Anti-mesothelin bispecific antibodies and methods of use thereof

Abstract

The present disclosure provides amino acid sequences (e.g., antibodies and bispecific antibodies) that are capable of binding mesothelin (MSLN). The present disclosure relates to bispecific antibodies and antigen-binding fragments thereof, conjugates, including immunoconjugates, having a specific binding to mesothelin, and related compositions and formulations. The disclosure also relates to methods of using mesothelin bispecific antibodies and antigen-binding fragments thereof to treat mesothelin-related diseases, disorders, and conditions, including cell proliferative diseases, such as cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to and claims priority from U.S. Provisional Patent Application Serial No. 63 / 429,821, filed on December 2, 2022, and the entire contents of which are incorporated herein by reference. Background Art

[0002] Mesothelin is a GPI-anchored glycoprotein that is normally expressed in the pleural cavity and other reproductive-related tissues. It has been shown to be a tumor-associated protein that is overexpressed in many types of cancer, including mesothelioma, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer (NSCLC), and breast cancer. Mesothelin is considered a candidate for cancer-targeted therapy [1]. Various forms of anti-mesothelin antibodies have been developed for preclinical and clinical evaluation [2-5]. These antibodies are derived from hybridomas or fully humanized phage libraries and have been tested in the form of naked antibodies, antibody-drug conjugates, immunotoxins, and CAR-T. Early clinical studies of mesothelin-targeted CAR-T cells have reported promising results; however, the development of other mesothelin-targeted therapies (e.g., cancer therapies) continues to be necessary.

[0003] The antibody m912[6] used in the CAR-T platform is derived from a fully human Fab library[7]. This antibody has nearly 100% germline sequence, but has poor affinity (reportedly no better than 20nM[7]). Despite this, this low-affinity antibody has shown good phase I clinical results in the CAR-T format[6]. It is well known that CAR-T therapy tolerates antibodies with relatively weak affinity, and the reasons for this are not fully understood. Due to this characteristic of CAR-T therapy, the affinity of m912 may not be good enough when integrated with other formats (such as ADCs or bispecific antibodies). Therefore, further development around this antibody and other antibodies and proteins that specifically bind to mesothelin is needed.

[0004] Described herein are specific binding proteins, antibodies, bispecific antibodies, monoclonal antibodies, and antigen-binding fragments and domains thereof that can bind mesothelin and can be used in methods of detecting, treating, and preventing mesothelin-related disorders, diseases, and related clinical conditions, including cancer. Summary of the Invention

[0005] The present disclosure generally provides an antibody (i.e., an isolated antibody, a bispecific antibody, and an antigen-binding fragment thereof) that binds to mesothelin (MSLN). In some aspects, the present disclosure provides an antibody, a bispecific antibody, an isolated antibody, and an antigen-binding fragment thereof that exhibits specific binding to a mesothelin epitope. In some embodiments, according to the aspects and embodiments provided herein, the antibody, bispecific antibody, or antigen-binding fragment thereof may comprise one or more heavy chain variable regions, one or more light chain variable regions, one or more CDR1s of light and / or heavy chain variable regions, one or more CDR2s of light and / or heavy chain variable regions, one or more CDR3s of light and / or heavy chain variable regions, or any combination thereof.

[0006] In embodiments, the antibody, bispecific antibody, or antigen-binding fragment thereof binds to an epitope within the human mesothelin protein sequence. In embodiments, the disclosure provides a bispecific antibody that binds to human mesothelin and a target sequence other than mesothelin.

[0007] In certain aspects, the present disclosure relates to an isolated antibody, bispecific antibody, or antigen-binding fragment thereof that binds to a mesothelin (MSLN) protein, wherein the antibody, bispecific antibody, or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising: CDR1, CDR2, and CDR3 of the amino acid sequence described herein; and a light chain variable region comprising: CDR1, CDR2, and CDR3 of the amino acid sequence described herein.

[0008] In some embodiments of this aspect, the present disclosure provides an isolated antibody, bispecific antibody, or antigen-binding fragment thereof comprising: (i) a heavy chain variable region having an amino acid sequence described herein; and (ii) a light chain variable region having an amino acid sequence described herein.

[0009] In some embodiments of the above aspects, the present disclosure provides an antigen-binding fragment comprising a single-chain Fv (scFv), a single-chain Fv-Fc (scFv-Fc), a single-chain antibody, a single-domain antibody, a Fab fragment, or a F(ab')2 fragment.

[0010] In some embodiments of the above aspects, the present disclosure provides antibodies or bispecific antibodies comprising an IgG, IgM, IgA, IgE, IgD, or IgY isotype.

[0011] In some embodiments of the above aspects, the present disclosure provides a monoclonal antibody (mAb) or a monoclonal bispecific antibody (mbsAb).

[0012] In some embodiments of the above aspects, the present disclosure provides an antibody, bispecific antibody, or antigen-binding fragment conjugated to a moiety. In some embodiments, the conjugated moiety comprises a therapeutic agent, an active agent, a solid support, an affinity agent, or a detectable label. In some further embodiments, the moiety comprises a cytotoxin. In some other embodiments, the moiety comprises an anticancer agent.

[0013] In some embodiments of the above aspects, the antibody, bispecific antibody, or antigen-binding fragment thereof has a K of about 1.0 pM to 200 nM. D Binds to mesothelin.

[0014] In some embodiments of the above aspects, the antibody, bispecific antibody, or antigen-binding fragment thereof inhibits cell proliferation with an IC50 of about 0.01-250 nM.

[0015] On the other hand, the present disclosure provides a bispecific antibody comprising an antibody, bispecific antibody or antigen-binding fragment thereof according to any one of the above aspects and embodiments, and an antibody, bispecific antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain a mesothelin epitope. In some further embodiments, the bispecific antibody comprises a mesothelin antibody and a therapeutic antibody. In a further embodiment, the bispecific antibody comprises an antibody, bispecific antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not contain a mesothelin epitope and induces an immune response. In a further embodiment, the bispecific antibody comprises a mesothelin antibody, bispecific antibody or fragment thereof according to the present disclosure, and an antibody, bispecific antibody or antigen-binding fragment thereof that specifically binds to an antigen other than mesothelin, comprising CD2, CD3, CD11a CD20, CD25 (IL2R), CD33, CD52, EGFR, VEGF, integrin-α3, GPIIb / IIIar, protein F, TNF-α, TNF-β, HER2 / Neu, C5 or IgE.

[0016] In any of the above aspects and embodiments, the isolated antibody, bispecific antibody, or antigen-binding fragment thereof can be conjugated to a solid support, affinity agent, or detectable agent.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of the above aspects and embodiments and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present disclosure provides a kit comprising a pharmaceutical composition or an antibody, a bispecific antibody, or an antigen-binding fragment thereof according to any of the above aspects and embodiments, optional reagents, and instructions for use.

[0019] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody, a bispecific antibody, or an antigen-binding fragment thereof that binds to a mesothelin protein, wherein the antibody, the bispecific antibody, or the antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising: CDR1, CDR2, and CDR3 of the amino acid sequence described herein; and a light chain variable region comprising: CDR1, CDR2, and CDR3 of the amino acid sequence described herein.

[0020] In some further embodiments of the above aspects, the method comprises administering an antibody, bispecific antibody, or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising an amino acid sequence as described herein.

[0021] In some embodiments of the above methods, the cancer comprises a solid tumor cell cancer.

[0022] In another aspect, the present disclosure provides a method for inhibiting the proliferation of a cell expressing mesothelin (e.g., abnormally expressing, overexpressing, etc.), the method comprising contacting the cell with an effective amount of an antibody, bispecific antibody, or antigen-binding fragment thereof according to the above aspects and embodiments. In some embodiments of the method, the cell comprises a cancer cell.

[0023] In another aspect, the present disclosure provides a method for detecting the presence of mesothelin in a biological sample, comprising contacting the biological sample with an effective amount of an antibody, bispecific antibody, or antigen-binding fragment thereof according to the above aspects and embodiments under conditions that allow the antibody, bispecific antibody, or antigen-binding fragment thereof to form a complex with mesothelin present in the sample, and measuring a detectable signal associated with the formation of the complex. In further embodiments of these methods, the antibody, bispecific antibody, or antigen-binding fragment thereof comprises a detectable label.

[0024] In any of the above aspects and embodiments relating to methods, some embodiments provide the subject to be a human, and / or the cell to be a human cell, and / or a biological sample from a human.

[0025] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody, bispecific antibody, or antigen-binding fragment thereof according to the above aspects and embodiments.

[0026] In another aspect, the present disclosure provides an isolated recombinant cell that produces an antibody, bispecific antibody, or antigen-binding fragment thereof according to the above aspects and embodiments.

[0027] Other aspects and embodiments of the present disclosure will be apparent to those skilled in the art from this disclosure and the illustrative examples that follow. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figures AB. show the predicted CDR sequences for m912 VH (A) and VL (B). These predictions were obtained by submitting the m912 VH and VL sequences to the proABC-2 web server. Based on the probability of intermolecular contact with the antigen (pt), the putative CDR residues are highlighted in black bars. The amino acid sequences of the putative CDRs are shown below.

[0029] Figure 2 Figures AF depict consensus sequences represented by sequence logos from the m912 affinity maturation experiment. The library and parent CDR sequences used for panning are indicated at the top and bottom of the figure logo, respectively. The number of sequences used to generate each logo is also indicated. The logos represent panning results for a single HCDR3 library (AD), a pooled HCDR1 library (E), a LCDR1 library (F), a pooled LCDR3 library (G), and an expanded LCDR3 library (H).

[0030] Figure 3 AJ. ELISA analysis of soluble scFvs from clones selected from the m912 affinity maturation experiment is depicted. Selected clones used to construct combinatorial antibodies are highlighted in bold. AB. ELISA analysis of clones selected from the HCDR1 panning. C. Western blot analysis of scFv supernatants from m912 and selected HCDR1 clones. DE. ELISA analysis of clones selected from the HCDR3 panning. F. Western blot analysis of scFv supernatants from selected HCDR3 clones. G. ELISA analysis of clones selected from the LCDR3 panning. H. Western blot analysis of scFv supernatants from m912, HP4-44, LCDR3-2, and selected LCDR1 clones. IJ. ELISA analysis of clones selected from the LCDR1 panning.

[0031] Figure 4 This study describes affinity analysis of MSLN antibodies using Octet using a single concentration of mesothelin. Biosensors containing captured antibodies were then immersed in wells containing 125 nM mesothelin for 5 minutes, followed by a 5-minute dissociation in kinetic buffer. m912 was used as a reference in each set of experiments.

[0032] Figure 5AC. Describes flow cytometry analysis of nonspecific binding of MSLN antibodies to mesothelin-negative H929 cells. A. MSLN-positive OVCAR3 cells were stained using m912, HP1-A13, and HP4-44 antibodies. Cells stained with 647 anti-human IgG Fc secondary antibody served as a negative control. Cells were stained with 5 mg / mL of antibody or antibody supernatant. B. Representative plots of nonspecific binding of MSLN-48 to H929 cells compared to m912 and HP4-44 staining. C. Nonspecific binding of MSLN antibodies to H929 cells was ranked using the x-mean of the peaks.

[0033] Figure 6 .Depicts the expression of selected MSLN-[L]-scFv by Octet CD3 Affinity measurements of bsAbs were performed. Representative data from the measurements. ForteBio's Fc epitope anti-human capture (AHC) biosensor was used to load the bsAbs tested. The bsAbs were loaded onto the probe by immersing them in 2.5 μg / ml antibody followed by a 30 second baseline step. A probe loaded with a control antibody was used as a reference sensor. The antibody loaded probe was immersed in a well containing a series of concentrations of mesothelin (0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM) for 300 seconds and then dissociated in kinetic buffer for 300 seconds. The dissociation constant (K D ) and K on / K off is estimated by fitting a 1:1 binding model. The probe was regenerated for multiple rounds of measurements.

[0034] Figure 7 Depicted are the results of surface plasmon resonance (SPR) affinity measurements of MSLN-39 IgG. Measurements were performed by immobilizing the test antibody and then perfusing the corresponding concentration of mesothelin. KD and kinetic parameters are listed.

[0035] Figure 8 AD. Depicts MSLN-[L]-scFv CD3 Cell surface binding of bsAbs. A. Binding to MSLN-positive OVCAR3 cells. B. Binding to MSLN-negative H929 cells. C. Binding to MSLN-[L]-scFv CD3 OVCAR3 surface binding properties of bsAb. D.MSLN-[L]-scFv CD3 Jurkat surface binding properties of bsAbs.

[0036] Figure 9.Depicts the antigen and MSLN-[L]-scFv CD3 The assay is performed by coating CD3 / CD3 heterodimers on a plate, allowing MSLN-[L]-scFv to bind to the CD3 / CD3 heterodimers. CD3 The bsAb is captured by the CD3 arm. Simultaneous binding of MSLN to the second arm is detected using HRP-conjugated streptavidin, which recognizes the biotin on the Avi tag that complements MSLN.

[0037] Figure 10 AD. Depicts the expression of MSLN-[L]-scFv in the presence of Capan-2 (A) and OVCAR3 (C) and in the absence of Capan-2 (B) and OVCAR3 (D). CD3 T cell activation assay of bsAb. CD3 A hook effect was observed for the bsAb. Data points at high concentrations were masked for ease of fitting.

[0038] Figure 11 AB. Depicts the expression of MSLN-[L]-scFv in the presence of PBMC and Capan-2 / KILR cells. CD3 KILR cytotoxicity assay of bsAb. PBMCs from two donors (A and B) were mixed with Capan2 / KILR cells at a ratio of 15:1. CD3 Cytotoxicity was assessed by monitoring ePL release after 48 h of incubation in the presence of bsAb.

[0039] Figure 12 .Depicts the luciferase-based MSLN-[L]-scFv in the presence of PBMCs and NCI-N87-Luc cells CD3 bsAb cytotoxicity assay.

[0040] Figure 13 AB. Depicts the interaction of soluble MSLN with MSLN-[L]-scFv CD3 The effect of bsAb cytotoxicity. This study used a luciferase-based cytotoxicity assay on NCI-N87 cells without and with MSLN supplementation. CD3 The bsAbs HP4-44 (A) and MSLN-39 (B) were assayed.

[0041] Figure 14 AD. Depicts MSLN-[L]-scFv CD3Inhibition of tumor growth by bsAb in the NCI-N87 / PBMC co-transplant model. A. Schematic diagram of the tumor cell inoculation and antibody treatment protocol. B. Tumor growth was measured throughout the study in the PBS group, the 0.2 mg / kg HPN536 group, and two HP4-44 and MSLN-39 bsAb samples at doses of 0.2 mg / kg, 0.02 mg / kg, and 0.002 mg / kg, respectively. Data points represent the mean ± standard deviation (SD) of six mice. Statistical analysis was based on a two-tailed t-test, *P < 0.05 and ***P < 0.0001 compared to the PBS group. C. Images of exfoliated tumors from each mouse in each group at the end of the study. D. Body weights were measured throughout the study.

[0042] Figure 15 . Depicts MSLN-[L]-scFv CD3 Pharmacokinetic study of bsAbs. Male BALB / c mice were divided into two groups (n=5 / group). HP4-44 and MSLN-39 bsAbs were injected intravenously (iv) at a dose of 5 mg / kg to the mice. Blood was then collected from the mice at the following time points: 0 (pre-dose), 1 5 min, 6 h, 1, 2, 4, 7, 10, 15, 21, and 28 days. Antibody concentrations in serum were quantified using ELISA, in which human recombinant mesothelin (Acro Biosystems) was immobilized. Pharmacokinetic (PK) parameters were determined by a non-compartmental analysis model using PKsolver. DETAILED DESCRIPTION

[0043] The present disclosure generally relates to engineered antibodies, and in a specific embodiment, to bispecific antibodies with specific binding to mesothelin, which have a wide range of uses (i.e., diagnostic and therapeutic applications). The antibodies and bispecific antibodies disclosed herein enhance the potential therapeutic effect of targeting mesothelin in disease treatment methods (e.g., uses in cancer immunotherapy). In some embodiments, the present disclosure relates to an engineered form of the m912 antibody that improves its binding affinity to mesothelin. As disclosed herein, the engineered form of m912 has been integrated into a bispecific form that can effectively redirect cytotoxic T cells to tumors expressing mesothelin. The bispecific antibodies according to the exemplary aspects and embodiments disclosed herein can be used to effectively treat various types of cancers associated with abnormal and / or overexpression of mesothelin. The enhanced binding affinity exhibited by the antibodies, bispecific antibodies, and fragments thereof described herein provide other antibody forms that can be used to treat cancer, such as ADCs and immunotoxins.

[0044] It is to be understood that this disclosure is not limited to the particular proteins, nucleic acids, compositions, methods or process steps described below as exemplary and illustrative aspects and embodiments, as variations and modifications are within the scope of this description.

[0045] As used in this specification and the appended claims, the singular forms "-", "a", and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press, provide a general dictionary of many terms used in the present invention for technicians. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be represented by their commonly accepted single-letter codes. A. Antibodies

[0046] The term "antibody" or its plural form, also referred to as immunoglobulin, as used herein, covers full-length antibody sequences, including, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed by at least two different epitope binding fragments, bispecific antibodies, human antibodies and humanized antibodies. In some embodiments, the present disclosure specifically provides bispecific antibodies or antigen-binding fragments thereof. Antibody "fragments" (or "antigen-binding fragments," "binding fragments," "epitope-binding fragments," etc.) described herein generally refer to any antibody sequence that is smaller than the full-length antibody sequence but still exhibits specific binding activity to the target antigen. In exemplary embodiments, the antibody fragment generally comprises a combination of at least three heavy chain variable domain CDR sequences (HCDR1, HCDR2, HCDR3) and at least three light chain variable domain CDR sequences (LCDR1, LCDR2, LCDR3). Some non-limiting examples of antibody fragments include single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, camelized antibodies, antibody fragments that exhibit a desired biological activity (e.g., antigen-binding portion), disulfide-linked Fv (dsFv), anti-idiotypic (anti-Id) antibodies, intrabodies, and epitope-binding fragments of any of the above. In some embodiments, the present disclosure provides antibodies comprising immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules that contain at least one antigen-binding site). Antibodies and fragments thereof may also include peptide fusions with antibodies or portions thereof (e.g., proteins fused to an Fc domain).

[0047] According to some aspects and embodiments described herein, the present disclosure provides and relates to bispecific antibodies having specific binding activity to mesothelin and another target sequence.

[0048] The immunoglobulin molecule can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, e.g., G1m (f, z, a, or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2, or 3)). Antibodies and fragments thereof can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals, such as birds (e.g., chickens).

[0049] In some aspects, the present disclosure provides novel binding agents. In some exemplary embodiments, the novel binding protein is an IgG, scFv, Fab, monoclonal antibody (mAb) or single-chain Fv-Fc (scFv-Fc) antibody. A typical or conventional mAb comprises two heavy chain subunits and two light chain subunits. Each mAb heavy chain contains a variable domain (VH) that contributes to antigen binding and a variable domain (VH) composed of three or four subdomains (C H 1. C H 2. C H 3. C H 4) composed of a constant domain (CH). VH contains three complementarity determining regions (CDRs): HCDR1, HCDR2, and HCDR3. Each mAb light chain contains a variable domain (VL) and a constant domain (CL). VL contains three CDRs: LCDR1, LCDR2, and LCDR3. Two light chain constant domain isotypes are found in mammals: kappa (κ) and lambda (λ). Disulfide bonds bind each C H 1 domain is connected to a CL domain and C H The two domains are connected to each other. Five types of heavy chains (α, δ, ε, γ, and μ) are present in different classes of antibodies (IgA, IgD, IgE, IgG, and IgM). mAb heavy chains have a hinge region that confers structural flexibility and mobility.

[0050] The "Fc" region encompasses the domains of the constant region of the heavy chain of an immunoglobulin, including fragments, analogs, variants, mutants, or derivatives thereof. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The Fc region may be a native sequence Fc region or an altered Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, namely, C H 2 domains and C H The "Fv" region encompasses the VH and VL domains of an immunoglobulin. As used herein, an "scFv-Fc" antibody refers to a single VH domain and a single VL domain connected by a hinge region, and a C-terminal domain of a single CH domain. H 2 domains and C H 3-domain fusion protein.

[0051] As described herein, the antibodies, bispecific antibodies, and antigen-binding fragments thereof disclosed herein comprise a binding domain that binds to an epitope of mesothelin. "Binding domain" or "binding sequence" are used interchangeably with antibody fragments, bispecific antibody fragments, or "antigen-binding fragments" and herein refer to a portion of a specific binding sequence that is sufficient and capable of binding to or interacting with a target structure, antigen, or epitope. In certain aspects, the antibodies bind to a region of mesothelin that comprises an antigenic sequence or fragment. In other aspects, the antibodies bind to a domain of the mesothelin protein (e.g., Region I, Region II, or Region III).

[0052] In some embodiments, the antibodies, bispecific antibodies, and antigen-binding fragments thereof according to the present disclosure bind to mesothelin and can inhibit its binding interactions with other molecules and / or one or more biological functions. In some exemplary embodiments, the antibodies, bispecific antibodies, and antigen-binding fragments thereof can inhibit or antagonize one or more mesothelin-related biological pathways. According to such exemplary embodiments of the present disclosure, the antibodies, bispecific antibodies, or fragments thereof can prevent, inhibit, or reduce the binding and / or activity of mesothelin and can provide neutralizing activity against mesothelin and its downstream signaling partners and pathways.

[0053] In some embodiments, the antibodies and bispecific antibodies disclosed herein can be characterized by one or more of the following structural and / or functional properties: a. An amino acid sequence comprising a VH domain and a VL domain, wherein the VH domain comprises HCDR1, HCDR2 and HCDR3 identified by a SEQ ID NO as disclosed herein; and wherein the VL domain comprises LCDR1, LCDR2 and LCDR3 identified by a SEQ ID NO as disclosed herein; b. an amino acid sequence comprising a VH domain and / or a VL domain, wherein the VH domain has the amino acid sequence of SEQ ID NO disclosed herein; and the VL domain has the amino acid sequence of SEQ ID NO disclosed herein; c. having an amino acid sequence of HCDR1, HCDR2, HCDR3 and / or LCDR1, LCDR2, LCDR3, wherein the HCDR1 comprises SEQ ID NO: 85, 86, 97, 49, 53, 57, 61, 65, 69, 73, 77 or 81; the HCDR2 comprises SEQ ID NO: 50, 54, 58, 62, 66, 70, 74, 78, 82, 87, 88 or 98; the HCDR3 comprises SEQ ID NO: 51, 55, 59, 63, 67, 71, 75, 79, 83, 89, 90, 91, 99, 100 or 101; the LCDR1 comprises SEQ ID NO: 93; the LCDR2 comprises SEQ ID NO: 94; the LCDR3 comprises SEQ ID NO: 95 or 96. d. Binding specificity for mesothelin; e. Binding specificity for mesothelin and another target sequence; f. For mesothelin, the dissociation constant (K D ) in the range of 1 pM-500 nM, 10 pM-500 nM, 0.1 nM-500 nM, 1-450 nM, 1-400 nM, 1-300 nM, 1-200 nM, 1-100 nM, 1-50 nM, 1-25 nM, or 1-10 nM; g. For mesothelin, IC50 values ​​are in the range of 0.01–250 nM, 0.01–200 nM, 0.01–150 nM, 0.01–100 nM, 0.01–50 nM, 0.01–25 nM, 0.01–20 nM, or 0.01–10 nM; h. The ability to inhibit, reduce, prevent or disrupt mesothelin binding interactions can be measured by methods including, for example, filter binding assays, fluorescence spectroscopy, ELISA, isothermal titration calorimetry and surface plasmon resonance.

[0054] In some aspects, the antibody, bispecific antibody, or antigen-binding fragment thereof that is capable of inhibiting, reducing, preventing, or destroying the binding activity or biological activity of mesothelin, and / or is capable of neutralizing the activity of mesothelin, binds to an epitope present in the mesothelin protein.

[0055] In some aspects, an antibody, bispecific antibody, or antigen-binding fragment thereof that binds mesothelin comprises a VH domain having an amino acid sequence described herein.

[0056] In some aspects, the antibody, bispecific antibody, or antigen-binding fragment thereof that binds to mesothelin comprises a VL domain having an amino acid sequence described herein.

[0057] In certain embodiments of these aspects, the antibody or antigen-binding fragment thereof comprises a VH domain and a VL domain having a combination of heavy and light chain, or heavy and light chain CDR sequences identified in the Table and identified by SEQ ID NO.

[0058] In exemplary embodiments, the antibodies, bispecific antibodies, or fragments thereof disclosed herein exhibit binding specificity for the mesothelin protein and can inhibit, reduce, prevent, or disrupt mesothelin binding interactions. In further embodiments, the antibodies, bispecific antibodies, or fragments thereof bind to mesothelin and another protein target.

[0059] Those skilled in the art will appreciate that the sequences disclosed herein can be modified to a certain extent without impairing the ability of the antibody or fragment thereof to interact with mesothelin. In certain aspects, the antibody or bispecific antibody sequence variants retain the ability to interact with mesothelin, thereby disrupting, preventing, reducing, or inhibiting the binding interaction between mesothelin and one or more of its partners.

[0060] As used herein, a sequence "variant" refers to an antibody amino acid sequence comprising at least one amino acid insertion, deletion and / or substitution, wherein the resulting antibody maintains one or more of its functional characteristics as described herein. In embodiments, the sequence variant maintains the ability to specifically bind to mesothelin. Amino acid insertion variants are characterized by the insertion of one or more amino acids between two existing amino acids. Amino acid deletion variants are characterized by the deletion of one or more amino acids from the antibody sequence. Amino acid substitutions are characterized by the replacement of at least one amino acid in the sequence with another amino acid. In embodiments involving substitutions, the amino acid substitutions can be conservative substitutions (i.e., an amino acid from one amino acid family (acidic, basic, non-polar and uncharged, based on side chain characteristics, including size) is replaced with an amino acid from the same family).

[0061] In some aspects, the sequence identity between the variant antibody sequence and the antibodies disclosed herein will be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. "Sequence identity" refers to the percentage of amino acid residues that are identical to the sequences being compared. B. Labels, Conjugates, and Moieties

[0062] The binding proteins, antibodies, bispecific antibodies, or antigen-binding fragments thereof disclosed herein can be conjugated to therapeutic agents, solid supports, affinity agents, or detectable agents. For example, the amino acid sequences disclosed herein can be conjugated to markers for the purpose of diagnosis and other tests in which the amino acid sequence (i.e., antibody and / or its associated target) is detectable. Markers include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles (microparticles and nanoparticles of various materials (polymers, magnetic materials, etc.)), haptens, enzymes, peptides, radioisotopes, etc., and combinations thereof.

[0063] In some aspects, the antibody or bispecific antibody is conjugated to a fluorophore. The selection of the fluorophore connected to the antibody will determine the absorption and fluorescence emission properties of the conjugated antibody. The physical properties of the fluorophore labels that can be used for antibodies and antibody-binding partners may include, but are not limited to, spectral properties (absorption, emission, and Stokes shift), fluorescence intensity, lifespan, polarization, and photobleaching rate or a combination thereof. All of these physical properties can be used to distinguish between a fluorophore and another fluorophore, thereby allowing multiple analysis. Other desired properties of fluorescent markers can include cell permeability and low toxicity, for example, if labeling of antibodies is to be performed in cells or organisms (for example, living animals).

[0064] In some aspects, the conjugated label may comprise an enzyme. In some embodiments, an enzyme is an ideal label because it can obtain amplification of the detectable signal and lead to an increase in the sensitivity of the assay. The enzyme itself does not produce a detectable reaction, but when it comes into contact with a suitable substrate, it acts to decompose the substrate so that the converted substrate produces a fluorescent, colorimetric or luminescent signal. The enzyme can amplify the detectable signal because an enzyme on the labeling reagent can cause a variety of substrates to be converted into a detectable signal. The enzyme substrate is selected to produce a preferred measurable product, such as colorimetric, fluorescent or chemiluminescent. Such substrates are widely used in the art and are well known to those skilled in the art, and include, for example, oxidoreductases (e.g., horseradish peroxidase) and substrates (e.g., 3,3'-diaminobenzidine (DAB)); phosphatases, such as acid phosphatase, alkaline phosphatase, and substrates such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, such as β-galactosidase, β-glucuronidase or β-glucosidase, and substrates such as 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal); other enzymes include hydrolases (e.g., cholinesterase and peptidase), oxidases (e.g., glucose oxidase and cytochrome oxidase), and reductases for which suitable substrates are known.

[0065] Enzymes that produce chemiluminescence and suitable substrates thereof are suitable for some assays. These include, but are not limited to, natural and recombinant forms of luciferase and aequorin. In addition, substrates that produce chemiluminescence for phosphatases, glycosidases, and oxidases, such as substrates containing stable dioxetanes, luminol, isoluminol, and acridinium esters, are also useful.

[0066] On the other hand, haptens such as biotin can also be used as labels. Biotin is useful because it can function in enzyme systems to further amplify the detectable signal, and it can be used as a label for affinity chromatography for separation. For detection purposes, an enzyme conjugate with affinity for biotin, such as avidin-HRP, is used. A peroxidase substrate is then added to generate a detectable signal.

[0067] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides, and the like.

[0068] In some aspects, fluorescent proteins can be conjugated to antibodies as labels. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliproteins and their derivatives. Fluorescent proteins, particularly phycobiliproteins, are particularly useful for producing tandem dye-labeled labeling reagents. These tandem dyes include fluorescent proteins and fluorophores to obtain a large Stokes shift, where the emission spectrum is further shifted from the wavelength of the fluorescent protein's absorption spectrum.

[0069] In certain aspects, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine ( 121 I. 123 I. 125 I. 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 111 In, 112 In, 113 mIn、 115 mIn), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 135 Xe), fluorine ( 18 F), 153 SM, 177 Lu, 159 Gd,149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 186 Re、 188 Re、 142 Pr, 105 Rh and 97 Such as.

[0070] In some aspects, the drug can be conjugated to an antibody or a bispecific antibody. For example, a bispecific antibody can be conjugated to a therapeutic moiety or an agent, such as an antiproliferative drug, an anticancer drug, a cytotoxic drug, or an antiviral drug. In some embodiments, the antibody can be combined with a target antigen, and the drug (such as an anticancer drug) is used to inhibit, inactivate, or kill cancer cells. Therefore, any drug known in the art can be conjugated to an antibody or its fragment according to the present disclosure. In certain embodiments, drugs and other molecules can be conjugated to an antibody or a bispecific antibody by site-specific conjugation. C. Antibody-encoding polynucleotides and antibody-producing recombinant cells

[0071] The present disclosure provides methods for producing antibodies, bispecific antibodies and antibody fragments. In certain aspects, the present disclosure provides a recombinant method for generating antibodies and / or its fragments. In some embodiments, the recombinant nucleic acid encoding an antibody or bispecific antibody or its fragment can be operably connected to one or more regulatory nucleotide sequences in an expression construct. In some embodiments, the nucleic acid sequence encoding the antibody light chain and heavy chain can be cloned in the same expression vector in any direction (for example, the light chain is before the heavy chain or vice versa), or can be cloned in two different vectors. If a kind of vector is used for expression, the two coding genes can have their own genetic elements (for example, promoter, RBS, leader sequence, terminator sequence, polyA etc.), or they can be cloned with a single set of genetic elements, but connected to a cistron element. The regulatory nucleotide sequence can be suitable for the host cell for expression. A variety of suitable expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Generally, one or more regulatory nucleotide sequences can include but are not limited to promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences and enhancer or activator sequences. It is contemplated that any known constitutive or inducible promoter is used in conjunction with the aspects and embodiments included in this disclosure. The promoter can be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct can be present in an episome such as a plasmid in the cell, or the expression construct can be inserted into the chromosome.

[0072] In some aspects, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are known and can vary with the host cell used. In some aspects, the present disclosure relates to an expression vector comprising a nucleotide sequence encoding a polypeptide, which is operably linked to at least one regulatory sequence. Regulatory sequences are generally known and can be selected to direct the expression of the encoded polypeptide. Therefore, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary non-limiting regulatory sequences are described in Goeddel; "Gene Expression Technology: Methods in Enzymology", Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed (e.g., antibody or fragment thereof). In addition, the copy number of the vector, the ability to control the copy number and the expression of any other protein encoded by the vector, such as an antibiotic marker, may be considered.

[0073] In some embodiments relating to the method for producing antibodies, host cells can be transfected with one or more expression vectors encoding the antibody or its fragment (e.g., a single vector encoding heavy and light chains, or two vectors, one encoding heavy chain, one encoding light chain), and can be cultured under appropriate conditions to allow polypeptide expression to occur. The antibody or its fragment can be secreted and separated from cells and / or cell culture media containing the antibody or its fragment. Alternatively, the antibody can be retained in the cytoplasm or membrane fraction, and the cells harvested can be cracked and subsequently purified and separated. Cell culture includes host cells, culture media and other by-products. Any suitable culture media for cell culture can be used in the production method. Using the common techniques of purifying proteins, particularly antibodies, including, for example, ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and immunoaffinity purification, antibodies and antibody fragments can be separated from cell culture media, host cells or both. In some aspects, antibodies can be produced as fusion proteins containing a domain (e.g., a His tag) that can promote their purification.

[0074] Recombinant nucleic acid can be produced by connecting the cloned gene or its part to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects or mammals) or both. Expression vectors for producing recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells such as Escherichia coli. In some aspects, mammalian expression vectors contain prokaryotic sequences that promote vector propagation in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfecting eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to promote replication and drug resistance selection in prokaryotes and eukaryotic cells. Alternatively, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP derived and p205) can be used for transient expression of proteins in eukaryotic cells. Various methods for preparing plasmids and transforming host organisms are well known in the art. For other suitable expression systems for prokaryotes and eukaryotic cells, and general recombination methods, see Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some cases, it may be necessary to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (eg, pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (eg, pAcUW1), and pBlueBac-derived vectors (eg, pBlueBac III containing β-gal).

[0075] The technology of preparing fusion gene is well-known.In essence, the connection of the various nucleic acid fragments encoding different polypeptide sequences is carried out according to routine techniques, and flat end or staggered end are used to connect, and restriction enzyme digestion is to provide suitable end, suitably fills sticky end, and alkaline phosphatase is handled to avoid undesirable connection, and enzymatic connection.On the other hand, fusion gene can be synthesized by routine techniques, including automatic DNA synthesizer.Or, the pcr amplification of gene fragment can be carried out using anchor primer, and this anchor primer produces complementary overhang between two continuous nucleic acid fragments, and this overhang can be annealed to produce chimeric gene sequence subsequently (referring to, for example, " contemporary molecular biology experimental method " (Current Protocols in Molecular Biology) people such as Ausubel write, John Wiley&Sons:1992).

[0076] In some aspects, expression vectors expressing any of the above nucleic acids can be used to express antibodies or bispecific antibodies in host cells. For example, antibodies can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0077] In some aspects, the heavy chain and light chain of antibody or bispecific antibody are expressed from a single promoter. In some aspects, the heavy chain and light chain of antibody or bispecific antibody are expressed from multiple promoters. In some aspects, the heavy chain and light chain of antibody are encoded on a single vector. In some aspects, the heavy chain and light chain of antibody or bispecific antibody are encoded on multiple vectors.

[0078] Mammalian cell lines that can be used as hosts for expressing recombinant antibodies are well known in the art, including many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), human epithelial kidney 293 cells, and many other cell lines. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure correct modification and processing of expressed antibodies or portions thereof. For this reason, eukaryotic host cells with the cellular machinery for appropriately processing primary transcripts, glycosylation, and gene product phosphorylation can be used. Such mammalian host cells include, but are not limited to, CHO, HEK293, VERO, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030, and HsS78Bst cells.

[0079] In some aspects, antibodies, bispecific antibodies and antibody fragments of the present disclosure are stably expressed in cell lines. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing antibodies or bispecific antibody molecules can be generated. Host cells can be transformed with suitable engineered vectors comprising expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selectable marker genes. After importing exogenous DNA, cells are grown in a nutrient medium for 1-2 days and then transferred to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the plasmid to be stably integrated into the cells in its chromosome to grow and form foci, which can be cloned and expanded into cell lines. Methods for producing stable cell lines in high yield are well known in the art, and reagents are typically commercially available.

[0080] In certain aspects, the antibodies, bispecific antibodies, and antibody fragments of the present disclosure are transiently expressed in cell lines. Transient transfection is a method in which a nucleic acid introduced into a cell is not integrated into the genome or chromosomal DNA of the cell, but is maintained in the cell as an extrachromosomal element, e.g., as an episome. The transcription process of the episomal nucleic acid is not affected, and the protein encoded by the episomal nucleic acid is produced.

[0081] The cell lines of stable transfection or transient transfection are maintained in cell culture medium and conditions well known in the art, resulting in expression and production of monoclonal antibodies. In some aspects, mammalian cell culture medium is based on commercially available culture medium formulations, including, for example, DMEM or Ham's F12. In other aspects, modified cell culture medium is to support the increase of cell growth and biological protein expression. As used herein, the terms "cell culture medium," "culture medium," and "culture medium formulation" refer to nutrient solutions for maintaining, growing, propagating, or amplifying cells in an artificial in vitro environment outside a multicellular organism or tissue. Cell culture medium can be optimized for specific cell culture purposes, including, for example, a cell culture growth medium formulated to promote cell growth or a cell culture production medium formulated to promote the production of recombinant proteins. The terms nutrients, ingredients, and components are used interchangeably herein to refer to the components that constitute a cell culture medium.

[0082] Once the antibody or bispecific antibody is produced, it can be purified by any method known in the art for purifying immunoglobulin molecules or other multimeric molecules, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for the specific antigen Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins.

[0083] When using recombinant technology, antibodies, bispecific antibodies and antibody fragments can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the molecule is produced intracellularly, as a first step, particulate debris, host cells or cleavage fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology, 10:163-167 (1992) describe a method for separating antibodies that are secreted into the periplasmic space of Escherichia coli. When the molecule is secreted into the culture medium, commercially available protein concentration filters are usually first used, such as Amicon or Millipore Pellicon ultrafiltration devices to concentrate the supernatant from this expression system. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit protein hydrolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

[0084] The compositions prepared by cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis and / or affinity chromatography alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain (if present) in the molecule, and it will be understood by those skilled in the art that this. The matrix to which the affinity ligand is attached is typically agarose, but other matrices are also available. Compared with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times. Depending on the molecule to be recovered, other techniques of protein purification such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin chromatography, SEPHAROSE chromatography on anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also available.

[0085] Regardless of how the antibody, bispecific antibody or antibody fragment is purified, a binding assay can be performed (before and / or after purification) to confirm functional binding activity. For example, an ELISA assay can be used, including a double ELISA assay. In some aspects, a first antigen is coated in a well and bound to the antigen to immobilize the antibody. A tagged second antigen is added to the well and detected. Only antibodies that are fixed by binding to both the first antigen and the second antigen will be detected. In some aspects, the present disclosure provides recombinant cell lines that can be deposited and maintained by an international depositary authority (i.e., an international depositary authority IDA) authorized under the provisions of the Budapest Treaty. D. Pharmaceutical preparations

[0086] In some aspects, the disclosure provides pharmaceutical compositions. Such pharmaceutical compositions can also be compositions comprising antibodies, bispecific antibodies and / or antibody fragments disclosed herein and pharmaceutically acceptable excipients. In some aspects, the pharmaceutical compositions of the disclosure are used as medicines (i.e., in methods for treating or preventing diseases or conditions (e.g., cancer or its clinically relevant symptoms) in subjects requiring treatment or prophylactic treatment). In some embodiments, the pharmaceutical composition can be a composition comprising nucleic acid molecules encoding antibodies disclosed herein.

[0087] In some aspects, antibodies, bispecific antibodies (or nucleic acid molecules encoding antibodies) can be formulated into pharmaceutical compositions with pharmaceutically acceptable carriers, excipients or stabilizers. In some aspects, such pharmaceutical compositions are suitable for administration to humans or non-human mammals or animals via any one or more routes of administration using methods known in the art. The route of administration and / or mode will vary according to the desired results. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations can generally contain salts, buffers, preservatives, compatible carriers and optional other therapeutic agents. Such pharmaceutically acceptable preparations can also contain compatible solid or liquid fillers, diluents or encapsulated materials suitable for administration to humans. Other expected carriers, excipients and / or additives that can be used for preparations described herein include, for example, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counterions such as sodium, etc. These and other known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as described in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers can be selected to suit the mode of administration, solubility, and / or desired stability.

[0088] Preparations as described herein include an activating agent (i.e., one or more antibodies, bispecific antibodies, or fragments thereof disclosed herein) at a concentration that produces a w / v suitable for the desired dose. In some aspects, the concentration of the activating agent in the preparation is from about 1 mg / ml to about 200 mg / ml, from about 1 mg / ml to about 100 mg / ml, from about 1 mg / ml to about 50 mg / ml, or from about 1 mg / ml to about 25 mg / ml. In some aspects, the concentration of the activating agent in the preparation can vary between about 0.1%-about 75% by gross weight. In some aspects, the concentration range of the activating agent is 0.003-1.0 moles.

[0089] When used for in vivo administration, the preparation should be sterile. The preparation can be sterilized by various sterilization methods, including aseptic filtration, radiation, etc. In one aspect, the preparation is filter-sterilized using a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be prepared according to conventional pharmaceutical practices, such as described in "Remington: The Science & Practice of Pharmacy", 21st edition, Lippincott Williams & Wilkins, (2005).

[0090] Thus, in embodiments, pharmaceutical formulations as described herein encompass therapeutic compositions and can be formulated for specific routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0091] The preparation can be in unit dosage form and can be prepared by any known method. The actual dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain the amount of the active ingredient that is effective to achieve the desired therapeutic response for a specific patient, composition and mode of administration without being toxic to the patient (e.g., a "therapeutically effective amount"). The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition used, the route of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and previous medical history of the treated patient, and similar factors well known in the medical field. A suitable dosage can be about 0.0001-about 100 mg / kg body weight or greater, for example, about 0.1, 1, 10 or 50 mg / kg body weight, wherein about 1-about 10 mg / kg body weight is suitable.

[0092] In some embodiments of the present disclosure, the formulations may be suitable for diagnostic and research use. The concentration of the active agent in such formulations and the presence or absence of excipients and / or pyrogens may be modified or selected based on the specific application and intended use. E. Method

[0093] The antibodies, bispecific antibodies, and fragments thereof described herein can be used in methods for preventing, inhibiting, or reducing the activity and / or expression of mesothelin in a subject. In this regard, the antibodies, bispecific antibodies, and fragments thereof disclosed herein can provide a neutralizing effect against mesothelin. In some aspects, the antibodies or fragments disclosed herein can be used to treat a mesothelin-related condition, disease, or disorder in a subject in need of treatment. In some aspects, the antibodies, bispecific antibodies, and fragments disclosed herein can be used to prevent a mesothelin-related condition, disease, or disorder in a subject who may be at risk of developing a condition, disease, or disorder. In some aspects, the antibodies or bispecific antibodies can be used to treat a cancer associated with mesothelin activity and / or expression in a subject in need of treatment.

[0094] In some aspects, the present disclosure relates to methods for treating, preventing, diagnosing, or monitoring a condition, disease, or disorder characterized by mesothelin, which in some embodiments is cancer. In these aspects, the antibodies, bispecific antibodies, compositions, and methods described herein can be used to treat a subject having a mesothelin-associated condition, disease, or disorder (e.g., a cancer characterized by mesothelin expression and / or activity).

[0095] In an embodiment, the present disclosure provides a method of treating a disease comprising an antibody, bispecific antibody, or antigen-binding fragment thereof according to the present disclosure, which can provide effective treatment or "disease control" (DC). Disease control can be complete remission (CR), partial remission (PR), or stable disease (SD).

[0096] A "complete response" (CR) is the disappearance of all tumors or lesions (whether measurable or not) with no new tumors or lesions forming. This can be confirmed by repeated, serial assessments (e.g., at least a defined number of weeks (e.g., 4 weeks) from the date of the first record). The appearance of new, non-measurable tumors or lesions excludes a CR.

[0097] A "partial response" (PR) is a reduction in tumor burden of ≥50% relative to baseline. Confirmation can be obtained by serial repeated assessments (e.g., at least a defined number of weeks (e.g., 4 weeks) from the date of the first record).

[0098] "Progressive disease" (PD) is defined as an increase in tumor burden of ≥25% relative to the lowest recorded value (nadir). Confirmation can be obtained using serial repeated assessments (e.g., at least a defined number of weeks (e.g., 4 weeks) from the date of the first record). New, non-measurable lesions do not fall within the definition of PD.

[0099] "Stable disease" (SD) means that the criteria for CR, PR, or PD are not met. SD indicates that a 50% reduction in tumor burden relative to baseline and a 25% increase relative to the nadir cannot be established.

[0100] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In the aspects and embodiments disclosed herein, the disease is typically cancer, including, for example, a solid tumor cancer. In some embodiments, the cancer may include ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, gastroesophageal cancer, or mesothelioma.

[0101] In some embodiments of various aspects, the present disclosure relates to determining the presence, expression level and / or activity of mesothelin in a sample obtained from a subject who may have cancer, which can be used to identify patients who may respond to treatment with an antibody according to the present disclosure. In some embodiments, the assay may comprise detecting mRNA encoding mesothelin and may be used to identify patients who may respond to treatment with a mesothelin antibody or bispecific antibody. In some embodiments, the methods disclosed herein comprise detecting the amount and / or activity of mesothelin to determine the responsiveness of cancer to a mesothelin-based immunotherapy. In some further embodiments, the methods disclosed herein comprise detecting the amount and / or activity of mesothelin protein by contacting a sample (i.e., a patient sample) with an antibody, bispecific antibody, or antigen-binding fragment thereof according to the present disclosure, and detecting the presence (e.g., the amount and / or activity of mesothelin) in the sample. In such embodiments, the method can be used to assess or determine the responsiveness of cancer to an immunotherapy comprising a mesothelin antibody or bispecific antibody. The methods disclosed herein may comprise analysis of a blood sample, a biopsy, or an immunohistochemical technique applied to an individual tissue and / or tumor sample. In some embodiments of this aspect, a second biomarker associated with cancer can be detected simultaneously with or separately from the detection of mesothelin.

[0102] In one aspect, the present disclosure provides a method of treating cancer in a patient comprising administering to the patient an antibody, bispecific antibody, or antigen-binding fragment thereof as disclosed herein, wherein the patient in need of treatment is identified by detecting mesothelin in a sample obtained from the patient.

[0103] As used herein, "treating" refers to administering an antibody or antigen-binding fragment thereof as described herein or a composition to a subject to eliminate or alleviate the clinical signs of a condition, disease, or disorder; to prevent, inhibit, or slow the progression of a condition, disease, or disorder in a subject; and / or to reduce the number, frequency, or severity of clinical symptoms and / or recurrence of a condition, disease, or disorder (e.g., cancer and / or cancer recurrence) in a subject currently suffering from or previously suffering from a condition, disease, or disorder. Specifically, the term "treating a disease" (e.g., a disease associated with cell proliferation, cancer, etc.) includes halting, ameliorating, alleviating, shortening the duration, slowing or inhibiting progression or worsening, or delaying the onset of a disease or its symptoms in a subject suffering from the disease.

[0104] In some embodiments, the present disclosure provides methods for preventing a disease in a subject (i.e., preventing a condition, disease, or disorder, preventing the onset of a condition, disease, or disorder, or clinical symptoms of a disease). In some further embodiments of the method related to preventing a disease, the subject may be at risk of one or more mesothelin-related conditions, diseases, or disorders. "At risk" or having an "increased risk" means that the subject is identified as having a higher than normal chance of developing a condition, disease, or disorder and / or clinical symptoms associated therewith compared to the general population. In some embodiments, a subject who has had or currently has a condition, disease, or disorder, or is of a certain age, is a subject at increased risk of developing a condition, disease, or disorder. In some aspects, a subject who exhibits increased mesothelin expression or activity may also be considered to have an increased risk of developing a condition, disease, or disorder. In some embodiments, the subject may also be immunocompromised. As used herein, "immunocompromised" refers to a subject whose immune system is weakened or whose ability to fight infection or other disease is reduced due to a genetic disorder or disease, infection, environmental disorder or disease, or other environmental factors.

[0105] The term "immunotherapy" refers to a treatment involving a specific immune response. As used herein, the terms "protect," "prevent," "prophylactic," "preventative," or "protective" refer to preventing the onset and / or spread of a condition, disease, or disorder and its clinical symptoms in an individual, and in certain embodiments, to minimizing the chance of further clinical symptoms or disease progression in a subject. For example, as described above, a person at risk for a condition, disease, or disorder associated with mesothelin may be a candidate for prophylactic therapy to prevent such condition, disease, or disorder or its associated clinical symptoms.

[0106] In certain embodiments, prophylactic administration of immunotherapy, such as prophylactic administration of an antibody, bispecific antibody, or composition comprising the same disclosed herein, can protect a recipient from a condition, disease, or disorder associated with mesothelin, or reduce the risk of the recipient developing such a condition, disease, or disorder. In some alternative embodiments, prophylactic administration can reduce the chance that a recipient will develop an increased severity of a condition, disease, or disorder associated with mesothelin.

[0107] Therapeutic administration of immunotherapy, such as the antibodies, bispecific antibodies, or compositions comprising the antibodies, bispecific antibodies, or fragments thereof disclosed herein, can inhibit the progression of a condition, disease, or disorder and / or clinical symptoms associated with the condition, disease, or disorder. Such methods may include embodiments comprising reducing or inhibiting the expression of mesothelin, the activity of mesothelin, and / or the total amount of cells associated with mesothelin expression, thereby preferably eliminating the condition, disease, or disorder and associated clinical symptoms.

[0108] In certain aspects, the present disclosure provides treatment methods comprising administering a mesothelin antibody, bispecific antibody, or antigen-binding fragment thereof in combination or conjunction with other cancer therapies.

[0109] The terms "subject," "individual," or "patient" are interchangeable and refer to vertebrates, preferably mammals. For example, in the context of the present disclosure, mammals are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals such as animals in zoos. The term "animal" as used herein also includes humans. The term "subject" may also include patients, i.e., animals, and in certain embodiments, humans suffering from a disease associated with mesothelin. A subject may also include a patient at risk of suffering from a condition, disease, or disorder associated with mesothelin. In certain embodiments, the subject, individual, or patient is a human.

[0110] The methods comprising the antibodies or binding fragments thereof described herein and compositions comprising the antibodies or binding fragments thereof can be administered by any conventional route, including by injection or infusion, orally, buccally, sublingually, transdermally, intraocularly, intranasally, by aerosol, by implant or depot, or intrarectally. In some embodiments, administration can be, for example, by injection, e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally.

[0111] The antibody, bispecific antibody or its fragment and the composition comprising them are administered in an effective amount. An "effective amount" includes an amount to achieve a desired reaction or desired effect, and can be in the form of a single dose or multiple doses. In the case of treating a specific disease or a specific clinical condition, the expected response refers to suppressing the progression of the disease or symptom. This suppression can include slowing down the progression of the disease / clinical symptoms, and in some embodiments, can also include interrupting or reversing the progression of the disease / clinical symptoms. The treatment of a disease or condition can also be to delay or prevent the onset of the disease or condition in a subject suffering from an active disease (e.g., cancer).

[0112] The effective amount of the composition of the present invention depends on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, body shape and weight), the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Therefore, the dose of the composition of the present invention administered can depend on various combinations of such parameters. In embodiments where the initial amount administered to the patient is insufficient, further administration of higher amounts, more frequent doses, or different / more localized routes of administration may be used.

[0113] According to the above aspects, in some embodiments, the present disclosure relates to a method of monitoring a condition, disease, or disorder associated with mesothelin in a subject, wherein the method comprises detecting and / or determining or monitoring the level of (i) mesothelin nucleic acid, (ii) a mesothelin antigen or portion thereof, (iii) an antibody to mesothelin or portion thereof, and / or (iv) cells comprising mesothelin in a biological sample isolated from the patient. In some further embodiments, the condition, disease, or disorder associated with mesothelin comprises cancer.

[0114] According to the above aspects, in some embodiments, the present disclosure relates to a method for diagnosing a condition, disease, or disorder characterized by mesothelin expression. In some embodiments, the method comprises detecting and / or determining the amount of (i) mesothelin nucleic acid, (ii) a mesothelin antigen or portion thereof, (iii) an antibody to mesothelin or portion thereof, and / or (iv) cells containing mesothelin in a biological sample isolated from a patient. In some further embodiments, the condition, disease, or disorder associated with mesothelin expression comprises cancer.

[0115] In some embodiments of the above aspects, the detecting comprises: (i) contacting a biological sample with an antibody, bispecific antibody, or antigen-binding fragment thereof that specifically binds to mesothelin or a portion thereof, an antibody to mesothelin or a portion thereof, and / or a cell that expresses at least a portion of a mesothelin antigen as disclosed herein; and (ii) detecting the formation of a complex between the antibody, or antigen-binding fragment thereof, and mesothelin or a portion thereof, an antibody to mesothelin or a portion thereof, and / or a cell that expresses at least a portion of a mesothelin antigen. In some embodiments, the biological sample isolated from the patient is compared to a reference sample (e.g., a comparable biological sample obtained from a healthy subject with normal mesothelin expression levels).

[0116] In another aspect, the present disclosure relates to a method for determining the regression, course, or onset of a disease characterized by mesothelin. In some embodiments, the disease can be identified according to a variety of methods provided herein. In some embodiments, the method comprises monitoring one or more parameters of a sample from a patient suffering from or suspected of suffering from the disease, the parameter being selected from the group consisting of: (i) the amount of nucleic acid associated with mesothelin, (ii) the amount of expressed mesothelin antigen or portion thereof, (iii) the amount of antibodies against the mesothelin antigen or portion thereof, and / or (iv) the amount of cells associated with mesothelin expression. In some embodiments, the method comprises determining the parameter in a first sample at a first time point and determining the parameter in another sample at a second time point, wherein the progression of the disease is determined by comparing the two samples.

[0117] The term "sample" may include any sample that is useful or usable according to the methods described herein. In some embodiments, the sample may be a biological sample, such as a sample from a tissue, which may include, for example, a body fluid and / or cell sample that can be obtained by any conventional method (e.g., by tumor / tissue biopsy, collection of blood, bronchial aspirate, sputum, urine, feces, or other body fluids). In some embodiments, the term "sample" may include processed samples, such as fractions or isolates of biological samples, such as nucleic acid and peptide / protein isolates.

[0118] According to some embodiments, one or more antibodies, bispecific antibodies, constructs or fragments described herein can be used to detect mesothelin antigen or cells expressing mesothelin antigen or fragments thereof, or to determine or monitor the amount of mesothelin antigen or cells associated with at least a portion of a mesothelin antigen. Methods of detection comprising incorporating an antibody, bispecific antibody or fragment thereof disclosed herein can include a detectable label. In some embodiments, the detectable label or marker is a fluorescent marker, a colorimetric marker, a radioactive marker or an enzyme marker, or other markers disclosed herein or known in the art.

[0119] As described above, the methods provided herein can reduce, slow, or halt tumor growth, and in certain aspects, such reduction or slowing can be statistically significant. The reduction in tumor growth can be measured by comparing the patient's baseline tumor growth, the expected tumor growth, the expected tumor growth based on a large patient population, or the tumor growth of a control population.

[0120] In certain aspects, administration of a mesothelin antibody, bispecific antibody, or antigen-binding fragment thereof can improve overall survival (OS). In other aspects, administration of a mesothelin antibody, bispecific antibody, or antigen-binding fragment thereof can achieve stable disease (SD). F. Kit

[0121] Another aspect of the present disclosure is a kit. On the one hand, the kit comprises any sequence, compound and pharmaceutical preparation or composition or pharmaceutical composition of nucleic acid, polypeptide, expression vector or host cell as generally described above, and instructions or labels for appropriate use or administration. Optionally, the kit may also include one or more container reagents, reactants and / or syringes or other devices for delivery or use. The present disclosure contemplates that all or any subset of components for conducting research tests, diagnostic tests and / or for administering a therapeutically effective amount may be included in the kit. Similarly, the kit may include instructions for preparing amino acid sequences / polypeptides, such as by culturing host cells expressing nucleic acids encoding antibodies of the present disclosure (or their constructs or antigen-binding fragments) under suitable conditions. As another example, a kit for therapeutic administration of antibodies of the present disclosure may include a solution of a pharmaceutical preparation or a lyophilized formulation containing the antibody, and instructions for administering the composition to patients in need and / or for reconstructing a lyophilized product.

[0122] The present disclosure also includes finished packaged and labeled pharmaceutical products. The product includes a suitable unit dosage form in a suitable bottle or container, such as a glass vial or other hermetically sealed container. In the case of a dosage form suitable for parenteral administration, the active ingredient, such as the above-mentioned antibody, is sterile and suitable for administration as a particle-free solution. In some aspects, the formulation is suitable for intravenous administration, such as intravenous infusion to humans or animals.

[0123] In a specific aspect, the formulations of the present disclosure are formulated as sterile liquids in single-dose vials. Exemplary containers include, but are not limited to, vials, bottles, prefilled syringes, IV bags, and blister packs (containing one or more pills). Optionally, associated with such containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, reflecting approval of the manufacture, use, or sale agency for human diagnosis and / or administration.

[0124] As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transport. Furthermore, the products of the present disclosure include instructions for use or other informational materials that inform the physician, technician, or patient how to appropriately prevent or treat the disease or condition in question. In other words, the article of manufacture includes instructions that describe or suggest a dosing regimen, including, but not limited to, actual dosages, monitoring procedures, and other monitoring information.

[0125] The kit for diagnostic testing can include a solution containing an antibody or antigen-binding fragment thereof of the present disclosure, or a lyophilized formulation of an antibody or fragment thereof, wherein the antibody or fragment specifically binds to one or more targets, and reagents for detecting such antibodies. The antibodies can be labeled according to methods known in the art and described herein, including but not limited to markers such as small molecule fluorescent tags, proteins such as biotin, GFP or other fluorescent proteins, or epitope sequences such as his or myc. Similarly, a primary antibody for detecting the antibody can be included in the kit. The primary antibody can be directed against a sequence on the antibody or against a marker, tag or epitope of the labeled antibody. The primary antibody can be labeled for detection in turn, or, if further amplification of the signal is desired, the primary antibody can be detected by a secondary antibody, which can also be included in the kit.

[0126] Kits for research use are also contemplated. For example, such a kit might be similar to a kit intended for diagnostic or therapeutic use, but also include a label specifying that the kit and its use are for research purposes only. G. Modification and / or Engineering

[0127] Another aspect of the present disclosure includes modification and / or engineering of antibodies, bispecific antibodies and / or their antigen-binding fragments as described herein. For example, antibodies, bispecific antibodies and / or their antigen-binding fragments include but are not limited to chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, double antibodies, multispecific antibodies, dual-specific antibodies (dual-specific antibody) and bispecific antibodies. Non-limiting embodiments of such constructs and antibody forms are described in the examples and throughout this disclosure. Disclosed antibodies, bispecific antibodies and their antigen-binding fragments can also be used to generate fusion proteins and / or for antibody-targeted cell fusion, such as fusion with immune fusion partners and cells.

[0128] The disclosed antibodies, bispecific antibodies, and antigen-binding fragments thereof can also be used to produce therapeutic immunoconjugates, wherein the disclosed antibodies, bispecific antibodies, or antigen-binding fragments thereof are conjugated to one or more therapeutic agents. Non-limiting embodiments of such immunoconjugates are described in the present disclosure. In addition, for example, the disclosed antibodies, bispecific antibodies, or antigen-binding fragments thereof can be used to produce antibody-drug conjugates (ADCs). Useful drugs include, but are not limited to, cell proliferation inhibitors, anticancer agents, agents that induce apoptosis, and combinations thereof.

[0129] As discussed herein, unless otherwise indicated, the practice of the methods disclosed herein employs available techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology familiar to those skilled in the art. Such techniques are fully explained in the literature, for example, in "Molecular Cloning: A Laboratory Manual," Second Edition; "Oligonucleotide Synthesis"; "Animal Cell Culture"; "Methods in Enzymology"; "Handbook of Experimental Immunology"; "Gene Transfer Vectors for Mammalian Cells"; "Current Protocols in Molecular Biology"; "PCR: The Polymerase Chain Reaction"; "Current Protocols in Immunology."

[0130] The following examples illustrate some of the above aspects and embodiments but are not intended to limit the scope of the claimed invention. Example Example 1. Affinity maturation of the m912 antibody by phage display

[0131] We began affinity maturation by defining the CDR sequences of the m912 variable region. We submitted the VH and VL sequences of m912 (Table 1) to the ProABC-2 web server for prediction. ProABC-2 uses machine learning methods to predict the paratope of antibodies. The probability of residues contacting the antigen through hydrogen bonds, hydrophobic interactions, or other non-bonded interactions is calculated and visualized. Figure 1 The recently solved structure of m912 confirmed the accuracy of the predicted CDR loops. Table 1. VH and VL sequences of MSLN antibody m912

[0132] A comprehensive affinity maturation process was initiated by random mutagenesis of the predicted CDRs in the variable region of m912. A total of 16 libraries were constructed, of which 8 were HCDRs and 8 were LCDRs (Table 2). The number of libraries for a single CDR depends on the length of the predicted CDR. The longer the CDR, the more libraries that can be specified. Each library typically covers 4-5 residues. If a CDR requires two or more libraries to cover, the libraries are combined for panning. Given that HCDR3 plays a very important role in the interaction with its cognate antigen, we focused on HCDR3 by constructing high-density overlapping libraries. For HCDR3, we constructed three overlapping libraries covering 6 predicted residues (Table 2) and panned them separately. Based on the panning results, additional libraries can be constructed to obtain the best consensus sequence. Table 2. Library construction (oligonucleotides) for affinity maturation of the m912 antibody.

[0133] A pair of 5' phosphorylated oligonucleotides were used to construct the library and the entire plasmid was amplified (Table 2). The forward primer was synthesized as a degenerate oligonucleotide containing 4-5 NNS codons to randomly mutate the target sequence. PCR was performed using Q5 high-fidelity DNA polymerase (New England BioLabs, M0491L) for 30 cycles. The PCR product was digested with DpnI to remove the template plasmid DNA and then extracted from a 0.8% agarose gel (ThermoFisher, G501808). The purified PCR product was then self-ligated and electroporated into TG1 electrocompetent cells (Lucigen, 60502). The coverage of each library was at least 100 times the computational complexity. For example, for a library covering 4 residues, the targeting complexity should be >100x 20^4 (i.e., 1.6x 10^7).

[0134] After electroporation, TG1 cells were spread onto large panning plates (ThermoFisher, 240845) and grown overnight in a 30°C incubator. The next day, the bacterial lawn was scraped and resuspended in 10 mL of 2YT medium supplemented with 100 mg / mL carbenicillin and 15% glycerol. A small amount of the bacterial suspension was inoculated into 100 mL of 2YT medium supplemented with carbenicillin and 2% glucose until the OD reached 0. 600 The culture was incubated at 37°C with shaking at 250 rpm until the OD 600The pH value reached ~0.5. Bacteria in the logarithmic growth phase were infected with M13KO7 helper phage (ThermoFisher, 18311019) and then cultured overnight at 30°C in the presence of kanamycin and carbenicillin. The phage library was precipitated from the collected supernatant with 1 / 5 volume of PEG solution (containing 20% ​​PEG-8000 and 2.5M NaCl).

[0135] Panning was performed in solution. Biotinylated mesothelin (Sino Biological, 13128-H01H-B50-B) was incubated with M-280 streptavidin magnetic Dynabeads (ThermoFisher, 1 1205D) and the phage library. The panning process was performed for two or three rounds, during which the mesothelin concentration was gradually reduced. The mesothelin concentration used in the first round of panning started at 20nM, and subsequent rounds used 1 / 10 of the mesothelin concentration in the previous round. Unbound phage was washed away with PBST (PBS solution containing 0.1% Tween-20). Bound phage was eluted with 0.1N HCl and rescued using logarithmic phase TG1 cells. TG1 cells were then plated at an appropriate density onto 2YT agar plates containing 100mg / mL carbenicillin and 2% glucose to identify and sequence individual clones.

[0136] Figure 2 The results of panning for the m912 project using WebLogo are summarized. Panning was performed on HCDR1, HCDR3, LCDR1, and LCDR3, rather than on HCDR2 or LCDR2, resulting in sequence enrichment and consensus sequences. For CDRs covered by two or more libraries, panning was performed by combining the libraries except for HCDR3. The three HCDR3 libraries were panned separately. From the HP3-1 panning, the negatively charged aspartic acid (D) was enriched at positions 1 and 2, while the hydrophobic residues valine (V) and leucine (L) were enriched at positions 3 and 4 ( Figure 2 A). This pattern was retained during the HP3-2 panning process even when glycine (G) at the fifth position was replaced by threonine (T) or serine (S) ( Figure 2 B). For HP3-3 panning, glutamate (E) and glycine (G) are enriched at positions 5 and 6 ( Figure 2 C). Based on the panning results, we can infer that position 1 of HCDR3 prefers negatively charged residues, while position 6 prefers glycine over alanine. Given that HCDR3 contains only 6 residues, a fourth library (HP3-4) was constructed by assigning a negatively charged residue to the first position and randomizing the last five residues (Table 1). After three rounds of panning, a consensus sequence was generated in which glycine (G) was indeed enriched primarily at position 6, while positions 2-5 appeared to be less restrictive ( Figure 2 D) We have thoroughly examined every position of HCDR3 using the HP3-4 library. Therefore, we are confident that the sequences enriched from the HP3-4 library should contain the optimal sequence of HCDR3.

[0137] According to the IMGT definition, LCDR3 is predicted to be the sequence from residue 89 to residue 98 in m912 VL. However, LCDR3 may be at least two amino acids shorter than the prediction based on proABC-2 ( Figure 1 B). To avoid potential off-target effects, we constructed four libraries (LP3-1, LP3-2, LP-3, and LP3-4) to cover the ten residues of generalized LCDR3, with four residues randomly assigned to each library. Through panning, we found that LP3-3 was mainly enriched from the pool of four libraries and generated a consensus sequence of DAXY ( Figure 2 G). The panning results indicated that the STPL sequence targeted by the LP3-3 library was likely part of a mutagenic hotspot. To explore whether the two amino acids before STPL could be mutated, we constructed a fifth library (LP3-5) by randomizing the SYSTPL sequence, but only allowing tyrosine (Y) or phenylalanine (F) at the position of tyrosine (Y). Panning of this extended library did not change the consensus sequence of DAXY; however, position 1 was more prone to proline (P) ( Figure 2 H). These results support the predictions of proABC-2 and demonstrate the value of this tool in CDR prediction.

[0138] To rank candidate sequences from each panning, we prepared soluble scFvs for ELISA analysis. Soluble scFvs were prepared by seeding individual clones into 100 ml of 2YT+carbenicillin+2% glucose medium in a 96-well plate. To evaluate the binding ability of the candidate compared to the parental m912 antibody, we also seeded the three parental clones on the same plate. The plate was shaken overnight at 37°C at 250 rpm. The next morning, 5 mL of the overnight culture was transferred to a new 96-well plate containing 100 mL of fresh 2YT+carbenicillin medium. After incubation at 37°C with shaking for 2 hours, an additional 50 mL of 2YT+carbenicillin and 3 mM IPTG were added to the plate, and incubation at 37°C with shaking continued for 3-4 hours. Soluble scFv was released from the periplasm by adding 50 mL of 2YT carbenicillin medium containing 1000 units of polymyxin B sulfate (Sigma, P0972) and incubating with shaking for another 1 hour at 37°C. The culture plate was centrifuged and the supernatant containing the released scFv was used for ELISA and Western blot analysis.

[0139] ELISA screening was performed as follows: recombinant mesothelin (PeproTech, 100-63) was coated on Immulon 4HBX plates (ThermoFisher, 3855) at 1 mg / mL in PBS overnight at 4°C. The plates were blocked with 4% milk powder dissolved in PBST (PBS + 0.1% Tween) and then incubated with 1:1 diluted scFv supernatant for 1 hour at room temperature. Bound scFv was detected using 1 mg / mL anti-myc antibody (ThermoFisher, MA1-980) and 0.5 mg / mL anti-mouse Fc secondary antibody (R&D Systems, HAF007). The plates were washed three times with PBST between each incubation step. The plates were developed by adding one-step UltraTMB-ELISA substrate (ThermoFisher, 34029). The color reaction was stopped by adding 2M sulfuric acid. The OD was read on a SpectraMax M3 microplate reader (Molecular Devices, CA, USA). 405 value.

[0140] Although most clones screened by scFv ELISA showed better binding than the parental m912, we usually selected 10-20 clones for further validation. The scFv ELISA assay was performed by adding 1:2 serial dilutions of scFv supernatant onto mesothelin-coated plates. The m912 scFv supernatant was used as a control. We also performed Western blotting experiments to detect scFv at the protein level. Soluble scFv was detected using c-Myc monoclonal antibody (9E10) (ThermoFisher, 13-2500). We gave priority to clones with stronger binding signals in scFv ELISA and weaker signals in Western blotting experiments. Figure 3 As shown, we screened 4 clones from 20 HCDR1 candidate clones ( Figure 3 A-3C), 3 clones were screened from 14 HCDR3 candidate clones ( Figure 3 D-3F), and one clone was selected from seven LCDR1 candidate clones ( Figure 3 G and 3H), and two clones were selected from 12 LCDR3 candidate clones ( Figure 3 1 and 3J) for the preparation of combination antibodies. In addition, we also screened out two clones with low to medium binding activity from HCDR3 panning. The sequences of the selected clones are listed in Table 3 for further evaluation. Table 3. Beneficial mutations identified by panning and soluble scFv ELISA. Example 2. Incorporation of beneficial CDR mutations into IgG

[0141] The CDR sequences selected in Table 3 were subcloned into corresponding expression vectors and reintegrated into the heavy and light chains of IgG. HP1-A13 and HP4-44 were selected to produce antibodies with previously determined nanomolar affinity. In order to produce antibodies with higher affinity, four best clones (HCDR1-2, HCDR1-3, HCDR1-9, and HCDR1-14) from the HCDR1 library were combined with three best clones (HCDR3-6, HCDR3-7, and HCDR3-8) from the HCDR3 library by overlapping PCR to generate 12 HCDR combination constructs. Similarly, two clones (LCDR3-2 and LP5-1) from the LCDR3 library were combined with a clone (LCDR1-6) from the LCDR1 library by overlapping PCR to generate two LCDR combination constructs. The 12 HCDR combination constructs were then co-transfected with two LCDR3 constructs and two LCDR1 / LCDR3 combination constructs to generate 48 combination antibodies (Table 4). Transfection was performed using the PEI transfection method in a 24-deep-well plate (Axygen, P-DW-10ML-24-C) containing 2.5 mL of 293F cells (ThermoFisher, R79007). Example 3. Screening for higher affinity binders based on kinetic analysis of a single antigen concentration

[0142] The culture medium on day 6 post-transfection was harvested by centrifugation and its expression titer was determined using Octet HTX (Forte Biosciences, TX, USA) (Table 3). To quickly scan the binding spectrum of these antibodies, we performed kinetic analysis in Octet using a single concentration of 125 nM mesothelin. The kinetic experiment first used an anti-human Fc (AHC) biosensor to capture the antibody, followed by two baseline steps of 30 seconds each in kinetic buffer. The biosensor with captured antibodies was then immersed in a well containing 125 nM mesothelin for 5 minutes, followed by dissociation in kinetic buffer for 5 minutes. The parent antibody m912 and two HCDR3 antibodies HP1-A13 and HP4-44 were included in the experiment. Figure 4 As shown, the off-rates of most antibodies were enhanced compared to the parental antibody m912. Example 4. Testing for non-specific binding on MSLN negative cell lines

[0143] To investigate whether these antibodies have nonspecific binding, we performed flow cytometric analysis on the MSLN-negative cell line NCI-H929 (ATCC, CRL-9068) using an Attune NxT flow cytometer (ThermoFisher). H929 cells were harvested from suspension cultures and blocked with 1% FBS in PBS for 1 hour on ice. Approximately 3x10^5 cells were incubated with 5 mg / mL antibodies on ice for 1 hour and then stained with Alexa Fluor 500. The cells were washed and stained with 647 anti-human Fc antibody (ThermoFisher, A21445) at a dilution of 1:1000 for 30 minutes. After washing, the cells were stained with SYTOX Green (ThermoFisher, S7020) at a dilution of 1:50000 and analyzed by flow cytometry. The negative response of H929 cells to mesothelin was confirmed using HP4-44 antibody, which positively stained OVCAR3 cells (ATCC, HTB-161) ( Figure 5 A), but negative staining for H929 cells ( Figure 5 B). Ranking of nonspecific binding of 48 combinatorial antibodies and parental m912 and HP4-44 to H929 cells using peak x-mean ( Figure 5 C). Although many antibodies did not bind to H929 cells or had low binding levels, a considerable number of antibodies showed varying degrees of binding to H929 cells, especially those containing mutations from HCDR1-14 and HCDR3-8 ( Figure 5 B and 5C, Table 4). Nonspecific binding can be due to sticky paratopes and / or aggregation properties of the antibody. Nevertheless, nonspecific binding screening helps us to quickly exclude low-quality antibodies from our combined antibody pool. Example 5. Conversion to IgG-[L]-scFv CO3 Bispecific formats

[0144] A total of 11 combination antibodies were screened and converted into IgG-[L]-scFv CD3 Bispecific format (Table 5). Santich et al. (2020) first reported the IgG-[L]-scFv format, in which the GD2 antibody fused to the C-terminus of the light chain with huOKT3-scFv showed excellent anti-tumor activity both in vitro and in vivo in mice

[11] . In this bispecific format, the N297G and K322A double mutations were introduced at the Fc terminus to eliminate the binding activity of Fc receptors and complement, and the light chain was connected to huOKT3-scFv using a 3xG4S linker. Parent m912 and two antibodies with low to medium affinity (HP1-A13 and HP4-44) from HCDR3 panning were also converted (Table 5).

[0145] The resulting constructs were transfected into 125 mL 293F cultures and their expression titers were determined on day 6 after PEI transfection (Table 6). Table 6. MSLN-[L]-scFv expressed in 125 ml freestyle 293f cells CD3 The titer was determined using Octet. bsAb Titer (mg / L) <![CDATA[m912-[L]-scFv CD3 ]]> 36.4 <![CDATA[HP1-A13-scFv CD3 ]]> 43.3 <![CDATA[HP4-44-[L]-scFv CD3 ]]> 39.2 <![CDATA[MSLN-4-[L]-scFv CD3 ]]> 73.2 <![CDATA[MSLN-21-[L]-scFv CD3 ]]> 16.8 <![CDATA[MSLN-28-[L]-scFv CD3 ]]> 50.1 <![CDATA[MSLN-30-[L]-scFv CD3 ]]> 30.5 <![CDATA[MSLN-31-[L]-scFv CD3 ]]> 38.0 <![CDATA[MSLN-32-[L]-scFv CD3 ]]> 36.5 <![CDATA[MSLN-33-[L]-scFv CD3 ]]> 46.4 <![CDATA[MSLN-39-[L]-scFv CD3 ]]> 8.1 <![CDATA[MSLN-40-[L]-scFv CD3 ]]> 36.4 <![CDATA[MSLN-42-[L]-scFv CD3 ]]> 12.2 <![CDATA[MSLN-45-[L]-scFv CD3 ]]> 16.6 Example 6. MSLN-[L]-scFv CD3 Binding affinity of bsAb

[0146] MSLN-[L]-scFv was evaluated using Octet as described above. CD3 The binding affinity of bsAb. Figure 6 As shown in Figure 5, the affinity of m912 is too low to be estimated. Using a 1:1 binding model, the dissociation constants (KD) of HP1-A13 bsAb and HP4-44 bsAb were estimated to be 28.7 nM and 2.9 nM, respectively. The KD of MSLN-30, MSLN-39, and MSLN-42 bsAbs were D The estimated concentration was 1-2 pM (Table 7), but these estimates may not be accurate due to the detection limit of Octet. Therefore, we measured one of the leads (MSLN-39) in IgG form by surface plasmon resonance (SPR) using Biacore 3000 (GE Healthcare). The MSLN-39 antibody was immobilized on the sensor chip via the Fc antibody, and then mesothelin was added to the sensor chip in five concentrations ranging from 0.625 nM to 10 nM. The data were collected and fitted globally using a four-parameter nonlinear curve fitting model. Figure 7 As shown, the K of MSLN-39IgG was measured. D It is 56.4pM. Table 7. Selected MSLN-[L]-scFvs by Octet CD3 Summary of parameters for bsAb kinetic experiments. BsAb KD(M) Ka(1 / Ms) Kd(1 / s) <![CDATA[m912-[L]-scFv CD3 ]]> NA NA NA <![CDATA[HP1-A13-[L]-scFv CD3 ]]> 2.87E-08 1.04E+05 2.97E-03 <![CDATA[HP4-44-[L]-scFv CD3 ]]> 2.91E-09 2.78E+05 8.09E-04 <![CDATA[MSLN-30-[L]-scFv CD3 ]]> 1.71E-12 2.21E+05 2.44E-07 <![CDATA[MSLN-31-[L]-scFv CD3 ]]> 1.18E-12 2.58E+05 3.03E-07 <![CDATA[MSLN-39-[L]-scFv CD3 ]]> 1.07E-12 3.79E+05 4.05E-07 <![CDATA[MSLN-42-[L]-scFv CD3 ]]> 1.12E-12 1.75E+05 1.96E-07 (NA)m912-[L]-scFv CD3 The signal from the bsAb was too weak to determine the binding affinity. Example 7. MSLN-[L]-scFv CD3 Cell surface binding of bsAb

[0147] Evaluation of MSLN-[L]-scFv by flow cytometry CD3 Surface binding of bsAbs to the mesothelin-positive cell line OVCAR3. Again, all our bsAbs specifically bound to OVCAR3 cells but not to control H929 cells ( Figure 8 A and 8B). Binding was dose-dependent ( Figure 8 C), all bsAbs tested showed better binding than the parental m912 bsAb (EC50 of 572 ng / mL). HP1-A13 bsAb with a KD of 28.7 nM showed intermediate binding, EC50 of 572 ng / mL. 50 Other affinity-enhanced bsAbs (including HP4-44bsAb) showed better binding to OVCAR3 cells, with EC 50 The range was between 9.6 and 15.7 ng / mL (Table 8). We also measured the binding of bsAb to Jurkat cells ( Figure 8 D) As expected, the overall binding of our bsAbs to CD3 was similar to each other but much weaker than the binding to MSLN (Table 8). Table 8. MSLN-[L]-scFv by assay CD3 Binding parameters of bsAbs to the surface of OVCAR3 and Jurkat cells. Example 8. MSLN-[L]-scFv CD3 bsAb binds to mesothelin and CD3 simultaneously

[0148] To detect MSLN-[L]-scFv CD3 To detect the simultaneous binding of bsAb to mesothelin and CD3, we developed a sandwich ELISA assay in which CD3e and CD3d heterodimers (ACRO Biosystems, Cat. No. CDD-H52Wa) were coated on a plate to capture MSLN-[L]-scFv with a CD3 arm. CD3bsAb. By adding biotinylated MSLN (with an Avi tag from R&D Systems, Cat. No. AVI10679-050), we were able to detect simultaneous binding of MSLN to the MSLN arm using HRP-conjugated streptavidin (Jackson Immuno Research, Cat. No. 016-030-084). Two MSLN-[L]-scFvs were tested in the assay. CD3 bsAb samples (HP4-44 and MSLN-39) showed simultaneous antigen binding with similar EC 50 ( Figure 9 ). Example 9. MSLN-[L]-scFv CD3 In vitro T cell activation with bsAbs

[0149] In order to study MSLN-[L]-scFv CD3 To investigate the activation of MSLN-specific T cells by bsAbs, we used a genetically engineered Jurkat cell line that expresses a luciferase reporter gene driven by an NFAT response element. When these cells are exposed to anti-TCR / CD3 stimulators, receptor-mediated signaling induces luminescence through activation of nuclear transcription factors. The assay was performed by transfecting Jurkat cells with MSLN-[L]-scFv CD3 The bsAbs were incubated in the absence and presence of OVCAR3 cells or Capan-2 cells. Figure 10 As shown, MSLN-[L]-scFv CD3 The bsAb significantly induced T cell activation in a dose-dependent manner ( Figure 10 A and 10C), whereas almost no activation was observed in the absence of target cells ( Figure 10 B and 10D). The potency of the bsAb we observed appears to correlate well with its affinity. K of the m912 bsAb D The KD of HP1-A13 bsAb was 28.7 nM, showing moderate activity, and the EC of HP1-A13 bsAb against Capan-2 and OVCAR3 was 72 nM, showing low activity against T cells. 50 The affinity of HP4-44 bsAb is about 3nM, and the EC 50 The affinity was significantly increased to 10 ng / mL (Table 9). 50 There was almost no improvement. On the contrary, B max increased significantly ( Figure 10A and 10C). One possible explanation is that the accessibility of the epitope on the target cell is limited, which may lead to saturated binding. The increased Bmax of T cell activation may be due to an increased dissociation constant. Our bispecific molecules can maintain T cells and target cells for longer periods of time, thereby improving maximum activation. Table 9. MSLN-[L]-scFv measured by T cell activation assay CD3 Efficacy of bsAbs. Example 10. Analysis of MSLN-[L]-scFv using KILR cytotoxicity assay CD3 bsAb

[0150] The same panel of MSLN-[L]-scFv was further analyzed by KILR cytotoxicity assay CD3 bsAb, in which target Capan-2 / KILR cells are incubated with PBMC in the presence of our bsAb. The target Capan-2 / KILR cell line is generated by stably transfecting the KILR gene expressing an ePL-tagged reporter protein (DiscoverX). After the cells are killed, the released e-PL will emit light in the presence of the KILR detection reagent. Figure 11 As shown, HP4-44, MSLN-30, MSLN-31, MSLN-33, MSLN-39, and MSLN-42 bsAbs showed similar potency, EC 50 EC close to T cell activation assay 50 (Table 9) HP1-A13 bsAb had low potency in T cell activation assay and KILR assay, EC 50 The EC of m912 in the KILR test was medium. 50 It appears to be better than in the T cell activation assay. Experiments performed using PBMCs from two donors produced comparable results (Table 10). Table 10. MSLN-[L]-scFv measured by KILR cytotoxicity assay CD3 Efficacy of bsAbs. Example 11. Analysis of MSLN-[L]-seFv using a fluorescence-based cytotoxicity assay CD3 bsAb

[0151] MSLN-[L]-scFv was also evaluated CD3Cytotoxicity of bsAb on the gastric cancer cell line NCI-N87, which constitutively expresses firefly luciferase. T cell-directed killing was measured by quantifying target cell viability, which is directly correlated with luciferase activity

[12] . Figure 12 As shown, two MSLN-[L]-scFv CD3 The bsAb samples (HP4-44 and MSLN-39) showed similar activities in the assay, which was consistent with the results of the T cell activation assay in Example 9 and the KILR cytotoxicity assay in Example 10. Example 12. Soluble Mesothelin vs. MSLN-[L]-seFv CD3 Impact of bsAbs

[0152] Mesothelin is continuously shed from the surface of cancer cells and forms a soluble protein reservoir in pleural effusions, ascites, and blood. Serum MSLN can serve as a decoy receptor for neutralizing antibody drugs

[13] . To evaluate the effect of soluble MSLN on MSLN-[L]-scFv CD3 To investigate the effect of bsAb on the cytotoxicity of lipopolysaccharide (LSLN), we used a luciferase-based cytotoxicity assay in the presence of soluble MSLN. Figure 13 As shown in Figure 3, we observed that HP4-44 bsAb was less sensitive to soluble MSLN compared to MSLN-39. At 200 ng / mL soluble MSLN, the IC50 value of MSLN-39 bsAb increased 27-fold, while the increase for HP4-44 bsAb was only 2.5-fold. These results are consistent with the notion that high-affinity antibodies are more sensitive to soluble antigens. Example 13. MSLN-[L]-seFv CD3 In vivo antitumor effects of bsAb in a co-transplantation model

[0153] The in vivo anti-tumor activity was evaluated in NOG-SCID mice using the NCI-N87 / PBMC co-transplantation model. PBMC (1.67x10 6 ) and human gastric cancer cells NCI-N87 (5x10 6 ) were pre-mixed and sc-implanted into female NOD / SCID mice (n=5 / group). CD3 The bsAbs (HP4-44 and MSLN-39) were tested, along with a positive control, HPN536. HPN536 is a bispecific molecule developed by Harpoon, Inc., in which four scFv domains are linked in series and capable of binding to MSLN, HSA, and CD3. Figure 14The regimen shown in A was performed with vehicle control (PBS), HPN536 (0.2 mg / kg), or three different dose levels (0.002, 0.02, and 0.2 mg / kg) of MSLN-[L]-scFv CD3 Mice were treated with bsAbs by iv. Due to its short half-life, HPN536 was injected 10 times daily. One mouse in the PBS group was too ill to be included in the analysis. Compared with PBS, both HPN536 and HP4-44 bsAbs at 0.2 mg / kg showed significant inhibition of tumor growth, while MSLN-39 bsAb, although with better affinity, showed only moderate inhibition ( Figure 14 B and 14C). The reduction in tumor volume in the HP-44bsAb-treated group was more pronounced at a dose of 0.2 mg / kg. The tumor in one mouse in this group completely disappeared ( Figure 14 C). There was no difference in body weight between the treated and control groups ( Figure 14 D). Example 14. MSLN-[L]-seFv CD3 Pharmacokinetics of bsAbs in mice

[0154] A single-dose pharmacokinetic study was conducted in BALB / c male mice at a dose of 5 mg / kg intravenously. Figure 15 As shown, both HP4-44 and MSLN-39 bsAbs exhibited favorable PK properties, with half-lives (t½) of 8.68 and 6.58 days, respectively. The maximum serum concentration (Cmax) of HP4-44 bsAb was 118.01 mg / mL, while that of MSLN-39 bsAb was 86.44 mg / mL. The area under the curve (AUC, a measure of systemic exposure) and total clearance (CL) of HP4-44 bsAb were 626.34 μg / day / mL and 7.16 mL / day / kg, respectively, while the AUC and CL of MSLN-39 bsAb were 349.95 μg / day / mL and 13.62 mL / day / kg, respectively. The shorter half-life of MSLN-39 bsAb may be related to faster drug clearance from the body. The smaller steady-state volume of distribution (Vss) of HP4-44 bsAb suggests that its tissue penetration may be lower than that of MSLN-39 bsAb. Together, these results indicate that HP4-44bsAb has superior clearance and systemic exposure profiles to MSLN-39bsAb in mice.

[0155] As shown in the present disclosure and exemplary embodiments, bispecific antibodies comprising regions that specifically bind to mesothelin can be used to target and fine-tune therapeutic interventions associated with mesothelin-related diseases or conditions. The Examples illustrate exemplary embodiments comprising bispecific antibodies comprising an anti-CD3 scFv linked to the C-terminus of a mesothelin antibody light chain variable region. The bispecific antibodies exemplified herein exhibit excellent anti-tumor activity both in vitro and in co-transplanted mouse disease models. The bispecific antibodies exemplified herein demonstrate that binding of the constructs may be less sensitive to soluble mesothelin. In addition, the bispecific antibodies exemplified herein exhibit excellent in vivo half-lives in animal models. References 1. Pastan, I. and R. Hassan, Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer Res, 2014.74(11):p.2907-12. 2. Molloy, ME, et al., Preclinical Characterization of HPN536, aTrispecific, T-Cell-Activating Protein Construct for the Treatment of Mesothelin-Expressing Solid Tumors. Clin Cancer Res, 2021.27(5):p.1452-1462. 3. Golfier, S., et al., Anetumab ravtansine: a novel mesothelin-targetingantibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect. Mol Cancer Ther, 2014.13(6):p.1537-48. 4.Hassan,R.,et al.,Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAb-009in patients with mesothelin-expressing cancers.Clin Cancer Res,2010.16(24):p.6132-8. 5.Hassan,R.,et al.,Phase 1study of the immunotoxin LMB-100in patientswith mesothelioma and other solid tumors expressing mesothelin.Cancer,2020.126(22):p.4936-4947. 6.Adusumilli,P.S.,et al.,A Phase I Trial of Regional Mesothelin-Targeted CAR T-cell Therapy in Patients with Malignant Pleural Disease,inCombination with the Anti-PD-1AgentPembrolizumab.Cancer Discov,2021.11(11):p.2748-2763. 7.Feng,Y.,et al.,A novel human monoclonal antibody that binds withhigh affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity.Mol Cancer Ther,2009.8(5):p.1113-8. 8.Ambrosetti,F.,et al.,proABC-2:PRediction of AntiBody contacts v2and its application to information-driven docking.Bioinformatics,2020.36(20):p.5107-5108. 9.Wang,C.,et al.,Design of a Novel Fab-Like Antibody Fragment withEnhanced Stability and Affinity for Clinical use.Small Methods,2022.6(2):p.e2100966. 10.Crooks,G.E.,et al.,WebLogo:a sequence logo generator.Genome Res,2004.14(6):p.1188-90. 11.Santich,B.H.,et al.,Interdomain spacing and spatial configurationdrive the potency of IgG-[L]-scFv T cell bispecific antibodies.Sci TranslMed,2020.12(534). 12.Nazarian,A.A.,et al.,Characterization of bispecific T-cell Engager(BiTE)antibodies with a high-capacity T-cell dependent cellular cytotoxicity(TDCC)assay.J Biomol Screen,2015.20(4):p.519-27. 13.Liu,X.,et al.,Multiple proteases are involved in mesothelinshedding by cancer cells.Commun Biol,2020.3(1):p.728.

Claims

1. An isolated antibody, bispecific antibody, or antigen-binding fragment thereof that binds to mesothelin, comprising: i. a heavy chain variable region comprising: a CDR1, CDR2, and CDR3 sequence from any of the sequences disclosed in Tables 2, 3, 4, or 5; and A light chain variable region comprising: a CDR1, CDR2 and CDR3 sequence from any of the sequences disclosed in Tables 2, 3, 4 or 5.

2. The isolated antibody, bispecific antibody, or antigen-binding fragment thereof according to claim 1, comprising: i. a heavy chain variable region comprising a sequence disclosed in Table 5; and A light chain variable region comprising the sequence disclosed in Table 5.

3. The isolated antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1-2, further comprising a conjugated moiety.

4. The isolated antibody, bispecific antibody, or antigen-binding fragment thereof of claim 3, wherein the conjugated moiety comprises a therapeutic agent, a solid support, an affinity agent, or a detectable label.

5. The isolated antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 3-4, wherein the conjugated moiety comprises an anticancer agent or a cytotoxin.

6. The isolated bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising an antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not comprise a mesothelin epitope.

7. The bispecific antibody of claim 6, wherein the antibody or antigen-binding fragment thereof that specifically binds to an antigen that does not comprise a mesothelin epitope binds to an antigen comprising CD2, CD3, CD11a CD20, CD25 (IL2R), CD28, CD33, CD47, CD52, EGFR, VEGF, integrin-α3, GPIIb / IIIar, protein F, TNF-α, TNF-β, HER2 / Neu, C5, or IgE.

8. A pharmaceutical composition comprising the isolated antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. A kit comprising the pharmaceutical composition of claim 8, or the antibody, bispecific antibody, or antigen-binding fragment thereof of any one of claims 1 to 7, optional reagents, and instructions for use.

10. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8.

11. The method of claim 10, wherein the cancer comprises a solid tumor cell cancer.

12. An isolated polynucleotide encoding the antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 7.

13. An isolated recombinant cell that produces the antibody, bispecific antibody, or antigen-binding fragment thereof according to any one of claims 1 to 7.

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