in vitro and in vivo.

[0009] Provided herein are single domain monoclonal antibodies that can bind, for example, specifically bind to mesothelin. In some embodiments, the monoclonal antibody comprises a complementary determining region (CDR) sequence of A101 or G8. Also provided herein are conjugates comprising the disclosed monoclonal antibodies. In some examples, CARs (as well as T cells and natural killer cells expressing CARs) comprising the monoclonal antibodies disclosed herein, immunoconjugates (such as immunotoxins), multispecific antibodies, antibody-drug conjugates (ADCs), antibody-nanoparticles, conjugates or fusion proteins are provided. The present invention also provides compositions comprising mesothelin-specific monoclonal antibodies and compositions of pharmaceutically acceptable carriers.

[0010] Also provided herein are nucleic acid molecules and vectors encoding mesothelin-specific monoclonal antibodies, CARs, immunoconjugates (such as immunotoxins), multispecific antibodies, and fusion proteins disclosed herein.

[0011] Further provided is a nucleic acid construct encoding both mesothelin-specific CAR and truncated human epithelial cell growth factor receptor (huEGFRt). The encoded CAR includes a mesothelin-specific single-domain monoclonal antibody fused to an extracellular hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain. huEGFRt includes two EGFR extracellular domains (structural domain III and structural domain IV) and an EGFR transmembrane domain, but lacks two membrane-terminal extracellular domains and all intracellular domains. In some embodiments, the nucleic acid molecule includes a nucleic acid encoding a first signal sequence in the 5' to 3' direction; a nucleic acid encoding a mesothelin-specific antibody; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second signal sequence; and a nucleic acid encoding huEGFRt. Also disclosed is a vector, such as a viral vector, which includes a nucleic acid molecule disclosed herein. Also disclosed are isolated cells, such as T lymphocytes, that co-express the disclosed CARs and huEGFRt.

[0012] Also provided are methods for treating mesothelin-positive cancer in a subject and methods for inhibiting tumor growth or metastasis of mesothelin-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a monoclonal antibody disclosed herein, or administering to the subject a CAR (or CAR T cell or CAR NK cell), an immune conjugate (such as an immunotoxin), an ADC, a multispecific antibody, an antibody-nanoparticle conjugate, or a fusion protein expressing a monoclonal antibody disclosed herein.

[0013] Further provided herein are methods of detecting expression of mesothelin in a sample. In some embodiments, the method comprises contacting the sample with a monoclonal antibody disclosed herein and contacting a detection antibody with the sample.

[0014] Also provided are methods of diagnosing a subject having a mesothelin-positive cancer. In some embodiments, the method comprises contacting a sample obtained from the subject with a monoclonal antibody disclosed herein and detecting binding of the antibody to the sample.

[0015] The foregoing and other objects and features of the present invention will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: Isolation of mesothelin-specific camelid single domain antibodies by phage display. The figure shows the results of an ELISA of polyclonal phage testing the output phage of each round of panning for binding to mouse mesothelin and human mesothelin. The bars on the left represent the panning used to isolate A101. The bars on the right represent the panning used to isolate G8. Both of these showed strong binding to human and mouse mesothelin.

[0017] Figure 2 :The purity and molecular weight of A101 and G8 were shown in SDS gel electrophoresis. A101 was 15.16 kDa, and G8 was 15.36 kDa.

[0018] Figure 3 : ELISA analysis of human and mouse mesothelin protein by A101 and G8 anti-mesothelin VHH antibodies.

[0019] Figure 4 : ELISA analysis of A101 and G8 anti-mesothelin VHH antibodies for mesothelin fragments with defined epitope binding. Both antibodies bound to mesothelin fragment I and full-length mesothelin.

[0020] Figures 5A-5C :Flow cytometric analysis of cell surface mesothelin expression using A101 antibody. ( Fig. 6A )M30 and H226 are human mesothelioma cell lines; OVCAR8 is a human ovarian cancer cell line; and Panc3.014 is a human pancreatic cancer cell line. ( Figure 5B ) KLM1 and T3M4 are human pancreatic cancer cell lines; and KMBC and OZ are human cholangiocarcinoma cell lines. ( Figure 5C )PDA95775, CREP133234 and CREP133239 are mouse pancreatic cancer cell lines. The peak labeled "1" indicates cell surface staining with subtype control, and the peak labeled "2" indicates cell surface staining of mesothelin. 10 μg / ml A101 was used for staining.

[0021] Figures 6A-6C :Flow cytometric analysis of cell surface mesothelin expression using G8 antibody. ( Fig. 6A )M30 and H226 are human mesothelioma cell lines; OVCAR8 is a human ovarian cancer cell line; and Panc3.014 is a human pancreatic cancer cell line. ( Figure 6B ) KLM1 and T3M4 are human pancreatic cancer cell lines; and KMBC and OZ are human cholangiocarcinoma cell lines. ( Figure 6C )PDA95775, CREP133234 and CREP133239 are mouse pancreatic cancer cell lines. The peak labeled "1" indicates cell surface staining with isotype control, and the peak labeled "2" indicates cell surface staining of mesothelin. 10 μg / ml of G8 was used for staining.

[0022] Figure 7A-7B :Generation of CAR T cells targeting mesothelin. ( Fig. 7A ) Schematic diagram of a lentiviral construct expressing a mesothelin-targeting CAR and a truncated human EGFR (huEGFRt) using a T2A ribosomal skipping sequence. ( Figure 7B ) Flow cytometric detection of huEGFRt expression was used to analyze the expression of mesothelin-targeting CAR on human T cells transduced with lentiviral particles.

[0023] Figure 8 : Killing of mesothelin-positive cells by T cells expressing CARs based on A101 or G8. Luciferase-expressing A431 (mesothelin-negative), H9 (mesothelin-overexpressing), KLM1 (pancreatic cancer), and H226 (mesothelioma) cells were co-cultured with mock (grey line), A101 or G8 CAR-truncated T cells (blue line) at the indicated E:T ratios for 20 h, and specific lysis was determined using a luminescence-based cytolysis assay.

[0024] Figures 9A-9C : CAR-T cells targeting mesothelin showed strong activity in mice bearing human mesothelin tumors. Fig. 9A ) Experimental schematic. 13 days after tumor cell inoculation, mock T cells or 20×10 6 CAR T cells were used to treat NSG mice bearing H226 tumors. Tumor burden was monitored by bioluminescence imaging. Fig. 9B )A101 CAR T cells demonstrated antitumor activity and showed a trend toward eradication of H226 xenograft tumors. ( Fig. 9C )right Figure 8 Bioluminescence was quantified in treated mice as in B.

[0025] Figures 10A-10B : Binding epitopes of A101 and G8 camelid single domain antibodies on human mesothelin (SEQ ID NO: 12). Fig. 10A ) produced human mesothelin fragments, including constructs encoding amino acid residues 296-390 (domain I), 391-486 (domain II), and 487-581 (domain III) of mesothelin, as well as constructs encoding smaller fragments within domain I: domain IAB (296-359), domain IBC (328-405), domain IA (296-337), domain IB (328-369), and domain IC (360-405). Domain IAB (269-359) mutants with E321A, W321A, or Y318A substitutions were also generated. ( Fig. 10B) A101 and G8 camelid single domain antibodies bind primarily to the N-terminal region I (296-390) of mesothelin, including the IAB domain (64 residues, 296-359) as determined by ELISA. When tyrosine residue 318 was mutated to alanine (Y318A), no A101 or G8 bound.

[0026] Fig.11 : A101 camelid antibody does not bind to mesothelin-negative cell lines. No binding of A101 (5 μg / ml) was detected on mesothelin-negative cell lines, indicating that A101 specifically binds to mesothelin.

[0027] Fig.12 : G8 camelid antibody does not bind to mesothelin-negative cell lines. No binding of G8 (5 μg / ml) was detected on mesothelin-negative cell lines, indicating that G8 specifically binds to mesothelin.

[0028] Figures 13A-13B : CAR T cells expressing A101 and G8 antibodies kill mesothelin-positive cell lines. ( Fig.13A ) Transduction efficiency of CAR T cells expressing A101 and G8 antibodies. Peripheral blood mononuclear cells (PBNC) of healthy donors were transduced with A101 or G8 camelid VHH. Cetuximab was used to detect CAR on the cell surface ( Fig. 13B )CAR T cell killing assay. Both A101 and G8CAR T cells can kill mesothelin-positive cell lines (H9, KLM1, and H226), but cannot kill mesothelin-negative cells (A431).

[0029] Figures 14A-14B : Testing of A101 CAR T cells in mice bearing peritoneal mesothelioma xenografts. 7-week-old female NSG mice were inoculated with 2MH226-luc cells. On day 3, animals were treated with mock T cells or A101 CAR T cells. ( Fig.14A ) Bioluminescence images of mock-treated and A101-treated mice. Fig. 14B ) Schematic diagram of the study (top) and graph showing the irradiance as a measure of tumor size in mock-treated and A101-treated animals (bottom). A101-CAR effectively inhibited the growth of H226 xenograft tumors in mice.

[0030] Sequence Listing

[0031] The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using the standard letter abbreviations for nucleotide bases and the three-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the displayed strand. The sequence listing is submitted as an ASCII text file, created on December 26, 2019, with a size of 14.4KB, and is incorporated herein by reference. In the attached sequence listing:

[0032] SEQ ID NO: 1 is the nucleotide sequence of the A101 antibody.

[0033] SEQ ID NO: 2 is the amino acid sequence of the A101 antibody.

[0034] SEQ ID NO: 3 is the nucleotide sequence of the G8 antibody.

[0035] SEQ ID NO: 4 is the amino acid sequence of the G8 antibody.

[0036] SEQ ID NO: 5 is an exemplary GMCSFRss amino acid sequence.

[0037] SEQ ID NO: 6 is an exemplary CD8 alpha hinge region amino acid sequence.

[0038] SEQ ID NO: 7 is an exemplary CD8 alpha transmembrane region amino acid sequence.

[0039] SEQ ID NO: 8 is an exemplary 4-1BB amino acid sequence.

[0040] SEQ ID NO: 9 is an exemplary CD3 zeta amino acid sequence.

[0041] SEQ ID NO: 10 is an exemplary self-cleaving T2A peptide amino acid sequence.

[0042] SEQ ID NO: 11 is an exemplary huEGFRt amino acid sequence.

[0043] SEQ ID NO: 12 is an exemplary human mesothelin amino acid sequence. DETAILED DESCRIPTION

[0044] I. Abbreviations

[0045] ADC Antibody-drug conjugate

[0046] ADCC Antibody-dependent cell-mediated cytotoxicity

[0047] CAR Chimeric Antigen Receptor

[0048] CDR complementarity determining region

[0049] CTL Cytotoxic T lymphocyte

[0050] E:T effector:target

[0051] EGF Epidermal Growth Factor

[0052] EGFR Epidermal Growth Factor Receptor

[0053] ELISA enzyme-linked immunosorbent assay

[0054] FACS fluorescence activated cell sorting

[0055] GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence

[0056] huEGFRt human truncated epidermal growth factor receptor

[0057] Ig immunoglobulin

[0058] NK Natural Killer

[0059] PE Pseudomonas exotoxin

[0060] PET Positron Emission Tomography

[0061] II. Overview of Terminology

[0062] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology are provided in Benjamin Lewin, Genes X, published by Jones & Bartlett, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley VCH, 2008, Volume 16; and other similar references.

[0063] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "antigen" includes both the singular and the plural, and may be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprising" means "including." It should be further understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values, unless otherwise indicated, given for nucleic acids or polypeptides are approximate and are used for descriptive purposes. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In the event of a conflict, the present specification, including the explanation of terminology, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. To facilitate review of the various embodiments, the following explanation of terminology is provided.

[0064] 4-1BB: A co-stimulatory molecule expressed by lymphocytes activated by the T cell receptor (TCR) and by other cells including natural killer cells. Ligation of 4-1BB induces a signaling cascade that can produce cytokines, express anti-apoptotic molecules, and enhance immune responses. An exemplary amino acid sequence of 4-1BB is described herein as SEQ ID NO: 8.

[0065] Administration: providing or administering an agent, such as an anti-mesothelin antibody provided herein, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, oral administration, injection (such as subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal injection, intravenous injection, and intratumor injection), sublingual administration, rectal administration, transdermal administration, intranasal administration, vaginal administration, and inhalation administration.

[0066] Antibody: A polypeptide ligand that includes at least one variable region that can recognize and bind (e.g., specifically recognize and specifically bind) an antigenic epitope. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each of which has a variable region, called a heavy (V) and a light (L) chain. H ) chain variable region and light (V L ) chain variable region. H Area and V L The heavy chain region is responsible for binding the antigen recognized by the antibody. There are five major classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecules IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary antibody produced by birds and reptiles that is similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0067] The variable region of an antibody includes a "framework" region and a hypervariable region, called a "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of the antigen. The framework region of an antibody is used to position and arrange the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be easily determined using any of a number of well-known numbering schemes, including Kabat et al., Protein Sequences of Immunological Significance, U.S. Department of Health and Human Services, 1991; "Kabat" numbering scheme), Chothia et al. (see Chothia and Lesk, Journal of Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273927-9481997; "Chothia" numbering scheme), those described by Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; Kunik et al., Nucleic Acids Res 40 (web server version): W521-5242012 subgroup CDRs), and the Immunogenetics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-92001; "IMGT" numbering scheme). The Kabat, Paratome, and IMGT databases can be maintained online.

[0068] "Single domain antibody" refers to an antibody with a single domain (the variable domain) that is capable of specifically binding to an antigen or antigenic epitope in the absence of additional antibody domains. Single domain antibodies include, for example, V H Domain antibodies, V NAR Antibodies, Camel V H H antibody and V L Domain antibodies. V NAR Antibodies are produced by cartilaginous fish such as nurse sharks, wahoo sharks, spiny dogfish and bamboo sharks. H H antibodies are produced by several species including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy chain antibodies that naturally lack light chains.

[0069] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by cells transfected with a coding sequence for a pre-existing single antibody. Monoclonal antibodies can be produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0070] "Chimeric antibodies" have framework residues from one species (eg, human) and CDRs (typically conferring antigen binding) from another species.

[0071] A "humanized" antibody is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor" and the human immunoglobulin that provides the framework is called the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. There is no constant region, but if present, they must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, such as about 95% or more identical. Therefore, all parts of the humanized immunoglobulin, possibly except the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding function or other immunoglobulin functions.

[0072] Antibody-drug conjugate (ADC): A molecule comprising an antibody (or an antigen-binding fragment of an antibody) conjugated (e.g., covalently bound) to a drug, such as a cytotoxic agent. ADCs can be used to specifically target drugs to cancer cells through specific binding of the antibody to tumor antigens expressed on the cell surface. Exemplary drugs used with ADCs include anti-microtubule agents (e.g., maytansinoids, auristatin E, and auristatin F) and inter-bond crosslinkers (e.g., pyrrolobenzodiazepines; PDBs). In some cases, the ADC is a bispecific ADC, which consists of two monoclonal antibodies or antigen-fragments thereof, each directed against a different antigen or epitope conjugated to a drug.

[0073] Antimicrotubule agents: A class of drugs that block cell growth by preventing mitosis. Antimicrotubule agents, also called "antimitotic agents," are used to treat cancer.

[0074] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another embodiment, binding affinity is determined by the antigen / antibody off-rate. In another embodiment, high binding affinity is determined by competitive emission immunoassay. In another embodiment, binding affinity is determined by ELISA. In other embodiments, antibody affinity is determined by flow cytometry or by surface plasmon reference. An antibody that "specifically binds" an antigen (such as mesothelin) is an antibody that binds to the antigen with a higher affinity but does not significantly bind to other unrelated antigens.

[0075] In some examples, an antibody or fragment thereof (such as an anti-mesothelin antibody provided herein) specifically binds to a target (such as mesothelin) with a binding constant that is at least 10 greater than the binding constant of another molecule in a sample or subject. 3 M -1 , 10 4 M -1 or 10 5 M -1 In some examples, the equilibrium constant (Kd) of the antibody (e.g., a monoclonal antibody) or a fragment thereof is 1 nM or less. For example, the antibody or fragment thereof binds to a target such as mesothelin with a binding affinity of at least about 0.1 x 10 -8 M, at least about 0.3x 10 - 8 M, at least about 0.5 x 10 -8 M, at least about 0.75 x 10 -8 M, at least about 1.0x 10 -8 M, at least about 1.3x 10 -8 M, at least about 1.5x 10 -8 M or at least approximately 2.0x 10 -8 M, at least about 2.5x 10 -8 , at least about 3.0x 10 -8 , at least about 3.5x 10 -8 , at least about 4.0x 10 -8 , at least about 4.5x 10 -8 or at least about 5.0x 10 -8 In certain embodiments, the dissociation constant (Kd) of a specific binding agent for binding to a target is ≤104 nM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 0 -13 M, for example 10 -9 M to 10 - 13 M). In one embodiment, Kd is determined by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen (see, e.g., Chen et al., J. Mol. Biol. 293:865-881, 1999). In another example, Kd is determined by surface plasmon resonance assay using a BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip with approximately 10 response units (RU).

[0076] Bispecific antibody: A recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies (e.g., two single domain antibodies) and is therefore capable of binding to two different antigens. In some embodiments, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, CTLs (e.g., CTL receptor components, such as CD3) or effector natural killer (NK) cells and tumor antigens. Similarly, a multispecific 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 (e.g., single domain antibodies).

[0077] Breast cancer: A cancer that forms in the tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that produce 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 high levels of the HER2 / neu protein. Triple-negative breast cancer is also called ER-negative PR-negative HER2 / neu breast cancer.

[0078] Chemotherapeutic agent: Any chemical agent that is therapeutically effective in treating a disease characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one embodiment, a chemotherapeutic agent is an agent used in treating mesothelin-positive tumors. In one embodiment, a chemotherapeutic agent is a radioactive compound. The following references disclose exemplary chemotherapeutic agents that can be used with the methods provided herein: Slapak and Kufe, Principles of Cancer Therapy, Harrison's Principles of Internal Medicine, 14th edition, Chapter 86; Perry et al., Chemotherapy, Abeloff's Clinical Oncology, 2nd edition, Chapter 17, Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds.): Handbook of Cancer Chemotherapy, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. An example is the administration of an antibody that binds mesothelin with a radioactive or chemical compound. In one example, the chemotherapeutic agent is biological, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody), such as an anti-mesothelin antibody provided herein, and other anti-cancer antibodies, such as anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-EGFR (e.g., cetuximab), or anti-VEGF (e.g., bevacizumab).

[0079] Chimeric Antigen Receptor (CAR): A chimeric molecule that includes an antigen binding portion (such as an scFv or a single domain antibody) and a signaling domain (such as a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, CAR consists of an antigen binding portion, a transmembrane domain, and an endodomain. The endodomain generally includes a signal chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the endodomain also includes an intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10. In some examples, CAR is bispecific or bicistronic. Bispecific CAR is a single CAR molecule that consists of two antigen binding domains (such as two scFvs or two single domain antibodies) that bind to different antigens, respectively. Bicistronic CAR refers to two complete CAR molecules, each containing an antigen binding portion that binds to a different antigen. In some cases, the bicistronic CAR construct expresses two complete CAR molecules connected by a cleavage joint. T cells or NK cells expressing bispecific or bicistronic CARs can bind to cells expressing two antigens targeted by the binding moiety (see, e.g., Qin et al., Blood, 130: 810, 2017; and WO / 2018 / 213337).

[0080] Cholangiocarcinoma: A cancer that develops in the cells lining the bile ducts in the liver.

[0081] Complementarity Determining Region (CDR): A highly variable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Single domain antibodies include three CDRs, referred to herein as CDR1, CDR2, and CDR3.

[0082] Conjugate: In the context of the present invention, 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, a toxin, a therapeutic agent, a detectable marker, a protein, a nucleic acid, a lipid, a nanoparticle, a photon absorber, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates". When the conjugate includes an antibody linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC". Other antibody conjugates include, for example, multispecific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0083] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of a protein (such as an antibody for mesothelin). For example, a monoclonal antibody that specifically binds to mesothelin may include up to about 1, at least about 2, at least about 5, and up to about 10, or up to about 15 conservative substitutions, and specifically binds to a 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 the antibody specifically binds to mesothelin. Non-conservative substitutions are those that reduce activity or binding to mesothelin.

[0084] Conservative amino acid substitution tables that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other.

[0085] 1) Alanine (A), serine (S), threonine (T);

[0086] 2) Aspartic acid (D), glutamic acid (E);

[0087] 3) Asparagine (N), glutamine (Q);

[0088] 4) Arginine (R), Lysine (K);

[0089] 5) isoleucine (I), leucine (L), methionine (M), and valine (V); and

[0090] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0091] Contact: Placed in direct physical connection; includes both solid and liquid phases.

[0092] Cytotoxic agent: A drug or compound that kills cells.

[0093] Cytotoxicity: A molecule such as an immunotoxin that is toxic to the cells that are intended to be targeted, as opposed to the cells of the rest of the organism. Conversely, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than the cells that are intended to be targeted by the targeting moiety of the immunotoxin, and the term "zootoxicity" refers to the toxicity of an immunotoxin to an animal, i.e., through the toxicity of the immunotoxin to cells other than those that are intended to be targeted by the immunotoxin.

[0094] Diagnostic: Identifying the presence, absence or nature of a pathological condition, such as mesothelin-positive cancer. Diagnostic methods vary in sensitivity and specificity. The "sensitivity" of a diagnostic assay refers to the percentage of diseased individuals for whom the test tests positive (percent true positives). The "specificity" of a diagnostic assay refers to 1 minus the false positive rate, where the false positive rate is defined as the proportion of those individuals who do not have the disease for whom the test tests positive. While a particular diagnostic assay may not provide a definitive diagnosis of a condition, it is sufficient if the assay provides a positive indication that aids in the diagnosis. "Prognosis" refers to the likelihood (e.g., severity) of developing a pathological condition, such as mesothelioma.

[0095] Diagnostic tumor imaging: Conjugating antibodies and their derivatives to positron-emitting radionuclides for positron emission tomography (PET) is a process often referred to as immuno-PET. Although full-length antibodies can be used as immuno-PET agents, their biological half-life requires waiting several days before imaging, resulting in increased non-targeted radiation doses. Smaller single-domain antibodies have biological half-lives suitable for same-day imaging.

[0096] Drug: Any compound used to treat, alleviate or prevent a disease or condition in a subject. In some embodiments herein, the drug is an anticancer agent, for example a cytotoxic agent, such as an antimitotic agent or an antimicrotubule agent.

[0097] Effector molecule: The portion of a chimeric molecule that is intended to produce a desired effect on the cells to which the chimeric molecule is targeted. Effector molecules are also referred to as effector moieties (EM), therapeutic agents, diagnostic agents, or similar terms. Therapeutic agents (or drugs) include compounds such 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 crosslinking with single or double DNA, and oligonucleotides that form triple chains. Alternatively, the molecule linked to the targeting moiety, such as an anti-mesothelin antibody, can be an encapsulation system, such as a liposome or micelle, which contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that is protected from direct exposure to the blood circulation system. Methods for preparing liposomes linked to antibodies or other therapeutic agents are known (see, for example, U.S. Pat. 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 for this purpose include radioactive isotopes, such as 35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H. 14 C. 15 N. 90 Y. 99 Tc, 111 In and 125 I. Chemiluminescent agents and enzymes.

[0098] Epitope: A type of determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (can elicit a specific immune response). Antibodies specifically bind to specific epitopes on polypeptides such as mesothelin.

[0099] Framework region: amino acid sequence inserted between CDRs. The framework region of an immunoglobulin molecule includes a light chain variable framework region and a heavy chain variable framework region.

[0100] Fusion protein: A protein that includes at least portions of two different (heterologous) proteins.

[0101] Heterologous: Originating from a different genetic source or species.

[0102] Immune response: The response of an immune system cell, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the response is a B cell response, which can result in the production of specific antibodies.

[0103] Immunoconjugate: A covalent link between an effector molecule and an antibody or a functional fragment thereof. The effector molecule can be, for example, a detectable marker, a photon absorber (such as IR700) or a toxin (to form an immunotoxin, such as an immunotoxin including a 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 its modified toxins, or other toxins that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that usually cause death through hepatotoxicity. However, PE and DT can be modified to be used as immunotoxins by removing the natural targeting components of the toxin (such as domain Ia of PE and chain B of DT) and replacing them with different targeting moieties such as antibodies. In one embodiment, the antibody is linked to the effector molecule. In another embodiment, the antibody linked to the effector molecule is further linked to a lipid or other molecule to increase its half-life in vivo. The connection can be chemically or recombinantly performed. In one embodiment, the connection is chemically carried out, wherein the reaction between the antibody portion and the effector molecule produces a divalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Because immunoconjugates are initially prepared from two molecules with different functionalities, such as antibodies and effector molecules, they are sometimes also referred to as "chimeric molecules". The term "chimeric molecule" as used herein therefore refers to a targeting portion, such as a ligand or an antibody, conjugated (coupled) to an effector molecule. The term "conjugated" or "connected" refers to making two polypeptides into a continuous polypeptide molecule.

[0104] Immunoliposome: A liposome with an antibody or antibody fragment bound to its surface. Immunoliposomes can deliver cytotoxic agents or other drugs to cells targeted by the antibody, such as tumor cells.

[0105] Interstrand cross-linker: A cytotoxic drug that covalently binds between the two strands of DNA, thereby preventing DNA replication and / or transcription.

[0106] Isolated: An "isolated" biological component such as a nucleic acid, protein (including antibodies) or organelle has been substantially separated or purified from other biological components of the environment (e.g., a cell) in which it is naturally present, such as other chromosomal and extrachromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell and chemically synthesized nucleic acids and proteins.

[0107] Label: A detectable compound or composition that can be conjugated directly or indirectly to another molecule, such as an antibody or protein, to facilitate detection of the molecule. Specific non-limiting examples of labels include fluorescent tags, enzymatic linkers, and radioactive isotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into an antibody. For example, a label is a detectable marker, such as the incorporation of a radiolabeled nucleic acid or the binding of a polypeptide with a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or having enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (such as 35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H. 14 C. 15 N. 90 Y. 99 Tc, 111 In and 125 I), fluorescent markers (such as fluorescent isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic markers (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by second reporters (leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, the labels are connected by spacer arms of various lengths to reduce possible steric hindrance.

[0108] Linker: In some cases, a linker is a peptide within a peptide binding fragment (such as an Fv fragment) that is used to indirectly bind the variable heavy chain to the variable light chain. A "linker" may also refer to a peptide used to connect a targeting moiety such as an antibody to an effector molecule such as a cytotoxin or a detectable marker. The terms "bind," "connect," "join," or "link" refer to making two polypeptides into a continuous polypeptide molecule or covalently attaching a radionuclide or other molecule to a polypeptide such as an antibody. The connection can be chemical or recombinant. "Chemical" refers to a reaction between the antibody portion and the effector molecule such that a covalent bond exists between the two molecules to form one molecule.

[0109] Lung cancer: Cancer that forms in the lung tissue, typically in the cells lining the airways. The two main types are small cell lung cancer and non-small cell lung cancer (NSCLC). These types can be diagnosed using microscopic examination.

[0110] Mesothelin: A 40 kDa cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. Human mesothelin protein is synthesized as a 70 kD precursor that is then proteolytically processed. The 30 kD amino terminus of mesothelin is secreted and is called megakaryocyte enhancing factor (Yamaguchi et al., J. Biol. Chem. 269: 805 808, 1994). The 40 kD carboxyl terminus is membrane-bound as mature mesothelin (Chang et al., Natl. Acad. Sci. USA 93: 136 140, 1996). Exemplary nucleic acid and amino acid sequences of mesothelin are described in PCT Publication WO 97 / 25,068; U.S. Pat. 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 Howdhury et al., Mol. Immunol. 34:9, 1997. An exemplary amino acid sequence of human mesothelin is set forth herein as SEQ ID NO:12 (see also GenBank Accession No. AAH09272). Mesothelin also refers to a mesothelin protein or polypeptide retained within a cell as well as secreted and / or isolated extracellular mesothelin protein.

[0111] Mesothelioma: A neoplasm derived from the cells lining the pleura and peritoneum that grows as thick sheets covering the internal organs. The lining consists of spindle cells or fibrous tissue that surround glandular spaces lined with dice-shaped cells. Mesothelioma usually originates from the lining tissue of the lungs, heart, or abdomen. In some cases, mesothelioma is caused by exposure to asbestos.

[0112] 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, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (such as triple-negative breast cancer), and ovarian cancer.

[0113] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, 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 continuous and, if necessary, connect two protein coding regions in the same reading frame.

[0114] Ovarian cancer: Cancer that develops in the tissue of the ovaries (one of the female reproductive glands where eggs are formed). Most ovarian cancers are either epithelial (cancer that begins in the cells on the surface of the ovaries) or malignant (cancer that begins in the egg cells).

[0115] Pancreatic cancer: A disease in which malignant (cancer) cells are found in the tissues of the pancreas. Also called exocrine cancer.

[0116] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is conventional. Compositions and preparations suitable for drug delivery of antibodies and other compositions disclosed herein are described in Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975. Generally, the nature of the carrier will depend on the specific mode of administration being adopted, for example, parenteral preparations generally include injectable liquids, which include pharmaceutically and physiologically acceptable liquids as carriers, such as water, saline, balanced salt solutions, water-soluble glycerol, etc. For solid compositions (such as powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0117] Photoimmunotherapy: A targeted cancer therapy that utilizes antigen-specific antibody-photoabsorber conjugates that can be activated by near-infrared light to kill targeted cells. The photon absorbers are typically based on phthalocyanine dyes, such as near-infrared (NIR) phthalocyanine dyes (e.g., 700DX, also known as IR700). The antibody (e.g., a mesothelin-specific antibody) binds to an appropriate cell surface antigen (e.g., mesothelin), and the photoactivated dye can cause lethal damage to the cell membrane after near-infrared light irradiation. Near-infrared light irradiation (690nm) can cause highly selective necrotic cancer cell death within a few minutes without damaging adjacent cells (e.g., see U.S. Application No. 2018 / 0236076). Therefore, the disclosed antibodies (e.g., A101 and G8 or fragments thereof) conjugated to IR700 are provided herein.

[0118] Prevent, treat, or alleviate disease: "Preventing" a disease means inhibiting the overall development of the disease. "Treatment" refers to therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing tumor burden or reducing the size of metastases. "Amelioration" refers to a decrease in the number or severity of signs or symptoms of a disease (e.g., cancer).

[0119] Prostate cancer: Cancer that develops in the tissues of the prostate (a gland in the male reproductive system located below the bladder and in front of the rectum). Prostate cancer usually occurs in older men.

[0120] 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 in its natural environment within a cell. In one embodiment, the preparation is purified so that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation. Substantially purified means purification from other proteins or cellular components. A substantially purified protein has a purity of at least 60%, 70%, 80%, 90%, 95% or 98%. Thus, in a specific non-limiting example, a substantially purified protein such as an antibody or antibody fragment is 90% free of other proteins or cellular components.

[0121] Pyrrolobenzodiazepines (PBDs): A class of sequence-selective DNA minor groove binding cross-linkers originally discovered in Streptomyces species. PDBs are significantly more effective than systemic chemotherapeutic drugs. The mechanism of action of PBDs is related to their ability to form adducts in the minor groove of DNA, thereby interfering with DNA processing. In the context of the present disclosure, PBDs include naturally occurring and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric, dimeric, and mixed PBDs (for review, see Gerratana, Med Res Rev 32(2):254-293, 2012).

[0122] Recombinant: A recombinant nucleic acid or protein is one that has a sequence that does not occur naturally or that is an artificial combination of two otherwise separate sequence fragments. This artificial combination is usually accomplished by chemical synthesis or by artificial manipulation of separate nucleic acid fragments, such as through genetic engineering techniques.

[0123] Sample (or biospecimen): A biological specimen obtained from a subject that contains genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsies, fine needle aspirates, surgical specimens, and autopsy material. In one example, the sample comprises a tumor biopsy. In one example, the sample comprises a fine needle aspirate.

[0124] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed as inter-sequence similarity, also known as sequence identity. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of polypeptides or nucleic acid molecules will have relatively high sequence identity when aligned using standard methods.

[0125] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described by Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Res. 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Akkad. Science. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, provide a detailed introduction to sequence alignment methods and homology calculations.

[0126] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from a number of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are available from the NCBI website on the Internet.

[0127] V of an antibody that specifically binds to mesothelin polypeptide HHomologues and variants of domains are generally characterized by having at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, counted using NCBI Blast 2.0 in the full-length comparison with the antibody amino acid sequence, with gap blastp set to default parameters. For comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function is adopted by using the default BLOSUM62 matrix (gap existence cost is 11, each residue gap cost is 1) set to default parameters. When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for alignment, using the PAM30 matrix to be set to default parameters (open gap 9, extended gap 1 penalty). When evaluated by this method, proteins with higher similarity to the reference sequence will show an increase in percentage identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities of less than the entire sequence, homologues and variants generally have at least 80% sequence identity within a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity through such short windows can be found on the NCBI website on the Internet. Those skilled in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain very important homologues beyond the ranges provided.

[0128] Small molecule: A molecule generally having a molecular weight of less than about 1000 Daltons, or in some embodiments less than about 500 Daltons, wherein the molecule is capable of modulating the activity of a target molecule to some measurable degree.

[0129] Squamous cell carcinoma: A malignant tumor that arises from stratified squamous epithelium, but may also occur in sites such as the bronchial mucosa where glandular or columnar epithelium normally exists. Squamous cell carcinoma is the most common type of skin cancer.

[0130] Gastric cancer: Cancer that forms in the tissues lining the stomach. Also called gastrointestinal cancer.

[0131] Subject: Living multicellular vertebrate organisms, this category includes human and veterinary subjects, including human and non-human mammals.

[0132] Synthetic: Produced by artificial means in a laboratory, e.g., synthetic nucleic acids or proteins (e.g., antibodies) can be chemically synthesized in a laboratory.

[0133] Therapeutically effective amount: The amount of a particular substance sufficient to achieve the desired effect in a subject being treated. For example, this can be the amount required to inhibit or suppress tumor growth. In one embodiment, a therapeutically effective amount is the amount required to eliminate, reduce tumor size, or prevent tumor metastasis, such as reducing 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 reducing 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% of the amount required, such as compared to the size / volume / number before treatment. When administered to a subject, a dose that has been shown to achieve the desired in vitro effect in target tissue concentrations (e.g., in tumors) is generally used.

[0134] Toxin: A molecule that is cytotoxic to cells. Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, serotonin or gelonin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that often cause death through hepatotoxicity. However, PE and DT can be modified to a form useful as an immunotoxin by removing the natural targeting component of the toxin (e.g., domain Ia of PE or chain B of DT) and replacing it with a different targeting moiety, for example as an antibody.

[0135] Vector: A nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector or an AAV vector.

[0136] III. Monoclonal antibodies specific for mesothelin

[0137] Described herein are two cross-species mesothelin-specific camel single-domain monoclonal antibodies separated by phage display. The disclosed mesothelin-specific antibodies A101 and G8 specifically bind to human and mouse mesothelin with high affinity. Chimeric antigen receptor (CAR) T cells composed of the disclosed antibodies can effectively kill mesothelin-positive tumor cells in vitro and in vivo. The nucleotide and amino acid sequences of A101 and G8 are provided below. Tables 1 and 2 list the amino acid positions of CDR1, CDR2, and CDR3 of each antibody, as determined using Kabat, IMGT, or Paratome, or a combination of all three. Those skilled in the art can easily determine CDR boundaries using alternative numbering schemes such as the Chothia numbering scheme.

[0138] A101 DNA (SEQ ID NO: 1)

[0139] CAGGTGCAGCTGGTGGAGTCTGGGGGCGGCACGGTGCAGGCTGGAGGGTCGCTGAAACTCGCCTGCGCAGCCTCTGGATTACCCAGAACGTACAATGTCATGGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGCGAGGGGGTCGCAATAATTTATACTACGACTGGAGCAACATACTATCGCGACTCCGTCAAGGGCCGGGCCACCATCTCCCAAGACAACGCCAAGAAGTCGGTGTCTCTCCAAATGAACAGCCTGAGGCCTGAGGACACGGCCATCTATTACTGTGTGGCTAGGCAACCCAATAGTGGTCCCTGGGAGTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0140] A101 Protein (SEQ ID NO: 2)

[0141]

[0142] (Underline = Kabat CDRs; Bold = IMGT CDRs; Italics = Paratome CDRs)

[0143] Table 1 Positions of CDRs in the A101 Amino Acid Sequence (SEQ ID NO: 2)

[0144] Numbering scheme CDR1 CDR2 CDR3 Kabat 31-35 50-66 99-108 IMGT 26-33 51-58 97-108 Paratome 27-35 47-61 97-108 combination 26-35 47-66 97-108

[0145] G8 DNA (SEQ ID NO: 3)

[0146] CAGGTAAAGCTGGAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAACTTCTGGATACACCAACAGTTACAAGTGGATGGGCTGGTTCCGCCAGGCTCCAGGACAAGAGCGCGAGGGGGTCGCAGTTATTTACACCGGTAATGATAGGACAT ACTATAGTGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAATATGATCTATCTGGACATGACGCGCCTGAGACCTGAGGACAGCGCTGTGTACGAGTGTGCCATCGGACATGATGGCGCATGGCGTTACTGGGGCCAGGGAACGCAGGTCACCGTCTCCTCA

[0147] G8 protein (SEQ ID NO: 4)

[0148]

[0149] (Underline = Kabat CDRs; Bold = IMGT CDRs; Italics = Paratome CDRs)

[0150] Table 2 Position of CDR in G8 amino acid sequence (SEQ ID NO: 4)

[0151] Numbering scheme CDR1 CDR2 CDR3 Kabat 31-35 50-66 99-106 IMGT 26-33 51-58 97-106 Paratome 27-35 47-60 98-106 combination 26-35 47-66 97-106

[0152] Provided herein are single domain (VHH) monoclonal antibodies that bind (e.g., specifically bind) to mesothelin (e.g., cell surface or soluble mesothelin). In some embodiments, the mesothelin is human mesothelin, mouse mesothelin, or both human and mouse mesothelin. In some embodiments, the antibody comprises at least a portion of an amino acid sequence as described herein as SEQ ID NO: 2 or SEQ ID NO: 4, e.g., one or more (e.g., all three) CDR sequences from SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 4 being determined by any numbering scheme such as IMGT, Kabat, Paratome, or Chothia.

[0153] In some embodiments, the monoclonal antibody comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 2. In some examples, the CDR sequences are determined using the IMGT, Kabat, Paratome, or Chothia numbering schemes, or a combination thereof. In a specific example, the CDR sequences are determined using a combination of Kabat, IMGT, and Paratome.

[0154] In some embodiments, the monoclonal antibody comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4. In some examples, the CDR sequences are determined using the IMGT, Kabat, Paratome, or Chothia numbering schemes, or a combination thereof. In specific examples, the CDR sequences are determined using a combination of Kabat, IMGT, and Paratome.

[0155] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the monoclonal antibody include residues 31-35, 50-66, and 99-108 of SEQ ID NO: 2; residues 26-33, 51-58, and 97-108 of SEQ ID NO: 2; residues 27-35, 47-61, and 97-108 of SEQ ID NO: 2; or residues 26-35, 47-66, and 97-108 of SEQ ID NO: 2. In some examples, the amino acid sequence of the monoclonal antibody 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. In specific, non-limiting examples, the sequence of the monoclonal antibody includes or consists of SEQ ID NO: 2.

[0156] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the monoclonal antibody include residues 31-35, 50-66, and 99-106 of SEQ ID NO: 4; residues 26-33, 51-58, and 97-106 of SEQ ID NO: 4; residues 27-35, 47-60, and 98-106 of SEQ ID NO: 4; or residues 26-35, 47-66, and 97-106 of SEQ ID NO: 4. In some examples, the amino acid sequence of the monoclonal antibody 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. In specific, non-limiting examples, the sequence of the monoclonal antibody includes or consists of SEQ ID NO: 4.

[0157] In some embodiments, the monoclonal antibody is a camelid antibody. In some embodiments, the monoclonal antibody is a humanized antibody. In some embodiments, the monoclonal antibody is a chimeric antibody.

[0158] Also provided herein is a chimeric antigen receptor (CAR) including a monoclonal antibody disclosed herein. In some embodiments, CAR further includes a hinge region, a transmembrane domain, a costimulatory signal portion, a signal domain, or any combination thereof. In a specific non-limiting example, the hinge region includes a CD8α hinge region, the transmembrane domain includes a CD8α transmembrane domain, and the costimulatory signal portion includes a 4-1BB signal portion and / or a signal domain includes a CD3ζ signal domain.

[0159] Further provided are cells expressing mesothelin-specific CARs. In some examples, the cells are T lymphocytes, such as CTLs. CARs and T cells expressing CARs are further described in Section IV.

[0160] Also provided herein are immunoconjugates comprising the monoclonal antibodies disclosed herein and effector molecules. In some embodiments, the effector molecule is a toxin, such as, but not limited to, a Pseudomonas exotoxin or a variant thereof, such as PE38. In other embodiments, the effector molecule is a detectable marker, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope. In other embodiments, the effector molecule is a photon absorber, such as IR700. Immunoconjugates comprising photon absorbers can be used for photoimmunotherapy. Immunoconjugates are further described in Section V.

[0161] Further provided herein are antibody-drug conjugates (ADCs) comprising drugs conjugated to the monoclonal antibodies disclosed herein. In some embodiments, the drug is a small molecule, such as an anti-microtubule agent, an anti-mitotic agent, and / or a cytotoxic agent. ADCs will be further described in Section VI.

[0162] Also provided herein are multispecific antibodies, which include a monoclonal antibody disclosed herein and at least one additional monoclonal antibody or its antigen-binding fragment. In some embodiments, the multispecific antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody. In some embodiments, at least one additional monoclonal antibody or its antigen-binding fragment specifically binds to a component of a T cell receptor or a natural killer (NK) cell activation receptor. Multispecific antibodies are further described in Section VII.

[0163] Further provided herein are antibody-nanoparticle conjugates comprising nanoparticles conjugated to monoclonal antibodies disclosed herein. In some embodiments, the nanoparticles comprise polymer nanoparticles, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, or vesicles. In some embodiments, the nanoparticles comprise a cytotoxic agent. Section VIII further describes antibody-nanoparticle conjugates.

[0164] Also provided herein is a fusion protein comprising a monoclonal antibody disclosed herein and a heterologous protein or peptide. In some embodiments, the heterologous protein is an Fc protein. In some examples, the Fc protein is a mouse Fc or human Fc protein.

[0165] Also provided are nucleic acid molecules encoding monoclonal antibodies disclosed herein. In some embodiments, the nucleic acid molecule has 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% identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some examples, the nucleic acid molecule includes SEQ ID NO: 1 or SEQ ID NO: 3 or a degenerate variant thereof, or consists of SEQ ID NO: 1 or SEQ ID NO: 3 or a degenerate variant thereof. Further provided are nucleic acid molecules encoding CAR, immune conjugates, multispecific antibodies, or fusion proteins disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter. Further provided herein are vectors comprising nucleic acid molecules.

[0166] Further provided herein is a nucleic acid construct expressing CAR and truncated human EGFR (huEGFRt). In some embodiments, the nucleic acid includes in the 5' to 3' direction: nucleic acid encoding the first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); nucleic acid encoding the mesothelin-specific monoclonal antibody disclosed herein; nucleic acid encoding the extracellular hinge region; nucleic acid encoding the transmembrane domain; nucleic acid encoding the intracellular costimulatory domain; nucleic acid encoding the intracellular signal domain; nucleic acid encoding the self-cleaved 2A peptide; nucleic acid encoding the second GMCSFRss; and nucleic acid encoding the truncated human epidermal growth factor receptor (huEGFRt). In some examples, the nucleic acid further includes a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' of the nucleic acid encoding the first GMCSFRss. In some examples, the hinge region includes the CD8 alpha hinge region. In some examples, the transmembrane domain includes the CD8 alpha transmembrane domain. In some examples, the costimulatory signal portion includes the 4-1BB signal portion. In some examples, the signal domain includes the CD3 ζ signal domain. In some examples, the amino acid sequence of the mesothelin-specific monoclonal antibody comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 4. Also provided are vectors comprising the nucleic acid constructs. In some embodiments, the vector is a lentiviral vector.

[0167] Also provided are isolated cells that co-express a mesothelin-specific CAR disclosed herein and huEGFRt. In some examples, the cell is a cytotoxic T lymphocyte (CTL).

[0168] Comprising a pharmaceutically acceptable carrier and a monoclonal antibody CAR, a separated cell (such as a cell expressing CAR, such as a CAR T cell or a CAR NK cell), an immunoconjugate, an ADC, a multispecific antibody, a composition of an antibody-nanoparticle conjugate, the present invention further provides a fusion protein disclosed herein. Compositions and uses thereof are further described in Section IX.

[0169] IV. Chimeric Antigen Receptor (CAR)

[0170] The disclosed monoclonal antibodies can also be used to produce CAR (also known as chimeric T cell receptors, artificial T cell receptors or chimeric immune receptors) and / or engineered to express CAR cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. Generally, CAR includes a binding portion, an extracellular hinge and a spacer element, a transmembrane region, and an inner domain that performs a signal transduction function (Cartellieri et al., J Biomed Biotechnol 2010: 956304, 2010; Dai et al., J Natl Cancer Inst 108 (7): djv439, 2016). In many cases, the binding portion is an antigen binding fragment of a monoclonal antibody, such as an antigen binding fragment of a scFv or a single domain antibody. The spacer / hinge region typically includes sequences from IgG subclasses, such as IgG1, IgG4, IgD, and CD8 domains. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8, or CD28. Several different endodomains have been used to generate CARs. For example, the endodomain can be composed of a signal chain with an ITAM, such as CD3ζ or FcεRIγ. In some cases, the endodomain also includes an intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, and / or DAP10.

[0171] CTLs, NK cells (or other immune cells) expressing CAR can be used to target specific cell types, such as mesothelin-positive tumor cells. Therefore, the monoclonal antibodies disclosed herein can be used to engineer CTLs or NK cells expressing CARs including mesothelin-specific monoclonal antibodies, thereby targeting engineered CTLs or NK cells to tumor cells expressing mesothelin. Engineered T cells have previously been used for adoptive therapy of certain types of cancer (see, e.g., Park et al., Mol Ther 15 (4): 825-833, 2007). Using T cells expressing CAR is more common than standard CTL-based immunotherapy, because CTLs expressing CAR are not restricted by HLA and can therefore be used for any patient with a tumor expressing a target antigen.

[0172] The present invention also contemplates bispecific or bicistronic CARs. In some embodiments, bispecific CARs include single domain antibodies specific for mesothelin (eg, A101 or G8) and single domain antibodies specific for different antigens. Similarly, bicistronic CARs include two CAR molecules expressed from the same construct, one of which is a mesothelin-targeted CAR and the second CAR targets a second antigen. See, e.g., Qin et al., Blood 130: 810, 2017; and WO / 2018 / 213337.

[0173] Therefore, CARs including mesothelin-specific antibodies are provided herein. Also provided are isolated nucleic acid molecules and vectors encoding CARs (including bispecific and bicistronic CARs), and host cells expressing CARs, bispecific CARs or bicistronic CARs, such as CTLs or NK cells. CTLs or NK cells expressing CARs composed of mesothelin-specific monoclonal antibodies can be used to treat cancers expressing mesothelin. In some embodiments herein, CAR is a bispecific CAR. In other embodiments herein, CAR is a bicistronic CAR.

[0174] In some embodiments, CAR includes a signal peptide sequence, for example, the N-terminus of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from a granulocyte-macrophage colony stimulating factor receptor (GMCSFR), an immunoglobulin light chain κ, or IL-2. Although the signal peptide sequence can promote the expression of CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not necessary for CAR to function. After CAR is expressed on the cell surface, the signal peptide sequence can be cut from CAR. Therefore, in some embodiments, CAR lacks a signal peptide sequence.

[0175] In some embodiments, the CAR disclosed herein is expressed from a construct (e.g., from a lentiviral vector) that also expresses a truncated human EGFR (huEGFRt). CAR and huEGFRt are separated by a self-cleaving peptide sequence (e.g., T2A) so that after expression in transduced cells, CAR is cleaved from huEGFRt.

[0176] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0177] GMCSFRss: MLLLVTSLLCELPHPAFLLIP (SEQ ID NO: 5)

[0178] NdeI:HM

[0179] Antigen binding: Mesothelin-specific antibody (such as SEQ ID NO: 2 or SEQ ID NO: 4)

[0180] SpeI:TS

[0181] CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 6)

[0182] CD8αTM:IYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:7)

[0183] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:8)

[0184] CD3ζ:

[0185] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:9)

[0186] T2A:EGRGSLLTCGDVEENPGP(SEQ ID NO:10)

[0187] GMCSFRss:MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:5)

[0188] huEGFRt:

[0189] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:11)

[0190] Human epidermal growth factor receptor is composed of four extracellular domains, one transmembrane domain and three intracellular domains. EGFR domains are found in the following order from N-terminal to C-terminal: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-near membrane domain-tyrosine kinase domain-C-terminal tail. Domain I and domain III are leucine-rich domains that participate in ligand binding. Domain II and domain IV are cysteine-rich domains that do not contact EGFR ligands. Domain II mediates the formation of homodimers or heterodimers together with similar domains from other EGFR family members, and domain IV can form disulfide bonds with domain II. EGFR TM domains pass through the cell membrane once and can play a role in protein dimerization. The intracellular domain includes the juxtamembrane domain, the tyrosine kinase domain, and the C-terminal tail, which mediate EGFR signal transduction (Wee and Wang, Cancers 9(52), doi:10.3390 / cancers9050052; Ferguson, Annu Rev Biophys 37:353-373, 2008; Wang et al., Blood 118(5):1255-1263, 2011).

[0191] A truncated form of human EGFR, referred to herein as "huEGFRt", includes only domain III, domain IV, and TM domains. Thus, huEGFRt lacks domain I, domain II, and all three intracellular domains. huEGFRt is unable to bind to EGF and lacks signaling activity. However, the molecule retains the ability to bind to specific EGFR-specific monoclonal antibodies such as FDA-approved cetuximab (PCT Publication No. WO2011 / 056894, which is incorporated herein by reference).

[0192] Transduction of T cells (or NK cells) with a construct (e.g., a lentiviral vector) encoding huEGFRt and a tumor antigen-specific CAR disclosed herein allows for the use of a labeled EGFR monoclonal antibody cetuximab (ERBITUX TM ) to select transduced T cells. For example, cetuximab can be labeled with biotin, and commercially available (e.g., from Miltenyi Biotec) anti-biotin magnetic beads can be used to select transduced T cells. Co-expression of huEGFRt also allows in vivo tracking of adoptively transferred CAR-expressing T cells (or NK cells). In addition, the binding of cetuximab to huEGFRt-expressing T cells induces cytotoxicity of ADCC effector cells, thereby providing a mechanism for eliminating transduced T cells in vivo (Wang et al., Blood 118 (5): 1255-1263, 2011), for example at the conclusion of treatment.

[0193] V. Immunoconjugates

[0194] The disclosed monoclonal antibodies may be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which the therapeutic agent is covalently linked to the antibody. Therapeutic agents are agents that have a specific biological activity against a specific target molecule or against cells bearing the target molecule. Those skilled in the art will appreciate that therapeutic agents may include various drugs such as vinblastine, daunomycin, etc., cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents containing pharmacological components (such as liposomes), radioactive agents such as 125 I. 32 P. 14 C. 3 H and 35 S, photon absorbers such as IR700, and other labels, targeting moieties and ligands.

[0195] The choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, a therapeutic agent may be a cytotoxin for causing death of a particular target cell (e.g., a tumor cell). Conversely, where it is desired to induce a non-lethal biological response (e.g., for detection), the therapeutic agent may be conjugated to a non-lethal agent or a liposome containing a non-lethal agent.

[0196] Using the therapeutic agents and antibodies described herein, a technician can easily construct multiple clones containing functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same effector portion or antibody sequence. Therefore, the present invention provides nucleic acids encoding antibodies and their conjugates and fusion proteins.

[0197] Effector molecules can be linked to the antibody of interest using a variety of methods. Both covalent and non-covalent linkages can be used. The procedure for linking the effector molecule to the antibody varies depending on the chemical structure of the effector molecule. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can react with suitable functional groups on the antibody to cause the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or connect additional reactive functional groups. Derivatization can involve the connection of any of many known linker molecules. The linker can be any molecule used to connect the antibody to the effector molecule. The linker can form a covalent bond with the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. If the antibody and effector molecule are polypeptides, the linker can be linked to the amino acids through their side groups (such as through disulfide bonds with cysteine) or to the α-carbon amino and carboxyl groups of the terminal amino acids.

[0198] In some cases, when the immunoconjugate reaches its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a cleavable linker near the target site. Cleavage of the linker to release the effector molecule from the antibody may be promoted by enzymatic activity or by conditions to which the immunoconjugate is subjected within the target cell or near the target site.

[0199] In view of the multitude of methods reported for linking various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for linking a given agent to an antibody or other polypeptide.

[0200] Antibodies disclosed herein can be derivatized or connected to another molecule (e.g., another peptide or protein). Generally, antibodies or portions thereof are derivatized so that binding to a target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally connected (by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities such as another antibody (e.g., a bispecific antibody or a double antibody), a detection agent, a photon absorber, a pharmaceutical agent, and / or a protein or peptide that can mediate the binding of an antibody or antibody portion to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0201] 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 produce bispecific antibodies). Suitable cross-linking agents include those that are heterobifunctional, having two different 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.

[0202] The antibody can be conjugated to a detectable marker; for example, a detectable marker that can be detected 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 inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used. The antibody or antigen binding fragment can also be conjugated to an enzyme for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme can use to produce a distinguishable reaction product. For example, when the horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine will produce a colored reaction product that can be detected visually. The antibody or antigen binding fragment can also be bound to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be bound to an enzyme or a fluorescent marker.

[0203] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese. Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). In some embodiments, the labels are connected by spacer arms of varying lengths to reduce possible steric hindrance.

[0204] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect the expression of the target antigen by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I.131 I.

[0205] The antibodies disclosed herein may also be conjugated to a photon absorber. In some embodiments, the photon absorber is a phthalocyanine dye, such as but not limited to 700DX (also known as "IR700"). Antibody-photoabsorber conjugates can be used in photoimmunotherapy.

[0206] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, for example to increase serum half-life or to increase tissue binding.

[0207] Toxins can be used with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and its subunits, and botulinum toxins A to F. These toxins can be easily obtained from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of toxins described herein (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is a Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length native (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, elimination of various amino acid deletions, single amino acid substitutions, and addition of one or more sequences at the carboxyl terminus in domains Ia, Ib, II, and III (e.g., see Siegall et al., J. Biol. Chem. 264: 14256-14261, 1989).

[0208] PE used with the monoclonal antibodies described herein may include native sequences, cytotoxic fragments of native sequences, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those that are cytotoxic in the presence or absence of subsequent proteolysis or other treatments in target cells. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional descriptions of PE and its variants, see, e.g., U.S. Patents 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication 2015 / 0099707; PCT Publications WO 99 / 51643 and WO 2014 / 052064; Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0209] 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, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCT Publications WO2007 / 016150, WO2009 / 032954, and WO2011 / 032022, incorporated herein by reference).

[0210] In some examples, 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 WO2009 / 032954). In other examples, PE is a variant designated PE-LR / 6X (PCT Publication WO 2011 / 032022). In other examples, the PE variant is a PE with reduced immunogenicity. In other examples, PE is a variant designated PE-LR / 8M (PCT Publication WO 2011 / 032022).

[0211] Modification of PE can occur in any variant of the cytotoxic fragments of PE described previously (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE may include any substitution, such as one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or the deletion of one or more T cell and / or B-cell epitopes. (See, e.g., U.S. Patent Application Publication 2015 / 0099707).

[0212] Expected PE forms also include PE with deimmunization forms, such as various versions lacking domain II (e.g., PE24). For example, PE with deimmunization forms is described in PCT Publications WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2007 / 016150, WO 2009 / 32904, WO 21205 / 3205, and WO 2012 / 170617.

[0213] Antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds on cells expressing tumors or viral antigens on their surfaces. Therefore, the antibodies of the present invention can be directly or through a joint connected to a drug that is pre-directly delivered to a cell expressing a cell surface antigen. This can be used for treatment, diagnosis or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioactive isotopes, photon absorbers, lipids, carbohydrates or recombinant viruses. Nucleic acid therapy and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides and triple-stranded oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA.

[0214] Alternatively, the molecule linked to the antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle, which contains a therapeutic composition, such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety preferably avoiding direct exposure to the circulatory system. Methods for preparing liposomes linked to antibodies are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,957,735; Connor et al., Pharm. Ther. 28: 341-365, 1985).

[0215] The antibodies described herein may also be covalently or non-covalently linked to a detectable marker. Detectable markers suitable for such uses include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful markers include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive markers (e.g., 3 H. 125I. 35 S. 14 C or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0216] Methods for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, and colorimetric labels are detected by simply observing the colored label.

[0217] VI. Antibody-drug conjugates (ADCs)

[0218] ADC is a compound composed of a tumor antigen-specific antibody (or its antigen-binding fragment) and a drug (usually a cytotoxic agent, such as an anti-microtubule agent or a cross-linking agent). Because ADCs are able to specifically target cancer cells, the drug is more effective than drugs used in standard chemotherapy. The IC50 of the most common cytotoxic drugs currently used with ADCs is 100 to 1000 times more potent than conventional chemotherapy drugs. Common cytotoxic drugs include anti-microtubule drugs such as maytansines and auristatins (such as auristatin E and auristatin F). Other cytotoxins used with ADCs include pyrrolobenzodiazepines (PDBs), which covalently bind to the minor groove of DNA to form interchain crosslinks. In many cases, the antibody to drug ratio in ADCs is 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10 (8; suppl 10): 3-7, 2012).

[0219] Antibodies and drugs can be connected by cleavable or non-cleavable joints. However, in some cases, it is necessary to have a joint that is stable in the circulation to prevent the systemic release of cytotoxic drugs that may cause significant off-target toxicity. Non-cleavable joints can prevent the release of cytotoxic agents before the ADC is internalized by target cells. Once entering the lysosome, the digestion of the antibody by lysosomal proteases leads to the release of cytotoxic agents (Bander, Clinical Advances in Hematology & Oncology 10 (8; Supplement 10): 3-7, 2012).

[0220] One method of site-specific and stable attachment of drugs to monoclonal antibodies is through glycan engineering. Monoclonal antibodies have a 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). By using a mutant β1,4-galactosyltransferase (Y289L-Gal-T1; U.S. Patent Application Publications 2007 / 0258986 and 2006 / 0084162, incorporated herein by reference), 2-keto-galactose is transferred to the free GlcNAc residue on the antibody heavy chain to provide a chemical treatment for attachment.

[0221] The oligosaccharide chains attached to monoclonal antibodies can be divided into three groups based on the terminal galactose residues—fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-G1), or completely degalactosylated (IgG-G0). Treatment of monoclonal antibodies with β1,4-galactosidase converts the antibodies to the IgG-G0 glycoform. Mutant β1,4-galactosyltransferase is able to transfer 2-ketogalactose or 2-azidogalactose from their respective UDP derivatives to the GlcNAc residues on the IgG-G1 and IgG-G0 glycoforms. Chemical manipulations on the transfer sugars enable a variety of molecules to be attached to monoclonal antibodies via the glycan residues (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0222] Provided herein is an ADC comprising a drug (e.g., a cytotoxic agent) that is bound to (e.g., specifically bound to) a monoclonal antibody that binds to mesothelin. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent, and / or an anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PDB, auristatin, maytansinoids, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, compositions, dolastatin, biscarboxamicin, enediynes, geldanamycin, indolebenzodiazepine dimers, puromycin, tubulysin, hemiasterlin, splice inhibitors or pladienolide, and stereoisomers, isosteres, analogs, and derivatives thereof that are cytotoxic.

[0223] In some embodiments, the ADC comprises 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, many BDs have been reported, including naturally occurring and synthetic analogs (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patents 6,884,799; 7,049,311; 7,067,526, 57,153; 7153; 7,528,126; and 7,557,099). As an example, PDB dimers recognize and bind to specific DNA sequences and have been shown to be useful as cytotoxic agents. PBD dimers have been linked to antibodies, and the resulting ADCs have been shown to have anticancer properties (see, e.g., US 2010 / 0203007). Exemplary attachment sites on PBD dimers include five-membered pyrrole rings, tethers between PBD units, and N10-C11 imine groups (see WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257 / US 36439; US 2011 / 0256157; and WO 2011 / 130598).

[0224] In some embodiments, ADC includes an antibody connected to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, which are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent 3,896,111). Subsequently, it was found that some microorganisms also produced maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patents 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.

[0225] In some embodiments, the ADC comprises an antibody linked to Aplysia or auristatin or an analog or derivative thereof (see U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatin is a derivative of the marine mollusk compound Aplysia-10. Aplysia and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer effects (U.S. Pat. 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-dolesorlysine-dolasalanine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolesorlysine-dolasalanine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Publication No. 2013 / 0129753).

[0226] In some embodiments, the ADC includes an antibody linked to one or more calicheamicin molecules. Antibiotics of the calicheamicin family and their analogs are capable of producing 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 calicheamicin drug moieties are described in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0227] In some embodiments, the ADC includes an anthracycline. Anthracyclines are antibiotic compounds with cytotoxic activity. It is believed that anthracyclines can operate to kill cells through a variety of different mechanisms, including inserting drug molecules into the DNA of cells, thereby inhibiting the synthesis of DNA-dependent nucleic acids; inducing the generation of free radicals, which then react with cellular macromolecules to cause damage to cells; and / or causing the interaction of drug molecules with cell membranes. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunorubicin, daunorubicin, doxorubicin, epirubicin, nimomycin, prorubicin, mitoxantrone and derivatives thereof. For example, PNU-159682 is an effective metabolite (or derivative) of nimorubicin (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 glycosylamino group of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0228] In some embodiments, the ADC may further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to connect one or more drug moieties to an antibody to form an ADC. In some embodiments, the ADC is prepared by using a linker having a reactive functional group for covalently linking the drug and 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 prepare an ADC.

[0229] In some examples, the linker has a functional group that can react with the free cysteine ​​present on the antibody to form a covalent bond. Exemplary linkers with such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, etc. activated esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0230] In some examples, the joint has a functional group that can react with the electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehydes and ketone carbonyl groups. In some cases, the heteroatom of the reactive functional group of the joint can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples include hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, carboxylic acid hydrazides, and aryl hydrazides.

[0231] In some examples, the linker is a cleavable linker, which facilitates the release of the drug. Examples of cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52: 127-131, 1992; U.S. Pat. No. 5,208,020).

[0232] The ADCs disclosed herein can be used to treat mesothelin-positive cancers alone or in combination with another therapeutic agent or with any standard therapy for cancer treatment, such as surgical resection of the tumor, chemotherapy, or radiation therapy.

[0233] VII. Multispecific Antibodies

[0234] Multispecific antibodies are recombinant proteins composed of two or more monoclonal antibodies (such as single domain antibodies) or antigen-binding fragments of two or more different monoclonal antibodies. For example, bispecific antibodies are composed of antigen-binding fragments of two different monoclonal antibodies (or two different single domain antibodies). Therefore, bispecific antibodies bind to two different antigens, and trispecific antibodies bind to three different antigens. Multispecific antibodies can be used for cancer immunotherapy by simultaneously targeting, for example, two CTLs (such as CTL receptor components, such as CD3) or effector natural killer (NK) cells and at least one tumor antigen. The mesothelin-specific monoclonal antibodies disclosed herein can be used to produce multispecific (such as bispecific or trispecific) antibodies targeting both mesothelin and CTL or targeting both mesothelin and NK cells, thereby providing a therapeutic means for treating cancers expressing mesothelin. In one example, the mesothelin-specific monoclonal antibodies disclosed herein are used to produce multispecific (such as bispecific or trispecific) antibodies targeting mesothelin and PD1, PDL1, EGFR or VEGF, thereby providing a therapeutic means for treating cancers expressing mesothelin.

[0235] Provided herein are multispecific (such as trispecific or bispecific) monoclonal antibodies including mesothelin-specific monoclonal antibodies. In some embodiments, the multispecific monoclonal antibodies further include monoclonal antibodies that specifically bind to components of T cell receptors such as CD3. In other embodiments, the multispecific monoclonal antibodies further include monoclonal antibodies that specifically bind to NK cell activation receptors such as CD16, Ly49 or CD94. Also provided are isolated nucleic acid molecules and vectors encoding multispecific antibodies, as well as host cells comprising nucleic acid molecules or vectors. Multispecific antibodies including mesothelin-specific antibodies can be used to treat cancers that express mesothelin. Therefore, provided herein are methods for treating subjects with cancer by selecting subjects with cancer that express mesothelin and administering to the subjects a therapeutically effective amount of multispecific antibodies targeting mesothelin.

[0236] VIII. Antibody-Nanoparticle Conjugates

[0237] The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic or other anticancer agents directly to tumor cells by binding the antibodies to mesothelin expressed on the surface of tumor cells. The use of nanoparticles reduces off-target side effects and can also improve the bioavailability of drugs and reduce the drug dose required to achieve a therapeutic effect. Nanoparticle formulations can be customized to suit the drug to be carried or encapsulated within the nanoparticles. For example, hydrophobic molecules can be incorporated into the core of the nanoparticle, while hydrophilic drugs can be carried in an aqueous core protected by a polymer or lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, vesicles, and polymer nanoparticles (Fay and Scott, Immunotherapy 3(3): 381-394, 2011).

[0238] Liposomes are one of the most common types of nanoparticles currently used for drug delivery. Antibodies conjugated to liposomes are often referred to as "immunoliposomes". The liposome component of immunoliposomes is usually a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, phospholipids are composed of a hydrophilic head group and two hydrophobic chains to be able to encapsulate hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly cleared from circulation by macrophages of the reticuloendothelial system (RES). In order to produce long-circulating liposomes, the composition, size and charge of the liposomes can be adjusted. The surface of the liposome can also be modified with glycolipids or sialic acid. For example, including polyethylene glycol (PEG) can significantly increase the circulation half-life. Liposomes for use as drug delivery agents, including for use in preparing immunoliposomes, have been described in the art (see, e.g., Paszko and Senge, Curr Med Chem 19(31):5239-5277, 2012; Immordino et al., Int J Nanomed 1(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655; 2010 / 00329981).

[0239] Niosomes are vesicles based on nonionic surfactants, and their structure is similar to liposomes. The membrane of the vesicle is composed only of nonionic surfactants, such as polyglycerol-alkyl ethers or N-palmitoyl glucosamine. The range of vesicles ranges from small unilamellar particles to large multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, low in toxicity, biodegradable and non-immunogenic, and improve the bioavailability of encapsulated drugs.

[0240] Dendrimers include a family of branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Typically, dendrimers consist of an initiator core surrounded by a layer of a selected polymer that is grafted onto the core to form a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamines) or poly(L-lysine). Dendrimers have been used in a variety of therapeutic and diagnostic applications, including for delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

[0241] Polymer micelles consist of aggregates of amphiphilic copolymers (composed of hydrophilic and hydrophobic monomer units) assembled into a hydrophobic core, surrounded by a hydrophilic polymer corona exposed to the aqueous environment. In many cases, the polymers used to prepare polymer micelles are heterobifunctional copolymers composed of hydrophilic blocks of PEG, polyvinylpyrrolidone, and hydrophobic poly(L-lactide) or poly(L-lysine) that form the core of the particle. Polymer micelles can be used to carry poorly soluble drugs. These nanoparticles have been used to encapsulate a variety of anticancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0242] Polymer nanoparticles include nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymers, while nanocapsules include an aqueous core. The formulation selected generally depends on the solubility of the therapeutic agent to be carried / encapsulated; poorly water-soluble drugs are more easily encapsulated in nanospheres, while water-soluble and unstable drugs, such as DNA and proteins, are more easily encapsulated in nanocapsules. Polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(esters), poly(alkyl cyanoacrylates), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0243] The antibody (or its fragment) can be conjugated to a suitable nanoparticle according to standard methods known in the art. For example, the conjugation can be covalent or non-covalent. In some embodiments where the nanoparticle is a liposome, the antibody is connected to a spatially stable long-circulating liposome via a PEG chain. The coupling of the antibody or antibody fragment to the liposome can also involve a thioester bond, such as by the reaction of a thiol and a maleimide group. Cross-linking agents can be used to generate sulfhydryl groups to connect the antibody to the nanoparticle (Paszko and Senge, Curr Med Chem 19 (31) 5239-5277, 2012).

[0244] IX. Compositions and Methods of Use

[0245] The composition provided includes one or more disclosed monoclonal antibodies that bind (e.g., specifically bind) to mesothelin in a carrier. Also provided are compositions including ADC, CAR (and CTL including CAR), multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates. The composition can be prepared in unit dosage form for administration to a subject. The amount and time of administration are determined by the treating clinician to achieve the desired result. Antibodies, ADC, CAR, CTL, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the antibody is formulated for parenteral administration, such as intravenous administration.

[0246] Compositions for administration may include solutions of antibodies, ADCs, CARs, CTLs, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates in pharmaceutically acceptable carriers such as aqueous carriers, and a variety of aqueous carriers, such as buffered saline, etc., may be used. These solutions are sterile and generally free of unwanted substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in these preparations may vary widely, and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the selected specific mode of administration and the needs of the subject.

[0247] Typical pharmaceutical compositions for intravenous administration include approximately 0.1 to 10 mg of antibody (or ADC, CAR, multispecific antibody, antibody-nanoparticle conjugate or immunoconjugate) per subject per day. A dosage of 0.1 to approximately 100 mg per subject per day can be used, particularly if the agent is applied to a hidden site rather than into the circulation or lymphatic system, such as into a body cavity or into the lumen of an organ. The actual method of preparing a administrable composition is known or apparent to those skilled in the art, and is described in more detail in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995), etc.

[0248] The single domain antibodies disclosed herein may also be administered by other routes, including by inhalation, oral, topical or intraocular routes. In some examples, the single domain antibody (or its conjugate) is administered by a fine needle.

[0249] Antibodies (or other therapeutic molecules) can be provided in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided as sterile solutions of known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, administered at a dose of 0.5 to 15 mg / kg body weight. TM Since its approval, antibody drugs have been marketed in the United States. Antibodies, ADCs, CARs, multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates can be administered by slow infusion rather than intravenous push or bolus injection. In one example, a higher loading dose is given, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg can be infused over a period of about 90 minutes, and then if the previous dose was a weekly maintenance dose of 2 mg / kg infused over 30 minutes, it would be 4-8 weeks. Well tolerated.

[0250] Controlled release parenteral formulations can be made into implants, oily injections, or microparticle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Microparticle systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the microparticles. Microparticles, microspheres, and microcapsules that are less than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. The diameter of capillaries is about 5 μm, so nanoparticles can only be injected intravenously. Microparticles are typically about 100 μm in diameter and can be administered by subcutaneous or intramuscular injection. See, e.g., 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).

[0251] Polymers can be used for controlled release of ions of the antibody-based compositions disclosed herein. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier for the 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. Technology 44 (2): 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled protein release (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many other systems for controlled delivery of therapeutic proteins are known (see U.S. Patents 5055303; 5188837; 4235871; 4501728; 4837028; 4957735; 5019369; 5055303; 5514670; 5413797; 5268164; 5004697; 4902505; 5506206; 5271961; 5254342 and 5534496).

[0252] A. Treatment methods

[0253] Antibodies, compositions, CARs (and CTLs expressing CARs), ADCs, multispecific (e.g., 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 mesothelin-positive cancers. In these applications, a therapeutically effective amount of the composition is administered to a subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to inhibit signs or symptoms of cancer. Suitable subjects may include those diagnosed with cancers expressing mesothelin, such as, but not limited to, mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (e.g., triple-negative breast cancer), or ovarian cancer.

[0254] Provided herein is a method for treating mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of mesothelin-specific antibodies, immunoconjugates, CARs (or CTLs expressing CARs), ADCs, multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or compositions disclosed herein. Also provided herein is a method for inhibiting tumor growth or metastasis of mesothelin-positive cancer in a subject by administering to the subject a therapeutically effective amount of mesothelin-specific antibodies, immunoconjugates, CARs (such as CTLs expressing CARs), ADCs, multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or compositions disclosed herein. In some embodiments, mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (such as triple-negative breast cancer), or ovarian cancer. In some examples, the method reduces the volume of a tumor (e.g., a metastasis) 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, compared to the volume before treatment. In some examples, the method reduces the size of a tumor (e.g., a metastasis) 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, compared to the volume before treatment. In some examples, the method reduces the number 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, compared to the number before treatment. In some examples, the method increases the prognosis of the subject, such as increasing the life expectancy of the subject by at least 4 months, at least 6 months, at least 8 months, at least 9 months, at least 12 months, at least 24 months, at least 36 months, or at least 60 months, for example, compared to the number before treatment. In some examples, a combination of these effects is achieved.

[0255] The therapeutically effective amount of the mesothelin-specific monoclonal antibody, ADC, CAR (e.g., CTL expressing CAR), multispecific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend on the severity of the disease, the type of disease and the general health of the patient. The therapeutically effective amount of the antibody-based composition is an amount that provides subjective relief of symptoms or objectively identifiable improvement as noted by a clinician or other qualified observer.

[0256] The administration of mesothelin-specific antibodies, ADCs, CARs, immunoconjugates, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, and compositions disclosed herein may also be accompanied by other anticancer agents or therapeutic treatments (such as surgical resection of tumors). Any suitable anticancer base may be administered in combination with antibodies, compositions, and immunoconjugates disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalated antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormones (e.g., antiandrogens), and antiangiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells (such as anti-PD1, anti-PDL1, anti-VEGF, and anti-EGFR antibodies).

[0257] 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, streptozotocin, or dacarbazine).

[0258] Non-limiting examples of antimetabolites include folate analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0259] 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).

[0260] Non-limiting examples of other agents include platinum coordination complexes (such as cis-diamine dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical suppressants (such as mitotane and aminoglutethimide).

[0261] Non-limiting examples of hormones and antagonists include cortical hormones (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as and tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include doxorubicin, melphalan, Ara-C, BiCNU, busulfan, CCNU, carboplatin, cisplatin, cyclophosphamide, daunorubicin, DTIC, 5-FU, fludarabine, Hydrea, idarubicin, ifosfamide, methotrexate, Mithramycin, mitoxantrone, mechlorethamine, paclitaxel (or other taxanes such as docetaxel), Velban, vincristine, VP-16, while some of the newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, and calcitriol.

[0262] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bromopyramide (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Research Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (Imreg from New Orleans, La), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0263] Another common treatment for some types of cancer is surgery, such as surgical removal of the cancer or a portion thereof. Another example of treatment is radiation therapy, such as the administration of radioactive material or energy to the tumor site (such as external beam radiation therapy) to help eradicate the tumor or shrink it prior to surgical removal.

[0264] B. Methods for Diagnosis and Detection

[0265] Provided herein are methods for detecting mesothelin proteins in vitro or in vivo. For example, the disclosed monoclonal antibodies can be used for in vivo tumor imaging. In order to use the disclosed antibodies as in vivo diagnostic reagents, the antibodies are labeled with detectable moieties such as radioactive isotopes, fluorescent markers, or positron-emitting radionuclides. As an example, the single domain antibodies disclosed herein can be conjugated to positron-emitting radionuclides for use in positron emission tomography (PET); this diagnostic process is often referred to as immuno-PET. Although full-length antibodies can make good immuno-PET reagents, their biological half-lives require waiting several days before imaging, which increases the associated non-target radiation dose. Smaller single domain antibodies have biological half-lives suitable for same-day imaging.

[0266] In other cases, mesothelin expression is detected in a biological sample. The sample can be any sample, including but not limited to tissue from a biopsy, autopsy, and pathological specimens. Biological samples also include tissue sections, for example, frozen sections taken for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0267] Provided herein is a method for determining whether a subject has mesothelin-positive cancer by contacting a sample from the subject with a mesothelin-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having mesothelin-positive cancer.

[0268] In another embodiment, a method is provided for confirming a diagnosis of mesothelin-positive cancer in a subject by contacting a sample from a subject diagnosed with mesothelin-positive cancer with a mesothelin-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample confirms a diagnosis of mesothelin-positive cancer in the subject.

[0269] In some examples of the disclosed methods, the monoclonal antibodies are directly labeled.

[0270] In other examples, the method further includes contacting a second antibody (detection antibody) that specifically binds to the monoclonal antibody with the sample; and detecting binding of the second antibody. An increase in binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects mesothelin-positive cancer in the subject or confirms a diagnosis of mesothelin-positive cancer in the subject.

[0271] In some instances, the cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, breast cancer (such as triple-negative breast cancer), or ovarian cancer.

[0272] In some examples, the control sample is a sample from a subject without cancer. In specific examples, the sample is a blood or tissue sample.

[0273] In some embodiments of the diagnostic and detection methods, an antibody that binds (e.g., specifically binds) mesothelin is directly labeled with a detectable marker. In another embodiment, an antibody that binds (e.g., specifically binds) mesothelin (primary antibody) is unlabeled, and a secondary antibody or other molecule that can bind to the antibody that specifically binds mesothelin is labeled. As is well known to those skilled in the art, a secondary antibody is selected that is capable of specifically binding to a primary antibody of a particular type and class. For example, if the primary antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0274] Suitable labels for antibodies or secondary antibodies 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, β-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 magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or 3 H.

[0275] In another embodiment, mesothelin in a biological sample can be measured by competitive immunoassay using a mesothelin protein standard labeled with a detectable substance and an unlabeled antibody that specifically binds to mesothelin. In this assay, a biological sample, a labeled mesothelin protein standard, and an antibody that specifically binds to mesothelin are combined, and the amount of the labeled mesothelin protein standard bound to the unlabeled antibody is measured. The amount of mesothelin in the biological sample is inversely proportional to the amount of the labeled mesothelin standard protein bound to the antibody that specifically binds to mesothelin.

[0276] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, the specifically bound antibodies can be used to detect the production of mesothelin in cells in cell culture. In another embodiment, the antibodies can be used to detect the amount of mesothelin in a biological sample such as a tissue sample or a blood or serum sample. In some examples, the mesothelin is a cell surface mesothelin. In other examples, the mesothelin protein is soluble (e.g., in a cell culture supernatant or a body fluid sample, such as a blood or serum sample).

[0277] In one embodiment, a kit for detecting mesothelin in a biological sample such as a blood sample or a tissue sample is provided. For example, in order to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. The kit for detecting mesothelin generally includes a monoclonal antibody that specifically binds to mesothelin, such as any monoclonal antibody disclosed herein, and may further include a labeled secondary antibody that can specifically bind to the anti-mesothelin antibody. In a specific embodiment, the anti-mesothelin antibody in the kit itself is labeled (e.g., with a fluorescent, radioactive or enzyme marker).

[0278] In one embodiment, the kit includes instructional materials disclosing methods of use of the antibody that binds mesothelin. The instructional materials may be written in electronic form (e.g., a computer floppy disk or CD) or in visual form (e.g., a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally include means for detecting the marker (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary marker example, such as a secondary antibody, etc.). The kit may also include buffers and other reagents routinely used in the practice of a particular method.

[0279] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassay may vary depending on the specific format used, the method of detecting mesothelin in a biological sample generally includes the step of contacting the biological sample with an antibody that specifically reacts with mesothelin under immunoreactive conditions. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0280] The antibodies disclosed herein can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. The antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse light scattering detection channels, and impedance channels, as well as other more complex detection levels, to separate or sort cells (see U.S. Patent No. 5,061,620). As disclosed herein, any monoclonal antibody that binds to mesothelin can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation.

[0281] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0282] Example

[0283] Example 1: Camelid VHH antibodies binding to human and mouse mesothelin

[0284] This example describes the identification and characterization of A101 and G8, two camel (dromedary) single domain (VHH) monoclonal antibodies that bind human and mouse mesothelin with high affinity.

[0285] Antibodies A101 and G8 were isolated from a camelid VHH phage display library by sequencing panning. Polyclonal phage ELISA was performed to assess the binding of phage clones to human and mouse mesothelin after each of four rounds of panning. After four rounds, phage display showed strong binding to human and mouse mesothelin ( Figure 1 ). VHH clones A101 and G8 were isolated from the phage library and further characterized. The purity and molecular weight of A101 and G8 are shown in Figure 2 The nucleotide and amino acid sequences of A101 are set forth herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The nucleotide and amino acid sequences of G8 are set forth herein as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0286] Binding of A101 and G8 antibodies to human and mouse mesothelin was assessed by ELISA ( Figure 3 ). R&D Mesothelin is a monomer with a His tag but no Fc. Fc-Mesothelin is a bivalent rabbit Fc fusion protein. Figure 3 As shown, the A101 and G8 antibodies bind to human and mouse mesothelin proteins.

[0287] To determine the mesothelin epitope bound by A101 and G8, a competitive ELISA was performed using full-length human mesothelin protein or mesothelin fragments: fragment 1 (residues 296-390), fragment 2 (residues 391-486), and fragment 3 (residues 487-598), numbered based on human mesothelin having an amino acid sequence of SEQ ID NO: 12 (see Kaneko et al., J Biol Chem 284(6): 3739-3749, 2009). The results demonstrated that both A101 and G8 bound to fragment 1 and full-length mesothelin, indicating that the epitope for each antibody is in the N-terminal fragment 1 ( Figure 4 ).

[0288] A101( Figures 5A-5C ) and G8( Figures 6A-6C) and mesothelin expressed on the cell surface were evaluated by flow cytometry. Binding experiments included human mesothelioma cancer cell lines M30 and H226, human ovarian cancer cell line OVCAR8, human pancreatic cell lines Panc3.014, KLM1 and T3M4, human cholangiocarcinoma cell lines KMBC and OZ, and mouse pancreatic cancer cell lines PDA95775, CREP133234 and CREP133239. Cells were exposed to 10 μg / ml A101 or G8, and the binding of the antibodies to the cell surface was determined by flow cytometry. Figures 5A-5C As shown in Figures 6A-6C, both antibodies were able to bind to mesothelin on the surface of all cell lines tested. Neither antibody was able to bind to mesothelin-negative cell lines ( Fig.11 and Fig.12 ).

[0289] A101 and G8 were used to generate mesothelin-targeted CAR T cells. Lentiviral constructs expressing CARs were generated, which included VHH antibody A101 or VHH antibody G8 and truncated human EGFR (huEGFRt) ( Fig. 7A ; see also Section IV above). To confirm successful transduction of the vector and expression of CAR in T cells, flow cytometry was used to detect the expression of huEGFRt. Figure 7B As shown, the transduction efficiency was approximately 40-50%.

[0290] Killing of mesothelin-positive cells by A101- or G8-based CAR T cells was evaluated using luciferase-expressing A431 (mesothelin-negative), H9 (mesothelin-overexpressing), KLM1 (pancreatic cancer), and H226 (mesothelioma) cells. Cells were co-cultured with A101- or G8-CAR-transduced T cells at E:T ratios ranging from 3.125:1 to 100:1 for 20 h, and specific lysis was measured using a luminescence-based cytolysis assay. Figure 8 As shown, mesothelin-targeted CAR T cells can induce specific lysis of all mesothelin-expressing cells (H9, KLM1, and H226) but not mesothelin-negative A431 cells.

[0291] Mesothelin-targeted CAR T cells based on antibodies A101 or G8 were evaluated in a mouse model of mesothelioma. NSG mice were inoculated intraperitoneally with H266 cells to establish tumors ( Fig. 9A On day 13 after tumor cell inoculation, mock T cells or 20×10 6 CAR T cells. Tumor burden was monitored by bioluminescence imaging for 27 days ( Fig. 9B ). Fig. 9CQuantification of bioluminescence in mice is shown in Figure 1. A101 CAR T cells demonstrated anti-tumor activity and showed a trend toward eradication of H226 xenograft tumors.

[0292] Example 2: Identification of A101 and G8 binding epitopes on human mesothelin

[0293] This example describes further analysis of the human mesothelin epitope bound by the A101 and G8 camelid VHH antibodies.

[0294] Based on SEQ ID NO: 12 ( Fig. 10A ) were used to generate several fragments of human mesothelin. These fragments include amino acid residues 296-390 (region I), 391-486 (region II), and 487-581 (region III) of human mesothelin, as well as smaller fragments within region I, particularly region IAB (296-359), region IBC (328-405), region IA (296-337), region IB (328-369), and region IC (360-405). The region IAB (296-359) is the binding region for the mesothelin binding partner MUC16 / CA125 (Kaneko et al., J Biol Chem 284 (6): 3739-3749, 2009). Mutants of the region IAB (269-359) with substitutions of E321A, W321A, or Y318A were also generated.

[0295] The binding of A101 and G8 to each mesothelin fragment was assessed by ELISA ( Fig. 10B ). The results showed that A101 and G8 camel single domain antibodies mainly bind to the N-terminal region I region (296-390), including the IAB domain (64 residues, 296-359), which is the region required for MUC16 binding, indicating that A101 and G8 antibodies can block the MUC16 / mesothelin interaction. In addition, when tyrosine residue 318 was mutated to alanine (Y318A), A101 and G8 binding was lost. Residue 318 is a key site for MUC16 binding, so MUC16 also does not bind to the Y318A mutant. These results indicate that both single domain antibodies bind to the ligand binding site of MUC16 / CA125.

[0296] Example 3: In vitro and in vivo activity of CAR T cells expressing A101 and G8

[0297] This example describes the in vitro cell killing of mesothelin-positive cells and growth inhibition of mesothelin tumors by A101 and G8 CAR T cells in xenograft animal models.

[0298] The generation of mesothelin-targeted CARs is described in Example 1. PBMCs from healthy donors were transduced with A101 or G8 CART cells. Cetuximab was used to detect CAR on the cell surface. The transduction efficiency of CAR T cells was greater than 60% ( Fig.13A ). Next, CAR T cells were evaluated in a CAR T cell killing assay using H9 (mesothelin overexpressing cell line); KLM1, a human pancreatic cancer line; H226, a human mesothelioma line; and A431, mesothelin-negative cells. Both A101 and G8 CAR T cells killed mesothelin-positive cell lines (H9, KLM1, and H226), but not mesothelin-negative cells (A431) ( Fig. 13B ). A101 CAR T cells showed better cytolytic activity than G8 CAR T cells in vitro.

[0299] A101 CAR T cells were tested in mice bearing human peritoneal mesothelioma xenografts. Seven-week-old female NSG mice were inoculated with 2MH226-luc cells. On day 3, animals were treated with mock T cells or A101 CAR T cells ( Fig. 14B , top). Tumor size was assessed by in vivo bioluminescence measurement using an IVIS imaging system. Figure 3 Bioluminescence images of mock-treated and A101-treated mice are shown in . Fig.14A The brightness of mock-treated and A101-treated animals is shown in Figure 2 (bottom). The results show that A101 CAR T cells effectively inhibited the growth of H226 xenograft tumors in mice.

[0300] In view of the many possible embodiments to which the principles of the disclosed subject matter can be applied, it should be recognized that the illustrated embodiments are merely examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Sequence Listing <110> The United States Government (represented by the Secretary of Health and Human Services) <120> Cross-species single-domain antibodies targeting mesothelin for the treatment of solid tumors <130> 4239-101800-02 <150> US 62 / 789,650 <151> 2019-01-08 <160> 12 <170> PatentIn Version 3.5 <210> 1 <211> 358 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (A101) <400> 1 caggtgcagc tggtggagtc tgggggcggc acggtgcagg ctggagggtc gctgaaactc 60 gcctgcgcag cctctggatt acccagaacg tacaatgtca tgggctggtt ccgccaggcc 120 ccagggaagg agcgcgaggg ggtcgcaata atttatacta cgactggagc aacatactat 180 cgcgactccg tcaagggccg ggccaccatc tcccaagaca acgccaagaa gtcggtgtct 240 ctccaaatga acagcctgag gcctgaggac acggccatct attactgtgt ggctaggcaa 300 cccaatagtg gtccctggga gtattggggc caggggaccc aggtcaccgt ctcctcaa 358 <210> 2 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide (A101) <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Thr Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Lys Leu Ala Cys Ala Ala Ser Gly Leu Pro Arg Thr Tyr Asn 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ile Ile Tyr Thr Thr Thr Gly Ala Thr Tyr Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Gln Asp Asn Ala Lys Lys Ser Val Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Val Ala Arg Gln Pro Asn Ser Gly Pro Trp Glu Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 3 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide (G8) <400> 3 caggtaaagc tggaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtacaa cttctggata caccaacagt tacaagtgga tgggctggtt ccgccaggct 120 ccaggacaag agcgcgaggg ggtcgcagtt atttacaccg gtaatgatag gacatactat 180 agtgactccg tgaagggccg attcaccatc tcccgagaca acgccaagaa tatgatctat 240 ctggacatga cgcgcctgag acctgaggac agcgctgtgt acgagtgtgc catcggacat 300 gatggcgcat ggcgttactg gggccaggga acgcaggtca ccgtctcctc a 351 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide (G8) <400> 4 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Tyr Thr Asn Ser Tyr Lys 20 25 30 Trp Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Tyr Thr Gly Asn Asp Arg Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Ile Tyr 65 70 75 80 Leu Asp Met Thr Arg Leu Arg Pro Glu Asp Ser Ala Val Tyr Glu Cys 85 90 95 Ala Ile Gly His Asp Gly Ala Trp Arg Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 22 <212> PRT <213> Homo sapiens <400> 5 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 6 <211> 45 <212> PRT <213> Homo sapiens <400> 6 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 7 <211> 21 <212> PRT <213> Homo sapiens <400> 7 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 8 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 9 <211> 112 <212> PRT <213> Homo sapiens <400> 9 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 11 <211> 335 <212> PRT <213> Homo sapiens <400> 11 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 <210> 12 <211> 622 <212> PRT <213> Homo sapiens <400> 12 Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45 Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60 Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu 65 70 75 80 Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu 85 90 95 Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro 100 105 110 Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro 115 120 125 Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile 130 135 140 Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln 145 150 155 160 Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175 Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185 190 Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu 195 200 205 Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg 210 215 220 Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp 225 230 235 240 Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly 245 250 255 Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg 260 265 270 Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile 275 280 285 Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300 Gly Lys Lys Ala Pro Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys 305 310 315 320 Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met 325 330 335 Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu 340 345 350 Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val 355 360 365 Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile 370 375 380 Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu 385 390 395 400 Val Asn Lys Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp 405 410 415 Arg Phe Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420 425 430 Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu 435 440 445 Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp 450 455 460 Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala 465 470 475 480 Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile 485 490 495 Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510 Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525 Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly 530 535 540 Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg 545 550 555 560 Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu 565 570 575 Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser 580 585 590 Met Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu Gly Pro Gly Pro 595 600 605 Val Leu Thr Val Leu Ala Leu Leu Leu Ala Ser Thr Leu Ala 610 615 620

Claims

1. A single domain monoclonal antibody that specifically binds to mesothelin, wherein: The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the Kabat numbering scheme are shown as residues 31-35, 50-66 and 99-108 of SEQ ID NO: 2, respectively; The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the IMGT numbering scheme are shown as residues 26-33, 51-58 and 97-108 of SEQ ID NO: 2, respectively; The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the Paratome numbering scheme are shown as residues 27-35, 47-61 and 97-108 of SEQ ID NO: 2, respectively; The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the Kabat numbering scheme are shown as residues 31-35, 50-66 and 99-106 of SEQ ID NO: 4, respectively; The monoclonal antibody has CDR1, CDR2 and CDR3 sequences as defined by using the IMGT numbering scheme as shown in residues 26-33, 51-58 and 97-106 of SEQ ID NO: 4, respectively; or The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined using the Paratome numbering scheme are shown as residues 27-35, 47-60 and 98-106 of SEQ ID NO: 4, respectively.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the monoclonal antibody consists of SEQ ID NO: 2 or SEQ ID NO:

4.

3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody is a camelid antibody. The monoclonal antibody according to claim 1 , wherein the monoclonal antibody is a humanized antibody. The monoclonal antibody according to claim 1 , wherein the monoclonal antibody is a chimeric antibody.

6. A chimeric antigen receptor (CAR), comprising the monoclonal antibody according to any one of claims 1 to 5, a hinge region, a transmembrane domain, a co-stimulatory signal portion and a signal domain.

7. The CAR of claim 6, wherein the hinge region comprises a CD8α hinge region.

8. The CAR of claim 6, wherein the transmembrane domain comprises a CD8α transmembrane domain.

9. The CAR according to any one of claims 6 to 8, wherein the co-stimulatory signal portion comprises a 4-1BB signal portion.

10. The CAR according to any one of claims 6-8, wherein the signaling domain comprises a CD3ζ signaling domain.

11. An isolated cell expressing the CAR of any one of claims 6-10.

12. The isolated cell according to claim 11, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

13. An immunoconjugate comprising the monoclonal antibody of any one of claims 1 to 5 and a toxin, a photon absorber, a fluorophore, an enzyme or a radioisotope.

14. The immunoconjugate of claim 13, wherein the toxin is Pseudomonas exotoxin or a variant thereof.

15. The immunoconjugate of claim 14, wherein the Pseudomonas exotoxin variant is PE38.

16. An antibody-drug conjugate (ADC), comprising a drug conjugated to the monoclonal antibody of any one of claims 1 to 5.

17. The ADC of claim 16, wherein the drug is a small molecule.

18. The ADC of claim 16 or claim 17, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and / or a cytotoxic agent.

19. A multispecific antibody comprising the monoclonal antibody of any one of claims 1 to 5 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

20. The multispecific antibody according to claim 19, which is a bispecific antibody. The multispecific antibody according to claim 19 , which is a trispecific antibody.

22. The multispecific antibody of any one of claims 19-21, wherein the at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activating receptor.

23. An antibody-nanoparticle conjugate comprising nanoparticles conjugated to the monoclonal antibody of any one of claims 1-5.

24. The antibody-nanoparticle conjugate of claim 23, wherein the nanoparticle comprises a polymer nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymer micelle, or a vesicle.

25. The antibody-nanoparticle conjugate of claim 23 or claim 24, wherein the nanoparticle comprises a cytotoxic agent.

26. A fusion protein comprising the monoclonal antibody according to any one of claims 1 to 5 and a heterologous protein or peptide. The fusion protein of claim 26 , wherein the heterologous protein is an Fc protein.

28. An isolated nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-5, the CAR of any one of claims 6-10, the immunoconjugate of any one of claims 13-15, the multispecific antibody of any one of claims 19-22, or the fusion protein of claim 26 or claim 27.

29. The isolated nucleic acid molecule according to claim 28, consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a degenerate variant thereof.

30. The isolated nucleic acid molecule of claim 28 or claim 29 operably linked to a promoter.

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

32. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising in the 5' to 3' direction: a nucleic acid encoding the first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); A nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 5; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and Nucleic acid encoding truncated human epithelial cell growth factor receptor (huEGFRt).

33. The nucleic acid molecule of claim 32, further comprising a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' of the nucleic acid encoding the first GMCSFRss.

34. A vector comprising the nucleic acid molecule of claim 32 or claim 33.

35. The vector of claim 34, wherein the vector is a lentiviral vector.

36. An isolated cell comprising the nucleic acid molecule of any one of claims 28-30, 32 and 33 or the vector of any one of claims 31, 34 and 35.

37. A composition comprising a pharmaceutically acceptable carrier and a monoclonal antibody as described in any one of claims 1-5, a CAR as described in any one of claims 6-10, an isolated cell as described in any one of claims 11, 12 and 36, an immunoconjugate as described in any one of claims 13-15, an ADC as described in any one of claims 16-18, a multispecific antibody as described in any one of claims 19-22, an antibody-nanoparticle conjugate as described in any one of claims 23-25, or a fusion protein as described in claim 26 or claim 27.

38. Use of the monoclonal antibody of any one of claims 1-5, the CAR of any one of claims 6-10, the isolated cell of any one of claims 11, 12 and 36, the immunoconjugate of any one of claims 13-15, the ADC of any one of claims 16-18, the multispecific antibody of any one of claims 19-22, the antibody-nanoparticle conjugate of any one of claims 23-25, or the fusion protein of claim 26 or claim 27, or the composition of claim 37 in the preparation of a medicament for treating mesothelin-positive cancer in a subject, wherein the mesothelin-positive cancer is mesothelioma or pancreatic cancer.

39. Use of the monoclonal antibody of any one of claims 1-5, the CAR of any one of claims 6-10, the isolated cell of any one of claims 11, 12 and 36, the immunoconjugate of any one of claims 13-15, the ADC of any one of claims 16-18, the multispecific antibody of any one of claims 19-22, the antibody-nanoparticle conjugate of any one of claims 23-25, or the fusion protein of claim 26 or claim 27, or the composition of claim 37 in the preparation of a medicament for treating mesothelin-positive cancer in a subject, wherein the mesothelin-positive cancer is ovarian cancer, in, In the monoclonal antibody, the CAR, the isolated cell, the immunoconjugate, the ADC, the multispecific antibody, the antibody-nanoparticle conjugate, the fusion protein or the composition: The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the Kabat numbering scheme are shown as residues 31-35, 50-66 and 99-108 of SEQ ID NO: 2, respectively; The monoclonal antibody has CDR1, CDR2 and CDR3 sequences as defined by using the IMGT numbering scheme as shown in residues 26-33, 51-58 and 97-108 of SEQ ID NO: 2, respectively; or The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined using the Paratome numbering scheme are shown as residues 27-35, 47-61 and 97-108 of SEQ ID NO: 2, respectively.

40. Use of the monoclonal antibody of any one of claims 1-5, the CAR of any one of claims 6-10, the isolated cell of any one of claims 11, 12 and 36, the immunoconjugate of any one of claims 13-15, the ADC of any one of claims 16-18, the multispecific antibody of any one of claims 19-22, the antibody-nanoparticle conjugate of any one of claims 23-25, or the fusion protein of claim 26 or claim 27, or the composition of claim 37 in the preparation of a medicament for inhibiting tumor growth or metastasis of mesothelin-positive cancer in a subject, wherein the mesothelin-positive cancer is mesothelioma or pancreatic cancer.

41. Use of the monoclonal antibody of any one of claims 1-5, the CAR of any one of claims 6-10, the isolated cell of any one of claims 11, 12 and 36, the immunoconjugate of any one of claims 13-15, the ADC of any one of claims 16-18, the multispecific antibody of any one of claims 19-22, the antibody-nanoparticle conjugate of any one of claims 23-25, or the fusion protein of claim 26 or claim 27, or the composition of claim 37 in the preparation of a medicament for inhibiting tumor growth or metastasis of mesothelin-positive cancer in a subject, wherein the mesothelin-positive cancer is ovarian cancer, in, In the monoclonal antibody, the CAR, the isolated cell, the immunoconjugate, the ADC, the multispecific antibody, the antibody-nanoparticle conjugate, the fusion protein or the composition: The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined by using the Kabat numbering scheme are shown as residues 31-35, 50-66 and 99-108 of SEQ ID NO: 2, respectively; The monoclonal antibody has CDR1, CDR2 and CDR3 sequences as defined by using the IMGT numbering scheme as shown in residues 26-33, 51-58 and 97-108 of SEQ ID NO: 2, respectively; or The CDR1, CDR2 and CDR3 sequences of the monoclonal antibody defined using the Paratome numbering scheme are shown as residues 27-35, 47-61 and 97-108 of SEQ ID NO: 2, respectively.

42. Use of the monoclonal antibody according to any one of claims 1 to 5 in the preparation of a reagent for detecting the expression of mesothelin in a sample.

43. The use according to claim 42, wherein the monoclonal antibody is directly labeled.

44. The use according to claim 42 or 43, wherein the sample is obtained from a subject suspected of having mesothelioma, pancreatic cancer, bile duct cancer or ovarian cancer.

45. The use according to claim 42 or 43, wherein the sample is a tumor biopsy.

46. ​​Use of the monoclonal antibody of any one of claims 1 to 5 in the preparation of a reagent for diagnosing a subject suffering from mesothelioma, pancreatic cancer, bile duct cancer or ovarian cancer, wherein the diagnosis comprises: contacting a sample obtained from the subject with the monoclonal antibody of any one of claims 1 to 5; as well as Binding of the antibody to the sample is detected, thereby diagnosing the subject with mesothelin-positive cancer.

47. The use according to claim 46, wherein the monoclonal antibody is directly labeled.

48. The use according to claim 46, wherein the diagnosis further comprises: contacting the monoclonal antibody with a detection antibody; as well as The binding of the detection antibody to the monoclonal antibody is detected, thereby diagnosing the subject with mesothelin-positive cancer.

49. The use according to any one of claims 46 to 48, wherein the sample is a tumor biopsy.

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