CN114423789B

CN114423789B - Antibodies to mesothelin and uses thereof

Info

Publication number: CN114423789B
Application number: CN202180004768.4A
Authority: CN
Inventors: 孝作祥; 彭佳萍; 周东文; 周炜
Original assignee: Zhejiang Shimai Pharmaceutical Co ltd
Current assignee: Zhejiang Shimai Pharmaceutical Co ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-09-30
Anticipated expiration: 2041-12-24
Also published as: CN114423789A; WO2023115528A1

Abstract

The present invention discloses antibodies against mesothelin and uses thereof, in particular monoclonal antibodies against mesothelin, bispecific antibodies against mesothelin and CD3, nucleic acids comprising nucleotide sequences encoding said antibodies, vectors comprising said nucleic acids and host cells comprising said nucleic acids or said vectors. Pharmaceutical compositions and conjugates comprising the antibodies, and methods of treatment using the antibodies are also disclosed.

Description

Antibodies to mesothelin and uses thereof

Technical Field

The present invention relates to antibodies directed to mesothelin and uses of such antibodies, particularly their use in the treatment of cancer.

Background

Mesothelin (MSLN) is encoded as a 71kDa precursor protein that is subsequently treated with the endoproteinase furin to produce a 31kDa abscission protein known as megakaryocyte enhancer factor (MPF) and a 40kDa fragment, mesothelin, which is linked to the cell membrane by a Glycosylphosphatidylinositol (GPI) anchor.

Mesothelin is a tumor differentiation antigen that is normally expressed in mesothelial cells of the pleural, peritoneal and pericardial linings, but is highly expressed in many human cancers, including almost all mesotheliomas and pancreatic cancers, as well as approximately 70% ovarian cancers and 50% lung adenocarcinomas.

Studies have shown that abnormal expression of mesothelin plays a central role in cancer cell proliferation, invasion and metastasis by activating the PI3K, ERK and MAPK signaling pathways. In the tumor context, mesothelin is known to bind to the ovarian cancer antigen MUC16/CA125, and MUC16/CA125 is a very large cell surface mucin shed into the serum and used to monitor response to ovarian cancer therapy. These two proteins are often co-expressed and the binding of mesothelin and MUC16 has been shown to induce intercellular adhesion and promote peritoneal spreading of ovarian cancer. In addition, it was reported that signaling mediated by the binding of MSLN and MUC16 increases the resistance of cells to anoikis, up-regulates matrix metalloproteinases that have important roles in cell invasion and metastasis, and induces the secretion of autocrine growth factors by constitutively activating nuclear factor kappa B (NF- κ B).

In view of its expression pattern in normal tissues and tumors, mesothelin has become an attractive target for cancer therapy.

Summary of The Invention

The present disclosure provides novel antibodies or antigen-binding fragments thereof that bind mesothelin, which may be in the form of monoclonal antibodies or bispecific antibodies, such as bispecific T cell engagers (bites). The antibodies disclosed herein are capable of binding to mesothelin and mediating killing of mesothelin-expressing target cells (e.g., various cancer cells) by effector cells.

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds mesothelin, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein VL comprises a heavy chain variable region (VH) having the amino acid sequences set forth in SEQ ID NOs: 1-3, and VH comprises an amino acid sequence as set forth in SEQ ID NOs: 6-8, and the HCDR 1-3 of the amino acid sequence shown in the specification.

In some embodiments of the presently disclosed antibodies or antigen-binding fragments thereof, VL comprises an amino acid sequence identical to SEQ ID NO: 4, and VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the VL comprises a sequence as set forth in SEQ ID NO: 4, and VH comprises the amino acid sequence as set forth in SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

In some embodiments, the antibody is of an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG 4.

In some embodiments, the antigen binding fragment may be selected from Fab, Fab ', F (ab') ₂ Fv, scFv and ds-scFv.

In some embodiments, the antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a light chain comprising a heavy chain variable region identical to SEQ ID NO: 5, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof, and 11 amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In other embodiments, the antibody may be a bispecific or multispecific antibody. In some embodiments, the antibody can be a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen or an immune cell antigen. In some embodiments, the second antigen may be a T cell antigen. In some embodiments, the T cell antigen may be selected from T Cell Receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG 2D.

In some embodiments, the second antigen is CD3, and the second antigen-binding region comprises a VL and a VH, wherein the VL comprises a VH having the amino acid sequence as set forth in SEQ ID NOs: 12-14, and the VH comprises an amino acid sequence set forth in SEQ ID NO: 17-19, or a pharmaceutically acceptable salt thereof.

In some embodiments, the second antigen-binding region comprises a VL comprising a sequence identical to SEQ ID NO: 15, and said VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the second antigen-binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 15 and the VH comprises the amino acid sequence shown as SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the VL of the second antigen-binding region is optionally linked to the C-terminus of the VL of the antibody that specifically binds mesothelin through a first linker, and the VH of the second antigen-binding region is optionally linked to the C-terminus of the VH of the antibody that specifically binds mesothelin through a second linker, wherein the first linker and the second linker are the same or different.

In some embodiments, the first linker comprises a sequence as set forth in SEQ ID NO:22 (GGGGSGGGGSGGGGS), and a second linker comprising an amino acid sequence as set forth in SEQ ID NO:23 (GSSGGGGSGGGGS).

In some embodiments, the bispecific antibody comprises a light chain comprising a heavy chain variable region that differs from the variable region of SEQ ID NO: 16, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE).

In another aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising a first mesothelin-binding antigen-binding region comprising a VL and a VH, and a second CD 3-binding antigen-binding region comprising a VL and a VH, wherein the VL of the first antigen-binding region comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1-3, and the VH of the first antigen-binding region comprises an amino acid sequence as set forth in SEQ ID NOs: 6-8, and HCDR 1-3 of the amino acid sequence shown in SEQ ID NO; and the VL of the second antigen-binding region comprises a vh having the amino acid sequence set forth in SEQ ID NO: 12-14, and the VH of the second antigen-binding region comprises an amino acid sequence set forth in SEQ ID NOs: 17-19, or a pharmaceutically acceptable salt thereof.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the VL of the first antigen-binding region comprises an amino acid sequence that is identical to SEQ ID NO: 4, and the VH of the first antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; and the VL of the second antigen binding region comprises a sequence identical to SEQ ID NO: 15, and the VH of the second antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the VL of the first antigen-binding region comprises an amino acid sequence as set forth in SEQ ID NO: 4, and the VH of the first antigen-binding region comprises the amino acid sequence set forth in SEQ ID NO: 9; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 15 and the VH of the second antigen-binding region comprises the amino acid sequence shown as SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the VL of the second antigen-binding region is optionally linked to the C-terminus of the VL of the first antigen-binding region by a first linker, and the VH of the second antigen-binding region is optionally linked to the C-terminus of the VH of the first antigen-binding region by a second linker, wherein the first linker and the second linker are the same or different. In some embodiments, the first linker comprises a sequence as set forth in SEQ ID NO:22 (GGGGSGGGGSGGGGS), and a second linker comprising an amino acid sequence as set forth in SEQ ID NO:23 (GSSGGGGSGGGGS).

In some embodiments, the bispecific antibody can be a bispecific T cell engager (BiTE).

In yet another aspect, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

In another aspect, the present disclosure provides a vector comprising a nucleic acid of the present disclosure.

In another aspect, the disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

In some embodiments of the presently disclosed pharmaceutical compositions, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent may be selected from an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the second therapeutic agent may be selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid.

In yet another aspect, the present disclosure provides a conjugate comprising an antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In some embodiments of the presently disclosed conjugates, the chemical moiety may be selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.

In some embodiments of the methods disclosed herein, the cancer is a mesothelin-positive cancer. In some embodiments, the cancer may be selected from mesothelioma, breast, pancreatic, lung, gastric, and ovarian cancer. In some embodiments, the cancer is Triple Negative Breast Cancer (TNBC).

In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent may be selected from an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the second therapeutic agent may be selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid.

In another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for the manufacture of a medicament for treating cancer in a subject. In some embodiments, the cancer is a mesothelin-positive cancer. In some embodiments, the cancer may be selected from mesothelioma, breast, pancreatic, lung, gastric, and ovarian cancer. In some embodiments, the cancer is Triple Negative Breast Cancer (TNBC).

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating cancer in a subject. In some embodiments, the cancer is a mesothelin-positive cancer. In some embodiments, the cancer may be selected from mesothelioma, breast cancer, pancreatic cancer, lung cancer, gastric cancer, and ovarian cancer. In some embodiments, the cancer is Triple Negative Breast Cancer (TNBC).

Drawings

An understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description that describes exemplary embodiments that utilize the principles of the invention and the accompanying drawings, in which:

figure 1 shows the binding of M8 Fab, M10 Fab and M38 Fab to recombinant human mesothelin as measured by ELISA. BSA was used as a negative control.

Figure 2 shows the binding of M8 Fab, M10 Fab and M38 Fab to mesothelin transiently transfected HEK293 cells as measured by flow cytometry. A commercial anti-mesothelin antibody was used as a positive Control (CTRL).

Figure 3 shows the binding of M10 Fab to cancer cell lines H226 and N87 as measured by flow cytometry.

Figure 4 shows the binding of M10 mAb to recombinant human mesothelin as measured by ELISA.

Figure 5 shows the binding of M10 mAb to cancer cell line H226 as measured by flow cytometry.

Figure 6 shows ADCC killing of H226 cells by M10 mAb in the presence of NK cells. An IgG4 isotype antibody was used as a negative control.

Figure 7A shows the binding of CMD007 to recombinant human CD3 as measured by ELISA.

Figure 7B shows the binding of CMD007 to recombinant human mesothelin as measured by ELISA.

Fig. 8A shows the binding of CMD007 to mesothelin-expressing cancer cell line H226 as measured by flow cytometry.

Fig. 8B shows the binding of CMD007 to Jurkat cells expressing CD3 as measured by flow cytometry.

FIG. 8C shows the binding of CMD007 to mesothelin-stably transfected cancer cells LS174T-MSLN (2B6) as measured by flow cytometry.

Fig. 8D shows the binding of CMD007 to the mesothelin-negative cancer cell line LS174T as measured by flow cytometry.

FIG. 9 shows CMD 007-induced T cell activation monitored by CD69 expression in the presence of mesothelin expressing cell lines H226 and LS174T-MSLN (2B 6). LS174T was used as a negative control for the cell line. CMD006-M2 was used as a negative control for the antibody.

FIG. 10 shows CMD 007-induced T cell activation monitored by CD25 expression in the presence of the mesothelin-expressing cell line LS174T-MSLN (2B 6). LS174T was used as a negative control for the cell line. CMD006-M2 was used as a negative control for the antibody.

FIG. 11 shows the killing effect of CMD007 on N87 cells in the presence of human PBMC. The ratio of target cells (N87) to effector cells (PBMC) was 1: 10. Antibodies of the IgG4 isotype were used as negative controls.

FIG. 12 shows the killing of LS174T-MSLN (2B6) cells by CMD007 in the presence of human PBMCs. The ratio of target cells (LS174T-MSLN (2B6)) to effector cells (PBMC) was 1: 5.

Figure 13 shows the killing effect of CMD007 on H226 cells in the presence of human PBMC. The ratio of target cells (H226) to effector cells (PBMC) was 1: 10. An IgG4 isotype antibody was used as a negative control (CTRL-IgG 4).

Figure 14 shows the concentration of CMD007 in serum of CMD 007-treated mice at 0.25 hours, 5 hours, 21 hours, 49 hours, and 69 hours post treatment.

FIG. 15A shows the inhibition of tumor growth in mice by either 50. mu.g/kg or 200. mu.g/kg CMD 007. 200 μ g/kg IgG4 isotype antibody was used as a negative control.

Figure 15B shows the body weight of mice in tumor growth inhibition assays. 200 μ g/kg IgG4 isotype antibody was used as a negative control.

FIG. 15C shows the rate of inhibition of tumor growth in mice by either 50. mu.g/kg or 200. mu.g/kg CMD 007.

Detailed Description

The above features and advantages and additional features and advantages of the present invention will be more clearly understood hereinafter from the following detailed description of embodiments taken in conjunction with the accompanying drawings. The embodiments described herein with reference to the drawings are illustrative, explanatory and are used for the general understanding of the present invention. The embodiments should not be construed as limiting the scope of the invention. The same or similar elements and elements having the same or similar functions are denoted by the same reference numerals throughout the description.

Unless otherwise stated or defined, all terms used have the ordinary meaning in the art that is well known to the skilled person. For example, reference is made to standard manuals, such as Leuenberger, H.G.W, Nagel, B. and Klbl, H. editors, "A multilingual collaboration of biological technical terms: (IUPAC Recommendations) ", Helvetica Chimica Acta (1995), Basel CH-4010, Switzerland; sambrook et al, "Molecular Cloning: a Laboratory Manual (2 nd edition), Vol.1-3, Cold spring harbor Laboratory Press (1989); authored by Ausubel et al, "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); roitt et al, "Immunology" (6 th edition), Mosby/Elsevier, Edinburgh (2001); and Janeway et al, "immunology" (6 th edition), Garland Science Publishing/churchlill Livingstone, new york (2005), and the general background art cited above.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and in some embodiments reference to "an antibody" includes a plurality of antibodies, and the like.

Unless otherwise stated or defined, the term "comprising" and its variants, such as "comprises" and "comprising", are understood to mean that the stated element or step or group of elements or steps is included, but not to exclude any other element or step or group of elements or steps.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind a particular antigen. Antibodies typically comprise a variable region and a constant region in each of the heavy and light chains. The variable regions of antibody heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as the first component C1q in the classical pathway of complement activation. Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form part of an antibody that binds to an antigen.

The "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region interspersed with three "complementarity determining regions" or "CDRs". The framework regions are used to adjust the CDRs for specific binding to the antigen epitope. The CDRs comprise the amino acid residues of the antibody that are primarily responsible for antigen binding. From amino-terminus to carboxy-terminus, both VL and VH domains comprise the following Framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. CDR1, 2, and 3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; CDR1, 2 and 3 of the VH domain are also referred to herein as HCDR1, HCDR2 and HCDR3, respectively.

The amino acid assignments for each of the VL and VH domains are according to any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD,1987 and 1991)); chothia definition (Chothia & Lesk, J.mol.biol.196: 901-917, 1987; Chothia et al, Nature 342: 878-883, 1989); (ii) complexation of Chothia Kabat CDRs, wherein CDR-H1 is a complexation of Chothia and Kabat CDRs; definition of AbM used by Oxford Molecular's antibody modeling software; and Martin et al (world wide web bio fo. org. uk/abs). Kabat provides a widely used numbering convention (Kabat numbering system), wherein corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use Kabat defined CDRs.

The term "antibody" as used herein is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two antigen-binding regions. The antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of the antibodies, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

As used herein, the term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (e.g., mesothelin protein). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab')2 fragment which is a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bond; (iii) fab 'fragments, which are essentially Fab but have a partial hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed.,3.sup. rd. ed. 1993); (iv) Fd fragments consisting of VH and CH1 domains; (v) Fd' fragments having VH and CH1 domains and one or more cysteine residues located C-terminal to the CH1 domain, (vi) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (vii) dAb fragments (Ward et al (1989) Nature 341: 544-, in this protein chain, the VL and VH regions pair to form monovalent molecules (known as single chain fv (ScFv); see, e.g., Bird et al (1988) Science 242, 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide and a modified version of any of the foregoing fragments that retain antigen binding activity form an antigen binding region.

These antigen binding fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments screened for utility in the same manner as intact antibodies.

As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. Affinity is expressed by the equilibrium constant (KD) for dissociation of an antigen from an antibody and is a measure of the strength of binding between an antigenic determinant and the antigen-binding site of an antibody: the smaller the KD value, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1/KD.

Avidity is a measure of the strength of binding between an antibody and an antigen of interest. Avidity relates to the affinity between an antigenic determinant and the antigen binding site of an antibody and the number of relevant binding sites present on an antibody. Typically, the antibody will be at 10 ^-5 To 10 ^-12 M or less dissociation constant (KD), preferably at 10 ^-7 To 10 ^-12 M is less, more preferably 10 ^-8 To 10 ^- ¹² M has a dissociation constant (KD) binding, and/or by at least 10 ⁷ M ^-1 Preferably at least 10 ⁸ M ^-1 More preferably at least 10 ⁹ M ^-1 E.g. at least 10 ¹² M ^-1 Binding affinity of (3). Generally any is considered to be greater than 10 ^-4 K of M _D Values represent nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant may be determined by any suitable means known per se, including for example scatchard analysis and/or competitive binding assays such as Radioimmunoassays (RIA), Enzyme Immunoassays (EIA) and sandwich competition assays and different variants known per se in the art.

The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes may be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed by consecutive amino acids (also known as linear epitopes) are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost in the processing of denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the minimum binding site of an antibody and is therefore the specific target of the antibody or antigen binding fragment thereof.

As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at identical positions after alignment. For example, an "amino acid sequence identical to SEQ ID NO: y is X% identical means that the amino acid sequence is identical to SEQ ID NO: y and is expressed as the percentage identity of X% of the residues in the amino acid sequence to SEQ ID NO: the sequence residues disclosed in Y are identical.

Such calculations are typically performed using a computer program. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller,1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al, 1997), BLASTP, BLASTN, or GCG (Devereux et al, 1984).

Furthermore, in determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which has little or no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, WO 04/037999, GB-A-2357768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferably) the type and/or combination of such substitutions may be selected in accordance with the relevant teachings from WO 04/037999 and WO 98/49185 and the further references cited therein.

Such conservative substitution is preferably a substitution in which one amino acid in the following groups (a) to (e) is substituted with another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (c) polar, positively charged residues: his, Arg and Lys; (d) large aliphatic, nonpolar residues: met, Leu, He, Val and Cys; and (e) aromatic residues: phe, Tyr, and Trp.

Particularly preferred conservative substitutions are as follows: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; ile to Leu or to Val; leu to Ile or to Val; lys to Arg, to Gln, or to Glu; met to Leu, to Tyr, or to Ile; phe to Met, to Leu or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.

Any amino acid substitution described herein as applied to a polypeptide may also be based on an analysis of the frequency of amino acid variation between homologous proteins of different species developed by Schulz et al, Principles of Protein Structure, Springer-Verlag,1978, based on Chou and Fasman, Biochemistry 13: 211,1974 and adv. enzymol., 47: 45-149,1978, and based on Eisenberg et al, Proc.nat. Acad Sci.USA 81: 140-144, 1984; kyte & Doolittle, J mol. biol.157: 105, 132,1981, and Goldman et al, Ann.Rev. Biophys.chem.15: 321-353,1986, which are incorporated herein by reference in their entirety.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each antibody comprising the population is identical except for a small number of mutations that may occur naturally. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.

The term "bispecific antibody" is to be understood in the context of the present invention as an antibody having two different antigen binding regions defined by different antibody sequences. This is understood to mean binding to different targets, but also to different epitopes of one target.

As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells, and thus can be used to target cancer cells.

As used herein, the term "CD 3" refers to a human CD3 protein complex that has five peptide chains, a gamma chain, a delta chain, an epsilon chain, a zeta chain, and an eta chain, and binds to the T cell receptor alpha and beta chains to form a TCR-CD3 complex. The term includes any CD3 variant, isoform and species homolog, which may be naturally expressed by cells including T cells, or by cells transfected with a gene or cDNA encoding the above chain.

As used herein, the term "bispecific T-cell engager" or "BiTE" refers to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen and the second of which binds to an antigen present on the surface of a target (see PCT publication WO 05/061547; Baeuuerle et al, 2008, Drugs of the Future 33: 137-. Thus, the BiTE of the present disclosure has an antigen-binding region that binds to mesothelin and a second antigen-binding region that targets a T cell antigen.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.

The term "host cell" as used herein refers to a cell into which an expression vector has been introduced.

The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and not deleterious to the recipient thereof in any substantial amount, and/or that such carrier or adjuvant is approved or available for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms "treatment", "treating" and the like refer to the administration of an agent or procedure in order to obtain an effect. These effects may be prophylactic in terms of completely or partially preventing a disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure affecting a disease and/or disease symptoms. As used herein, "treating" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease or disease symptoms (e.g., including diseases that may be associated with or caused by primary disease) in a subject susceptible to the disease but not yet diagnosed as diseased; (b) inhibiting the disease, i.e. arresting its development; (c) remission of the disease, i.e. causing regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; (iii) alleviating; reduce symptoms or make the disease condition more tolerable to the patient; slowing the rate of deterioration or decline; or a reduction in worsening end-point weakness. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of the physician's examination. Thus, the term "treating" includes administering an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or arrest or inhibit the development of a symptom or disorder associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, disease symptoms, or disease side effects in a subject.

The term "effective amount" as used herein refers to an amount sufficient to effect treatment of a disease when administered to a subject to treat such disease.

As used herein, the term "subject" refers to any mammalian subject for which diagnosis, treatment, or therapy is desired. "mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds mesothelin, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein VL comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NOs: 1-3, and VH comprises an amino acid sequence as set forth in SEQ ID NOs: 6-8, and (b) is the HCDR 1-3 of the amino acid sequence shown in the specification.

In some embodiments, the CDR sequences are defined according to the Kabat numbering system.

When the CDR sequences are defined according to the Kabat numbering system, the VL of the disclosed antibodies comprises a vh having the amino acid sequence as shown in SEQ ID NO: 1(SGDALPKQYAF), SEQ ID NO: 2(KDSERPS) and SEQ ID NO: 3(QSADSSATSVI), the VH of the antibody disclosed herein comprises the amino acid sequences LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 6(DYAMH), SEQ ID NO: 7(GISWNSGSIGYADSVKG) and SEQ ID NO: 8(DSGSSGWYGYFQH) and HCDR1, HCDR2 and HCDR 3.

In some embodiments of the presently disclosed antibodies or antigen-binding fragments thereof, VL comprises an amino acid sequence identical to SEQ ID NO: 4, and VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4, provided that the functional variant retains the ability to bind to mesothelin. In some embodiments, the VH comprises a sequence as set forth in SEQ ID NO: 9, provided that the functional variant retains the ability to bind to mesothelin.

A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of a parent polypeptide. For example, SEQ ID NO: 4 comprises or consists of a sequence identical to SEQ ID NO: 4, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. For example, SEQ ID NO: 98 comprises or consists of a functional variant identical to SEQ ID NO: 9 have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the nucleic acid sequence of SEQ ID NO: 4 comprises or consists of a sequence identical to SEQ ID NO: 4 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity and is obtained by insertion, deletion, and/or substitution of SEQ ID NO: 4, or one or more amino acids of seq id No. 4. In some embodiments, the nucleic acid sequence of SEQ ID NO: 9 comprises or consists of a sequence identical to SEQ ID NO: 9 has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity and is produced by insertion, deletion, and/or substitution of SEQ ID NO: 9, and 9, or a pharmaceutically acceptable salt thereof.

In the context of functional variants, the number of inserted, deleted and/or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is from 1% to 33%, more preferably is from 5% to 30%, more preferably is from 10% to 25%, more preferably is from 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of amino acids inserted, deleted and/or substituted may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of amino acids inserted, deleted and/or substituted is 1, 2, 3, 4, 5, 6 or 7.

In some embodiments, insertions, deletions, and/or substitutions may be made at the Framework (FR) regions, e.g., FR1, FR2, FR3, and/or FR 4.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such conservative substitution is preferably a substitution in which one amino acid in the following groups (a) to (e) is substituted with another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln; (c) polar, positively charged residues: his, Arg and Lys; (d) large aliphatic, non-polar residues: met, Leu, He, Val and Cys; and (e) aromatic residues: phe, Tyr, and Trp.

In a preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 4, VH comprises the amino acid sequence as set forth in SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

Depending on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulin molecules can be divided into five classes (isotype): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of an antibody can be classified into a lambda (lambda) chain and a kappa (kappa) chain according to the amino acid sequence of the light chain. The antibodies disclosed herein can be of any of the classes or subtypes described above.

In some embodiments, the antibody may be of an isotype selected from IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody may be a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG 4. In a preferred embodiment, the antibody is an IgG1 antibody.

The antibodies disclosed herein can be whole antibodies or antigen-binding fragments thereof. The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to mesothelin. Examples of antigen-binding fragments include, but are not limited to: a Fab fragment; a F (ab')2 fragment; a Fab' fragment; (ii) a fragment of Fd; (ii) a fragment of Fd'; (iv) Fv fragments; a scFv fragment; a dAb fragment; a single Complementarity Determining Region (CDR); a nanobody; a linear antibody comprising a pair of Fd fragments (VH-CH1-VH-CH1) in tandem, and a modified form of any of the foregoing fragments which retains antigen binding activity.

In some embodiments, the antigen binding fragment may be selected from Fab, Fab ', F (ab') ₂ Fv, scFv and ds-scFv. In preferred embodiments, the antigen binding fragment is a Fab or scFv. In another preferred embodiment, the antigen binding fragment is a Fab comprising a Fab having the amino acid sequence as set forth in SEQ ID NO: 5(SYVLTQPPSVSVSPGQTARITCSGDALPKQYAFWYQQKPGQAPVLVIYK DSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSATSVIFGG GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA DSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGS TVEKTVAPTECS) and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (QLVETGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDS GSSGWYGYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCGQAGHHHHHHGDYKDDDDKG). In some embodiments, the antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a light chain comprising a heavy chain variable region identical to SEQ ID NO: 5, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the light chain comprises a sequence as set forth in SEQ ID NO: 5, provided that the functional variant retains the ability to bind to mesothelin. In some embodiments, the heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO: 11, provided that the functional variant retains the ability to bind to mesothelin.

For example, SEQ ID NO: 5 comprises or consists of a sequence identical to SEQ ID NO: 5, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. For example, SEQ ID NO: 11 comprises or consists of a functional variant identical to SEQ ID NO: 11, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the number of amino acids inserted, deleted and/or substituted is preferably no more than 40%, more preferably no more than 35%, more preferably 1% to 33%, more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of amino acids to be inserted, deleted and/or substituted may be 1 to 50, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted and/or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

In some embodiments, the insertions, deletions, and/or substitutions may be in the Framework (FR) regions, e.g., FR1, FR2, FR3, and/or FR 4; and/or constant regions, such as at CL, CH1, CH2, and/or CH 3.

In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

In a preferred embodiment, the light chain comprises the sequence as set forth in SEQ ID NO: 5 and the heavy chain comprises the amino acid sequence shown as SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

In other embodiments, the antibody may be a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen or an immune cell antigen.

Many tumor-associated antigens have been identified in the art that are associated with a particular cancer. In some embodiments, a tumor-associated antigen is an antigen that can potentially elicit a significant tumor-specific immune response. Some of these antigens are encoded by, but not necessarily expressed by, normal cells. These antigens can be characterized as antigens that are normally silenced (i.e., not expressed) in normal cells, antigens that are expressed only at certain differentiation stages, and antigens that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutated P53), and fusion proteins resulting from internal deletions or chromosomal translocations. Other cancer antigens may be encoded by viral genes, such as genes carried by RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies thereto are known and/or commercially available and can be made by those skilled in the art.

Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, C-Met, CSPG4, IgM, C-type lectin-like molecule 1(CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutant p53, mutant ras, ROR1, GPC3, VEGFR2, and combinations thereof.

In some embodiments, the second antigen may be a T cell antigen. In some embodiments, the T cell antigen may be selected from T Cell Receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3 and the second antigen-binding region binds to any of the gamma, delta, epsilon, zeta and eta chains of CD 3.

In some embodiments, the second antigen is CD3 and the second antigen-binding region comprises a VL and a VH, wherein the VL comprises a VH having the amino acid sequence as set forth in SEQ ID NOs: 12-14, and the VH comprises an amino acid sequence set forth in SEQ ID NO: 17-19, or a pharmaceutically acceptable salt thereof.

In some embodiments, the CDR sequences are defined according to the Kabat numbering system. When using the CDR sequences defined by Kabat, the VL of the disclosed second antigen binding region comprises a CDR having the sequence as shown in SEQ ID NO: 12(RSSTGAVTTSNYAN), SEQ ID NO: 13 (ganrap) and SEQ ID NO: 14(ALWYSNLWV), and LCDR1, LCDR2, and LCDR3, the VH of the second antigen-binding region disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 17(GFTFNTY), SEQ ID NO: 18(RSKYNNYA) and SEQ ID NO: 19(HGNFGSSYVSYFAY) and HCDR1, HCDR2 and HCDR 3.

In some embodiments, the second antigen-binding region comprises a VL comprising a sequence identical to SEQ ID NO: 15, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the VL comprises a sequence as set forth in SEQ ID NO: 15, provided that the functional variant retains the ability to bind to CD 3. In some embodiments, the VH comprises a sequence as set forth in SEQ ID NO: 20, provided that the functional variant retains the ability to bind to CD 3.

For example, SEQ ID NO: 15 comprises or consists of a sequence identical to SEQ ID NO: 15, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. For example, SEQ ID NO: 20 comprises or consists of a sequence identical to SEQ ID NO: 20, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the number of amino acids inserted, deleted and/or substituted is preferably no more than 40%, more preferably no more than 35%, more preferably 1% to 33%, more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of amino acids inserted, deleted and/or substituted may be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and/or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

In a preferred embodiment, the second antigen-binding region comprises a VL and a VH, the VL comprising an amino acid sequence as set forth in SEQ ID NO: 15, the VH comprising the amino acid sequence shown as SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the VL of the second antigen-binding region is optionally linked to the C-terminus of the VL of the antibody that specifically binds mesothelin through a first linker, and the VH of the second antigen-binding region is optionally linked to the C-terminus of the VH of the antibody that specifically binds mesothelin through a second linker, wherein the first linker and the second linker are the same or different. In some embodiments, the first linker comprises a sequence as set forth in SEQ ID NO:22 (GGGGSGGGGSGGGGS), and a second linker comprising an amino acid sequence as set forth in SEQ ID NO:23 (GSSGGGGSGGGGS).

In some embodiments, the light chain comprises a sequence as set forth in SEQ ID NO: 16, provided that the functional variant retains the ability to bind to mesothelin and CD 3. In some embodiments, the heavy chain comprises a heavy chain as set forth in SEQ ID NO: 21, provided that the functional variant retains the ability to bind to mesothelin and CD 3.

For example, SEQ ID NO: 16 comprises or consists of a sequence identical to SEQ ID NO: 16 has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity. For example, SEQ ID NO: 21 comprises or consists of a sequence identical to SEQ ID NO: 21, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

In a preferred embodiment, the light chain comprises the sequence as set forth in SEQ ID NO: 16, and the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 21, or a pharmaceutically acceptable salt thereof.

In some embodiments, the bispecific antibody can be a bispecific T cell engager (BiTE). In some embodiments of the presently disclosed antibodies or antigen binding fragments thereof, the bispecific antibody is in the form of an HBiTE as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety). In HBiTE, the light chain comprises, from N-terminus to C-terminus, an anti-target VL domain, anti-CD 3 VL-CL, and monomeric human IgG1 Fc (e.g., mfc 7.2); the heavy chain comprises, from N-terminus to C-terminus, an anti-target VH domain, anti-CD 3 VH-CH1, and monomeric human IgG1 Fc (e.g., mfc 7.2). Monomer fc7.2 contains two amino acid mutations (T366L and Y407H) that inhibit Fc homodimerization.

In another aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising a first mesothelin-binding antigen-binding region comprising a VL and a VH, and a second CD 3-binding antigen-binding region comprising a VL and a VH, wherein the VL of the first antigen-binding region comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1-3, and the VH of the first antigen-binding region comprises an amino acid sequence as set forth in SEQ ID NOs: 6-8, and HCDR 1-3 of the amino acid sequence shown in the specification; and the VL of the second antigen-binding region comprises a vh region having the amino acid sequence set forth in SEQ ID NO: 12-14, and the VH of the second antigen-binding region comprises an amino acid sequence as set forth in SEQ ID NOs: 17-19, or a pharmaceutically acceptable salt thereof.

In some embodiments of the bispecific antibodies or antigen-binding fragments thereof disclosed herein, the VL of the first antigen-binding region comprises an amino acid sequence that is identical to SEQ ID NO: 4, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, and the VH of the first antigen-binding region comprises an amino acid sequence that is identical to SEQ ID NO: 9, an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; and the VL of the second antigen-binding region comprises a sequence identical to SEQ ID NO: 15, and the VH of the second antigen-binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

In some embodiments, the VL of the first antigen-binding region comprises a sequence as set forth in SEQ ID NO: 4, provided that the functional variant retains the ability to bind to mesothelin. In some embodiments, the VH of the first antigen-binding region comprises a sequence as set forth in SEQ ID NO: 9, provided that the functional variant retains the ability to bind to mesothelin. In some embodiments, the VL of the second antigen-binding region comprises a sequence as set forth in SEQ ID NO: 15, provided that the functional variant retains the ability to bind to CD 3. In some embodiments, the VH of the second antigen-binding region comprises a sequence as set forth in SEQ ID NO: 20, provided that the functional variant retains the ability to bind to CD 3.

SEQ ID NO: the functional variants of 4, 9, 15 and 20 may be those described above.

In a preferred embodiment, the VL of the first antigen-binding region comprises an amino acid sequence as set forth in SEQ ID NO: 4, and the VH of the first antigen-binding region comprises the amino acid sequence as set forth in SEQ ID NO: 9; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 15 and the VH of the second antigen-binding region comprises the amino acid sequence shown as SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the bispecific antibody comprises a single polypeptide chain comprising a first antigen-binding region and a second antigen-binding region, and optionally an Fc region.

The Fc region may be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one embodiment, the Fc region is deficient in effector function. For example, the Fc region may be of the IgG1 isotype, or of the non-IgG 1 type, such as IgG2, IgG3, or IgG4, which has been mutated such that its ability to mediate effector functions such as ADCC is reduced or even eliminated. Such mutations are described in Dall' Acqua WF et al, J Immunol.177 (2): 1129-1138(2006) and Hezareh M, J Virol; 75(24): 12161-12168(2001) have been described.

In one embodiment, the Fc region comprises a mutation that removes the acceptor site for Asn-linked glycosylation or is otherwise manipulated to alter glycosylation properties. For example, in the IgG1 Fc region, the N297Q mutation can be used to remove Asn-linked glycosylation sites. Thus, in a specific embodiment, the Fc region comprises the IgG1 wild-type sequence having the N297Q mutation.

In further embodiments, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding compounds to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al (2010) biotechnol.bioeng.105: 350, or by using FUT8 knock-out cells, such as Yamane-ohniki et al (2004) biotechnol.bioeng 87: 614. Alternatively, it is possible to use

Et al (1999) Nature Biotech 17: 176 to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al (2009) Cancer sci.100: 2411.

In some embodiments, the Fc region comprises a modification or mutation that inhibits homodimerization of Fc. In some embodiments, the Fc region comprises a variant of the human IgG1 Fc wild-type sequence. The variant may comprise amino acid substitutions at positions T366 and Y407(Kabat numbering) of human IgG 1. Preferably, T366 is substituted by L (leucine). Preferably, Y407 is substituted with I (isoleucine), F (phenylalanine), L (leucine), M (methionine), H (histidine), K (lysine), S (serine), Q (glutamine), T (threonine), W (tryptophan), a (alanine), G (glycine) or N (asparagine). More preferably, Y407 is substituted with histidine. In one embodiment, T366 is substituted with leucine and Y407 is substituted with histidine.

In some embodiments, the Fc region may be monomeric human IgG1 Fc (e.g., mfc7.2), as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.

In some embodiments, the bispecific antibody comprises a first polypeptide chain comprising a VL of a first antigen-binding region and a VL of a second antigen-binding region, and optionally an Fc region; and a second polypeptide chain comprising a VH of the first antigen-binding region and a VH of the second antigen-binding region, and optionally an Fc region. The Fc region may be those described above.

In some embodiments, the first polypeptide chain further comprises a light chain constant region (CL). In some embodiments, the first polypeptide chain comprises a monomeric human IgG1 Fc (e.g., mfc7.2) as described above. In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: VL of the first antigen-binding region, VL of the second antigen-binding region, CL and mfc 7.2.

In some embodiments, the second polypeptide chain further comprises a heavy chain constant region (CH), e.g., CH 1. In some embodiments, the second polypeptide chain comprises a monomeric human IgG1 Fc (e.g., mfc7.2) as described above. In some embodiments, the second polypeptide chain comprises, from N-terminus to C-terminus: VH of the first antigen-binding region, VH of the second antigen-binding region, CH1 and mfc 7.2.

SEQ ID NO: functional variants of 16 and 21 may be those described above.

In a preferred embodiment, the light chain comprises the sequence as set forth in SEQ ID NO: 16 and the heavy chain comprises the amino acid sequence shown as SEQ ID NO: 21, or a pharmaceutically acceptable salt thereof.

In some embodiments, the bispecific antibody may be a bispecific T cell engager (BiTE), preferably HBiTE as described above.

Any vector may be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an Epstein Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-related vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-Puro, pBABE-neo largetTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP _2.0, TtRMPVIR, pMSCV-IRES-mCheherFP, pRetrox GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λ GT10, λ GT11, λ ZapII (Stratagene), λ EMBL4 and λ NM1149 may also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, such as Sambrook et al, Molecular Cloning: a Laboratory Manual, third edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain replication systems that are functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, COlEl, 2. mu. plasmid, lambda, SV40, bovine papilloma virus, and the like.

Any cell can be used as a host cell for a nucleic acid or vector of the present disclosure. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example enterobacteriaceae (enterobacteriaceae), such as Escherichia (Escherichia), e.g., Escherichia coli (e.coli); enterobacter (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia species (Serratia), such as Serratia marcescens (Serratia marcescens); and Shigella (Shigella); bacillus (bacillus), such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (p. aeruginosa); and Streptomyces (Streptomyces). In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell comprises, for example, a CHO cell, such as a CHOs cell and a CHO-K1 cell, or a HEK293 cell, such as HEK293A, HEK293T, and HEK293 FS.

In some embodiments, carriers or excipients for use with the compositions disclosed herein include, but are not limited to, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methyl pyrrolidone, dimethyl sulfoxide, N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

In some embodiments of the presently disclosed pharmaceutical compositions, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent may be selected from an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the second therapeutic agent may be selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid, or any combination thereof.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenediones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cephalosporins, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.

In some embodiments of the presently disclosed conjugates, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulator, a radioactive compound, an enzyme (e.g., perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin. In some embodiments, the therapeutic agent can be, for example, a maytansine, a geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin), or a minor groove binding agent such as calicheamicin (calicheamicin).

Other suitable therapeutic agents include, for example, small molecule cytotoxic agents, i.e., compounds having a molecular weight of less than 700 daltons having the ability to kill mammalian cells. Such compounds may also contain toxic metals capable of having a cytotoxic effect. In addition, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, raschimycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium II, temozolomide, topotecan, trimethylbiguanide, auristatin E, vinca alkaloids, and doxorubicin; peptide cytotoxins, i.e., proteins or fragments thereof having the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, and rnases; radionuclides, i.e., labile isotopes of elements that decay with the simultaneous emission of one or more of alpha or beta particles or gamma rays, such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225, and astatine-213; chelating agents which can be used to facilitate the binding of these radionuclides to molecules or multimers thereof.

In some embodiments, the detectable moiety may be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion or fluorescent, a phosphorescent, or a chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

In some embodiments, the immunostimulatory molecule is an immune effector molecule that elicits an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN- γ, a chemokine such as IL-8, platelet factor 4, melanoma growth-stimulating protein, a complement activator; a viral/bacterial protein domain, or a viral/bacterial peptide.

In some embodiments of the methods disclosed herein, the cancer is a mesothelin-positive cancer. In some embodiments, the cancer may be selected from mesothelioma, liver cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, esophageal cancer, bladder cancer, prostate cancer, colorectal cancer, uterine cancer, cervical cancer, brain cancer, cervical cancer, stomach cancer, cholangiocarcinoma, chondrosarcoma, kidney cancer, thyroid cancer, skin cancer, melanoma, glioma, neuroblastoma, lymphoma, and myeloma. Preferably, the cancer is selected from the group consisting of mesothelioma, breast cancer, pancreatic cancer (pancreatic adenocarcinoma), lung cancer (such as lung mesothelioma, lung adenocarcinoma and lung squamous cell carcinoma), gastric cancer and ovarian cancer. In a preferred embodiment, the cancer is Triple Negative Breast Cancer (TNBC).

In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject being treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician may determine an effective amount to administer to a human or other subject to treat a medical condition. The precise amount required to be therapeutically effective may depend on a number of factors, such as the activity of the antibody and the route of administration.

The dosage of the antibodies, compositions, or conjugates described herein can be administered to the mammal once or in a series of sub-doses over a suitable period of time, e.g., once daily, half-week, weekly, bi-week, semi-month, bi-month, semi-year, or yearly as desired. Dosage units comprising an effective amount of an antibody, composition or conjugate may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more divided doses administered daily, as desired.

Suitable modes of administration may be selected by the physician. The route of administration may be parenteral, for example by injection, nasal, pulmonary or transdermal administration. Systemic or local administration can be carried out by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, or conjugate is selected for parenteral delivery, inhalation, or delivery through the digestive tract, e.g., oral. The administration dose and method may vary depending on the weight, age, condition, etc. of the subject, and may be appropriately selected.

In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In certain embodiments, the binding agent is administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In some preferred embodiments, the second therapeutic agent may be selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid, or any combination thereof.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents may include, for example, cytotoxic agents, antimetabolites (e.g., folic acid antagonists, purine analogs, pyrimidine analogs, etc.), topoisomerases inhibitors (e.g., camptothecin derivatives, anthracenediones, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc.), antimicrotubule agents (e.g., taxanes, vinca alkaloids), protein synthesis inhibitors (e.g., cephalosporins, camptothecin derivatives, quinoline alkaloids), alkylating agents (e.g., alkyl sulfonates, ethyleneimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc.), alkaloids, terpenoids, and kinase inhibitors.

In another aspect, the present disclosure provides a method of detecting mesothelin-positive cancer in a subject, comprising (i) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof disclosed herein, or a bispecific antibody or antigen-binding fragment thereof disclosed herein, or a conjugate disclosed herein; and (ii) detecting binding of the antibody or antigen-binding fragment thereof to mesothelin in the sample.

In some embodiments, the antibody or antigen-binding fragment thereof is linked to a detectable moiety. The detectable moiety may be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

In some embodiments, the cancer is a mesothelin-positive cancer. Preferably, the cancer is selected from mesothelioma, breast cancer, pancreatic cancer (pancreatic adenocarcinoma), lung cancer (such as lung mesothelioma, lung adenocarcinoma and lung squamous cell carcinoma), gastric cancer and ovarian cancer. In a preferred embodiment, the cancer is Triple Negative Breast Cancer (TNBC). In yet another aspect, the present disclosure provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of a pharmaceutical composition described herein, such as an antibody or antigen-binding fragment disclosed herein. Optionally, associated with such containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In a specific embodiment, the kit comprises a first container comprising an antibody or antigen-binding fragment disclosed herein. In a specific embodiment, the kit comprises a first container which is a vial containing the antibody or antigen-binding fragment as a lyophilized sterile powder under vacuum, and a second container comprising a pharmaceutically acceptable fluid.

In a specific embodiment, the invention provides an injection device comprising an antibody or antigen-binding fragment disclosed herein. In one embodiment, the injection device comprises the antibody in the form of a sterile solution. In a particular embodiment, the injection device is a syringe.

In yet another aspect, the present disclosure provides a kit for detecting the presence of a mesothelin antigen in a sample comprising an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, or a conjugate disclosed herein. Preferably, the antibody or antigen-binding fragment thereof is linked to a detectable moiety. The detectable moiety may be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

In another aspect, the present disclosure provides a use of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for the manufacture of a medicament for treating cancer in a subject. In some embodiments, the cancer is a mesothelin-positive cancer.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating cancer in a subject. In some embodiments, the cancer is an mesothelin-positive cancer.

In yet another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein, in the manufacture of a kit for detecting a mesothelin-positive cancer in a subject.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof disclosed herein, a bispecific antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein, for use in detecting a mesothelin-positive cancer in a subject.

In some embodiments of the uses disclosed herein, the mesothelin-positive cancer is preferably selected from mesothelioma, breast cancer, pancreatic cancer (pancreatic adenocarcinoma), lung cancer (such as lung mesothelioma, lung adenocarcinoma and lung squamous cell carcinoma), gastric cancer and ovarian cancer. In a preferred embodiment, the cancer is Triple Negative Breast Cancer (TNBC).

Examples

The following examples are presented to illustrate various embodiments of the invention and are not intended to limit the invention in any way. The present examples, as well as the methods described herein, presently represent preferred embodiments, are exemplary and are not intended as limitations on the scope of the invention. Variations and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Cell lines including H226 (human lung squamous cell carcinoma cell line), N87 (human gastric carcinoma cell line) and LS174T (human colorectal carcinoma cell line) were purchased from the national certified cell culture collection center.

By using Lipofectamine ^TM LTX reagent, PLUS ^TM Reagent (Thermo) and transfection-specific Medium Opti-MEM ^TM I (Gibco) the commercial MSLN recombinant plasmid pCMV-MSLN (Sino biological) was transiently transfected into LS174T cells to generate the stably mesothelin-expressing tumor cell line LS174T-MSLN (2B 6). Hygromycin B was added to the cell culture and positive clones were selected. After 2-3 weeks, individual positive clones were gradually isolated and verified by flow cytometry.

Human Mesothelin (MSLN) protein and cynomolgus monkey Mesothelin (MSLN) (296-. Anti-human IgG (gamma chain specific) -R-PE antibody, anti-human IgG (Fc specific) -peroxidase antibody and monoclonal antibody

Peroxidase was purchased from Sigma. M13KO7 helper phage was purchased from Invitrogen. Dynabeads ^TM Myone ^TM Streptavidin T1 was purchased from ThermoFisher Scientific. PE anti-His tag antibody was purchased from BioLegend.

Example 1 panning and screening of phage displayed Natural human Fab libraries to identify Mesothelin antibodies

As previously described (Zhu et al, J Virol 2006, 80: 891-899) (with minor modifications thereto, using 5, 1 and 0.2mg of antigen in the first, second and third rounds of panning, respectively), two large forms (scale, 10) with peripheral blood B cells from about 30 healthy individuals were used ¹¹ ) Phage display of natural human Fab libraries to select against magnetic beads (Dynabeads) ^TM Myone ^TM Streptavidin T1; thermo fisher Scientific) conjugated recombinant human mesothelin. After round 3 biopanning, a strong positive signal was observed by using polyclonal phage ELISA. The 3 rd round phages were then tested for specific binding. Three specific Fab clones, designated M8, M10 and M38, were identified by monoclonal enzyme-linked immunosorbent assay (SemELISA) based on soluble expression and DNA sequencing analysis. All three Fab clones had lambda light chains.

Will sixHistidine-tagged M8 Fab, M10 Fab and M38 Fab were expressed in E.coli strain HB2151 and purified from the soluble fraction of the periplasm using Ni-NTA resin. ELISA was then performed using standard protocols to measure binding affinity to recombinant human mesothelin (full-length extracellular domain). Briefly, recombinant human mesothelin (acrobic systems) was coated at 50ng per well on Corning EIA/RIA high binding 96-well plates (Corning corporation) overnight at 4 ℃ and blocked with 3% skim milk in PBS (ph 7.4). Five fold serial dilutions of antibody were added and incubated at room temperature for 2 hours. Plates were washed with PBS containing 0.05% tween 20. Bound antibody was detected by HRP conjugated anti-FLAG tag antibody (nano Biological). The assay was developed with TMB substrate (Solarbio) at room temperature and OD measured at 450nm with a microplate reader. The results showed that M8 Fab, M10 Fab and M38 Fab have EC, respectively ₅₀ Affinities of 127nM, 21nM and 15.7nM were obtained (FIG. 1).

To measure the binding of M8 Fab, M10 Fab, and M38 Fab to cell surface bound mesothelin, flow cytometry was performed on HEK293 cells transiently transfected with mesothelin. A commercial anti-mesothelin antibody (anti-mesothelin-FITC, Miltenyi, Cat #: 130-. The results showed that only the M10 Fab had the ability to bind to cell surface-bound mesothelin (figure 2).

The M10 Fab was then further tested for binding to cell surface bound mesothelin using cancer cell lines including H226 and N87. Each cell line (5X 10) ⁵ Individual cells) were incubated with Fab antibody (10 μ g/mL) on ice for 60 minutes. Cells were washed once with Pbs (PBSA) containing 0.1% bovine serum albumin and resuspended in 200mL PBSA. Then 2. mu.L of anti-His-PE conjugate (BioLegend) was added and incubated for 60 min. Cells were washed once with PBSA and then analyzed by flow cytometry. The results are shown in FIG. 3.

As can be seen from fig. 3, the M10 Fab bound well to H226 and N87 cells, indicating that the M10 Fab can bind well to mesothelin expressing cancer cell lines.

Example 2 construction and preliminary characterization of anti-mesothelin monoclonal antibody

The Fab clone M10 was used to construct an intact form of anti-human mesothelin monoclonal antibody IgG1(M10 mAb). Briefly, the heavy chain Fd fragment of M10 Fab was fused to the N-terminus of the human IgG1 Fc fragment. Both the light and heavy chains were constructed into vector pDin1, which was modified by the inventors from pDR12 to contain two Molecular Cloning Sites (MCS). Construction and preliminary characterization of the M10 mAb was performed as follows.

Cloning of anti-mesothelin monoclonal antibodies

To generate constructs of anti-mesothelin monoclonal antibodies, the following primers were used:

bnIgG20L1, 5 'GTGTAAGCTTACCATGGGTGTGCCCACTCAGGTCC TGGGGT 3' (sense) (SEQ ID NO: 24);

MSLN-IgG 1-VL-sense, 5 'CTTACAGATGCCAGATGTTCCTATGTGCT GACTCAG 3' (sense) (SEQ ID NO: 25);

MSLN-IgG 1-VL-inverted, 5 'CGGAATTCTTATGAACATTCTGTAGGGG CCAC 3' (antisense) (SEQ ID NO: 26);

MSLN-IgG1-VH-FP-Xba1,5 'TGTTCTAGAGCCACCATGGAATGGAG CTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCCCAG CTGGTGGAGA 3' (sense) (SEQ ID NO: 27);

MSLN-IgG1-VH-RP-OL,5 'ATGTGTGAGTTTTGTCACAAGATTTGG GCTCAACT 3' (antisense) (SEQ ID NO: 28);

MSLN-IgG1-FC-FP-OL,5 'GACAAAACTCACACATGCCCACCGT 3' (sense) (SEQ ID NO: 29);

MSLN-IgG1-FC-RP-Sal1,5 'CTGAGTCGACTTATTTACCCGGGGACA GGGAGA 3' (antisense) (SEQ ID NO: 30).

To generate light chains, the VL gene fragment was amplified from anti-mesothelin M10 Fab using the primer pair MSLN-IgG 1-VL-forward/MSLN-IgG 1-VL-reverse. The leader L peptide was fused to the 5' end of the VL PCR fragment by overlap PCR using the primer pair bnIgG20L1/MSLN-IgG 1-VL-inverse. The PCR product was cloned into the MCS of pdIn1 vector containing CL and IgG1 light chain Fc fragment by using the restriction enzyme EcoR I/Hind III.

To generate the heavy chain, the VH-CH1 gene fragment was amplified from anti-mesothelin M10 Fab using the primer pair MSLN-IgG1-VH-FP-Xba1/MSLN-IgG 1-VH-RP-OL. The primer pair MSLN-IgG 1-FC-FP-OL/MSLN-IgG 1-FC-RP-Sal1 was used to amplify the Fc domain from an unrelated IgG 1. To obtain full-length heavy chains, the two gene fragments were fused together by overlap PCR using the primer pair MSLN-IgG1-VH-FP-Xba1/MSLN-IgG1-FC-RP-Sal 1. The full-length heavy chain gene fragment was then cloned into a recombinant plasmid containing an anti-mesothelin light chain insert via XbaI and SalI restriction sites.

Protein expression, purification and preliminary characterization

Anti-mesothelin M10 mAb was expressed in 293FS or CHO-S cells. The plasmid and transfection reagent PEI were mixed in a ratio of 1:3 and then added dropwise to 293FS or CHO-S cell cultures. Cells continued to grow for 5-7 days after transfection. Cell cultures were harvested by centrifugation at 8000rpm for 20 minutes. The culture supernatant containing the target Protein was loaded onto Protein a Sepharose 4Fast Flow column chromatography (GE Healthcare) and purified according to the manufacturer's instructions.

The purified protein was subjected to SDS-PAGE. On non-reducing SDS-PAGE, M10 mAb showed an apparent molecular weight (aMW) of approximately 150 kDa. On reducing SDS-PAGE, the heavy and light chains had apparent molecular weights of approximately 55kDa and 30kDa, respectively (data not shown). The M10 mAb CDR sequences according to the Kabat numbering system are shown in table 1. The amino acid sequences of the light chain variable region (VL) and the heavy chain variable region (VH) are shown in Table 2. The complete light and heavy chain sequences of the M10 mAb are shown in table 3.

TABLE 1 CDR sequences of M10 mAb

LCDR1	SGDALPKQYAF(SEQ ID NO:1)
		LCDR2	KDSERPS(SEQ ID NO:2)
LCDR3	QSADSSATSVI(SEQ ID NO:3)
		HCDR1	DYAMH(SEQ ID NO:6)
HCDR2	GISWNSGSIGYADSVKG(SEQ ID NO:7)
		HCDR3	DSGSSGWYGYFQH(SEQ ID NO:8)

TABLE 2 VL and VH sequences of M10 mAb

TABLE 3 light and heavy chain sequences of M10 mAb

Example 3 binding of anti-mesothelin monoclonal antibody to mesothelin

ELISA was performed according to standard protocols to determine the binding affinity of anti-mesothelin M10 mAb to recombinant human mesothelin (AcroBiosystems). Briefly, recombinant human mesothelin was coated overnight at 4 ℃ on Corning EIA/RIA high binding 96-well plates (Corning corporation) at 50ng per well and blocked with 3% skim milk in PBS (ph 7.4). Five-fold serial dilutions of antibody were added and incubated at room temperature for 2 hours. Plates were washed with PBS containing 0.05% Tween 20. Bound antibody was detected by HRP conjugated streptavidin (nano Biological). The assay was developed with TMB substrate (Solarbio) at room temperature and monitored with a microplate reader at 450 nm. Half maximal binding (EC) was calculated by fitting the data to Langmuir adsorption isotherms ₅₀ ). The results are shown in FIG. 4.

The results indicate that M10 mAb can be administered at 0.2nMEC ₅₀ Binding to recombinant human mesothelin indicates that the M10 mAb has high binding affinity for human mesothelin.

Example 4 binding of anti-mesothelin monoclonal antibodies to cancer cell lines

To measure the binding capacity of the anti-mesothelin M10 mAb to cell surface-bound mesothelin, flow cytometry was performed with the mesothelin-positive cancer cell line H226. Will be about 5X10 ⁵ The individual cells were incubated with the antibody (10. mu.g/mL) on ice for 1 hour. Cells were washed once with Pbs (PBSA) containing 0.1% bovine serum albumin and resuspended in 100 μ L PBSA. Then 1 μ L of anti-human IgG (Fc-specific) -FITC conjugate (Sigma) was added and incubated for 30 min. Cells were washed once with PBSA and then used for flow cytometry analysis. The results are shown in FIG. 5.

The results indicated that M10 mAb binds well to H226, indicating that M10 mAb has good binding capacity for mesothelin-positive tumor cell line.

Example 5 ADCC killing of human cancer cell lines mediated by anti-mesothelin monoclonal antibodies

To evaluate ADCC killing by M10 mAb, H226 cells were used as target cells and NK cells as effector cells. Resuscitating frozen NK cells (

Biotechnologies, cat #: PB56-N-1CW) and in RPMI1640 complete medium containing 20% FBS, 1% penicillin/streptomycin and 50IU IL-2, at 5% CO ₂ The cells were incubated overnight at 37 ℃ in an incubator. Preparation of H226 cells to 1X 10 cells Using complete Medium ⁵ Concentration of individual cells/mL, 100. mu.L/well in 96-well plates, and 5% CO ₂ Was cultured overnight in a 37 ℃ incubator. The following day, a series of concentrations (400. mu.g/mL, 40. mu.g/mL and 0. mu.g/mL) of M10 mAb were prepared in complete medium with IgG4 isotype antibody as negative control. 50 μ L of antibody solution was added to a 96-well plate containing H226 cells. NK cells were harvested and diluted to 3X 10 ⁵ cells/mL, then 50 μ L/well into 96-well plates. The plates were kept at 5% CO ₂ The culture was carried out in an incubator at 37 ℃ for 72 hours. The final concentrations of antibody were 100. mu.g/mL, 10. mu.g/mL, and 0. mu.g/mL, respectively.After 72h incubation, the medium was removed and replaced with 100 μ L/well of fresh complete medium containing 10% CCK8, and the plates were incubated at 37 ℃ for 30 min. OD at 450nm was measured using an ELISA reader. The killing efficiency was calculated as follows:

(OD _{tumor + NK + 0. mu.g/mL mab} -OD _{Tumor + NK + x μ g/mL mab} )/OD _{Tumor + NK + 0. mu.g/mL mab} ×100％,

Wherein x represents 10 or 100.

ADCC killing of H226 cells by M10 mAb is shown in figure 6. The results indicate that the M10 mAb induced less than 10% killing of tumor cells at 10 μ g/mL, more than 30% killing of tumor cells at 100 μ g/mL, while the control antibody IgG4 isotype did not induce any ADCC at the highest concentration of 100 μ g/mL, indicating that ADCC is triggered by the specific binding of the M10 mAb to mesothelin-positive tumor cells H226 and the recruitment of NK cells by the Fc portion of the mAb. This indicates that the M10 mAb has a potent ability to induce ADCC killing of mesothelin-positive tumor cells.

Example 6 construction and preliminary characterization of anti-mesothelin bispecific antibodies

Bispecific T-cell engagers (BiTE) are a novel class of bispecific antibodies that direct cytotoxic T-cells to kill cancer cells by simultaneously binding to a tumor antigen and a T-cell antigen, such as the CD3 molecule on the surface of T-cells. HBiTE as described in PCT application number PCT/US2018/016524 (which is incorporated herein by reference in its entirety) is a specific form of BiTE. HBiTE has a light chain and a heavy chain that form heterodimers. The light chain comprises, from N-terminus to C-terminus, an anti-target (e.g., tumor antigen) VL domain, anti-CD 3 VL-CL, and monomeric human IgG1 Fc (e.g., mfc 7.2). The heavy chain comprises, from N-terminus to C-terminus, an anti-target VH domain, anti-CD 3 VH-CH1 and monomeric human IgG1 Fc (e.g., mfc 7.2). Monomer fc7.2 contains two amino acid mutations (T366L and Y407H) that inhibit Fc homodimerization. To generate MSLNXCD 3 HBiTE, the VL and VH domains of the above anti-mesothelin antibody were fused to the N-terminus of the VL and VH domains of anti-CD 3 Fab by linkers GGGGSGGGGSGGGGS (SEQ ID NO:22) and GSSGGGGSGGGGS (SEQ ID NO:23), respectively. The anti-CD 3 Fab was further fused to the N-terminus of mfc 7.2. The light and heavy chains were constructed into a single vector pDin1 for expression in mammalian cells. Construction and preliminary characterization of bispecific antibodies targeting mesothelin and CD3 (M10-based MSLN x CD3 HBiTE) were performed as follows.

Cloning of bispecific antibodies targeting mesothelin and CD3

To generate constructs based on M10 MSLN × CD3 HBiTE bispecific antibody, the following primers were used:

MSLN-VL-forward, 5' ACTACAGGTGTCCACTCCTCCTATGTGCTGACT CAGC 3 (sense) (SEQ ID NO: 31);

MSLN-VL-reverse, 5 'GTAGGATCCTAGGACGGTCAGCTTGGTC 3' (antisense) (SEQ ID NO: 32);

bnIgG20H1,5 'GTGTTCTAGAGCCGCCACCATGGAATGGAGCTGGG TCTTTC 3' (sense) (SEQ ID NO: 33);

MSLN-VH-Forward, 5 'GGCTTACAGATGCCAGATGTCAGCTGGTGGAG ACCGG 3' (sense) (SEQ ID NO: 34);

MSLN-VH-reverse, 5 'GATAGAGCTCCCTCCACCTGAGGAGACGGTG ACC 3' (antisense) (SEQ ID NO: 35).

To generate bispecific antibodies, gene segments of VL and VH domains were amplified from M10 Fab using primer pairs MSLN-VL-forward/MSLN-VL-reverse and MSLN-VH-forward/MSLN-VH-reverse, respectively. The PCR product was fused to the 3' end of the H and L leader sequences by overlap PCR using the primer pairs bnIgG20H 1/MSLN-VL-reverse and bnIgG20L 1/MSLN-VH-reverse, respectively. The H leader-VL gene fragment was digested with XbaI and BamHI and cloned into HBiTE-derived pDin1 vector containing anti-CD 3 hSP34 Fab and the entire Fc fragment. The L leader-VH gene fragment was then further cloned into a recombinant plasmid containing the H leader-VL insert by HindIII and SacI restriction sites. The MSLN x CD3 HBiTE based on M10 is named CMD 007.

Protein expression, purification and preliminary characterization

CMD007 was expressed in 293FS or CHO-S cells. The plasmid and transfection reagent PEI were mixed in a ratio of 1:3 and then added to 293FS or CHO-S cell cultures. Cells continued to grow for 5-7 days after transfection. Cell cultures were harvested by centrifugation at 8000rpm for 20 minutes. The culture supernatant containing the target Protein was loaded onto Protein a Sepharose 4Fast Flow column chromatography (GE Healthcare) and purified according to the manufacturer's instructions.

The purified protein was subjected to SDS-PAGE. CMD007 showed an apparent molecular weight (aMW) of about 120kDa on non-reducing SDS-PAGE. On reducing SDS-PAGE, the heavy and light chains were close to each other, with an apparent molecular weight of about 62kDa (data not shown). The CDR sequences of CMD007 according to the Kabat numbering system are shown in Table 4. The amino acid sequences of the light chain variable region (VL) and the heavy chain variable region (VH) are shown in Table 5. The light and heavy chain sequences of CMD007 are shown in table 6.

TABLE 4 CDR sequences of CMD007

TABLE 5 VL and VH sequences of CMD007

TABLE 6 light and heavy chain sequences of CMD007

Example 7 binding of bispecific antibody to mesothelin and CD3

To determine the binding affinity of bispecific antibody CMD007 for mesothelin and CD3, an ELISA experiment was performed as described in example 3, wherein human mesothelin or human CD3 protein was used for coating. The results are shown in FIGS. 7A to 7B.

The results showed that CMD007 has an EC of 12.1nM ₅₀ Bind human CD3 (FIG. 7A) with an EC of 8.3 nM ₅₀ Binding to human mesothelin (fig. 7B). These results indicate that CMD007 can bind to mesothelin and CD3 protein with an affinity suitable for use as BiTE to trigger T cell killing of tumor cells.

Example 8 binding of bispecific antibodies targeting mesothelin and CD3 to cancer cell lines

To determine the binding affinity of bispecific antibody CMD007 to cell surface expressed mesothelin and CD3, flow cytometry was performed using mesothelin-positive cancer cell line H226, CD 3-positive Jurkat cell line, mesothelin-negative cancer cell LS174T (negative control), and LS174T-MSLN stably transfected with mesothelin (2B 6). The procedure was similar to that described in example 4. The results are shown in FIGS. 8A to 8D.

The results showed that CMD007 bound well to H226, Jurkat and LS174T-MSLN (2B6) cells, but did not bind to mesothelin-negative LS174T cells. This indicates that CMD007 can bind to mesothelin-expressing cancer cells and CD 3-expressing cells.

Example 9 bispecific antibody mediated T cell activation

To assess the ability and specificity of CMD007 to activate human T cells in the presence of mesothelin-expressing tumor cells, target cells (one mesothelin-negative cell LS174T and two mesothelin-positive cells LS174T-MSLN (2B6) and H226) were plated at 1 × 10 in 100 μ L of RMPI1640 complete medium per well ⁴ (for H226) or 2X 10 ⁴ (for LS174T and LS174T-MSLN (2B6)) density of individual cells were seeded in 96-well plates. 50 μ L of CMD007, a 5-fold serial dilution, bispecific antibody CMD006-M2 (containing anti-CD 3 Fab, irrelevant TAA antibody variable fragment, and mFc7.2) was added to each well as a negative control. Then 50. mu.L of RMPI1640 complete medium per well at 1X 10 ⁵ Density of individual cells add effector cells (Jurkat cells), target cells: the effector cell ratio was 1:10 (H226: Jurkat) or 1:5(LS 174T: Jurkat and LS174T-MSLN (2B6): Jurkat). After 24 hours of incubation, using a BD FACS Calibur,assays were developed using the CD3 monoclonal antibody (17A2), human CD69 monoclonal antibody (FN50), anti-CD 25/IL2RA Antibody (APC), and mouse monoclonal antibody according to the manufacturer's instructions.

First, the expression of the early activation marker CD69 was determined using H226, LS174T-MSLN (2B6) and LS174T cells. CMD007 effectively activated human Jurkat cells (63%) in the presence of mesothelin-positive H226 cells. CMD007 also activated human T cells efficiently (65%) for LS174T-MSLN (2B6) cells. For negative controls in which CMD007 was incubated with Jurkat cells alone, or CMD007 was incubated with mesothelin-negative cells LS174T and Jurkat cells, or control antibody CMD006-M2 was incubated with Jurkat and LS174T-MSLN (2B6) cells, no expression of CD69 was detected (fig. 9). The results showed that T cells were activated only when CMD007 simultaneously bound CD3 antigen of Jurkat cells and mesothelin antigen of tumor cells.

Next, LS174T-MSLN (2B6) and LS174T cells were used to determine expression of the T cell activation marker CD 25. When CMD007 was incubated with mesothelin-positive LS174T-MSLN (2B6) cells and Jurkat cells, it was observed that about 45% of Jurkat cells expressed CD 25. For the negative control group in which CMD007 was incubated with mesothelin-negative LS174T cells and Jurkat cells or the control antibody CMD006-M2 was incubated with LS174T-MSLN (2B6) and Jurkat cells, no expression of CD25 was detected (fig. 10). These results indicate that T cells are activated only when CMD007 binds to Jurkat cells expressing CD3 and mesothelin-expressing tumor cells, indicating that T cell activation is specifically target tumor antigen dependent.

Example 10 bispecific antibody-mediated killing of human cancer cell lines

Bispecific T cell engagers can bind to both tumor antigens and T cell antigens (e.g., CD3 molecules on the surface of T cells) resulting in T cell aggregation and activation, ultimately leading to killing of tumor cells. To evaluate the killing efficiency of the bispecific antibody CMD007, a CCK8 assay was performed using three mesothelin expressing tumor cell lines H226, N87, and LS174T-MSLN (2B6) as target cells.

Target cells (H226 is 1X 10) ⁴ ，N87Is 3 x10 ⁴ LS174T-MSLN (2B6) is 2X 10 ⁴ ) The inoculation was overnight in 100. mu.L of RPMI1640 complete medium. At the same time, the frozen PBMC were thawed and seeded in 30mL RPMI1640 complete medium overnight. The following day PBMC (H226 and LS174T-MSLN (2B6) were added at 1X 10 in 50. mu.L RPMI1640 complete medium ⁵ N87 is 3X 10 ⁵ ). Then, 50 μ L of antibody (H226 and N87 serially diluted 5-fold from 4 μ g/mL, LS174T-MSLN (2B6) serially diluted 5-fold from 3.2 μ g/mL) was added to each well, and IgG4 isotype antibody was used as a negative control. After 48 hours of incubation, the medium was removed and 100. mu.L of RPMI1640 complete medium containing 20% CCK8 was added, in CO ₂ Incubate in the incubator for 30 minutes. Cell killing activity was measured using a microplate reader according to the manufacturer's instructions. The results are shown in FIGS. 11 to 13.

The results showed that almost 100% of the tumor cells N87 and LS174T-MSLN (2B6) were killed in the presence of CMD007 and PBMC. EC of CMD007 for killing of N87 ₅₀ EC for killing of LS174T-MSLN (2B6) by CMD007 at 1.381ng/mL (FIG. 11) ₅₀ It was 1.685ng/mL (FIG. 12). For H226 cells, CMD007 showed moderate killing efficiency, with about 50% of tumor cells killed in the presence of PBMC, and EC of CMD007 for H226 killing ₅₀ It was 0.812ng/mL (FIG. 13).

In summary, the results indicate that CMD007 has potent killing ability against multiple cancer cell lines expressing mesothelin.

Example 11 bispecific antibody mediated tumor growth inhibition in mice

Pharmacokinetic determination

Three NOD/SCID mice were administered intravenously 300 μ g of CMD007 on day 0. Plasma samples were collected at time points 0.25 hours, 5 hours, 21 hours, 49 hours, and 69 hours post-treatment for measurement of antibody serum concentrations by ELISA.

The results showed that the serum concentration of CMD007 was gradually decreased, but remained high until the endpoint, the calculated serum half-life (t) _1/2 ) About 57.3 hours (fig. 14).

In vivo tumor growth inhibition

Will effect cellsPBMC(100μL，1×10 ⁶ ) And mesothelin-expressing tumor cell LS174T-MSLN (2B6) (100. mu.L, 1X 10) ⁶ ) Mixed in 100 μ L matrigel and inoculated subcutaneously into the right abdomen of B-NDG mice. In the experimental group, mice were injected intravenously with CMD007 at 50. mu.g/kg or 200. mu.g/kg. An IgG4 isotype antibody of 200. mu.g/kg was used as a negative control. These mice were dosed twice weekly. Three weeks after treatment, mice were sacrificed and tumor volume and body weight of the mice were measured. The tumor growth inhibition rate was calculated using the following formula:

(average body weight of control group-average body weight of experimental group)/average body weight of control group.

The results showed that CMD007 showed effective inhibition of tumor growth in a dose-dependent manner (fig. 15A). There was only a small change in body weight of mice in all groups (fig. 15B). CMD007 at 200. mu.g/kg showed 95% tumor growth inhibition and CMD007 at 50. mu.g/kg showed 61% tumor growth inhibition (FIG. 15C).

In summary, the results indicate that CMD007 has a long serum half-life and can specifically and effectively inhibit the growth of mesothelin-expressing tumor cells, indicating its potential for the treatment of mesothelin-positive cancers.

While preferred embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention herein. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Sequence listing

<110> Zhejiang Shimai pharmaceutical Co., Ltd

<120> antibodies against mesothelin and uses thereof

<130> C21W0953

<160> 35

<170> PatentIn version 3.5

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Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala Phe

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Lys Asp Ser Glu Arg Pro Ser

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Gln Ser Ala Asp Ser Ser Ala Thr Ser Val Ile

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<223> M10 mAb VL

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Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

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Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Phe Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Ala Thr Ser

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 5

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<223> M10 mAb light chain

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Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

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Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Phe Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Ala Thr Ser

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

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Asp Tyr Ala Met His

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Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys

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Gly

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Asp Ser Gly Ser Ser Gly Trp Tyr Gly Tyr Phe Gln His

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<220>

<223> M10 mAb VH

<400> 9

Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly

35 40 45

Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Ser Gly Ser Ser Gly Trp Tyr Gly Tyr Phe Gln His Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

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<223> M10 Fab heavy chain

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Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly

35 40 45

Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Ser Gly Ser Ser Gly Trp Tyr Gly Tyr Phe Gln His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly

210 215 220

Gln Ala Gly His His His His His His Gly Asp Tyr Lys Asp Asp Asp

225 230 235 240

Asp Lys Gly

<210> 11

<211> 450

<212> PRT

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<220>

<223> M10 mAb heavy chain

<400> 11

Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly

35 40 45

Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Ser Gly Ser Ser Gly Trp Tyr Gly Tyr Phe Gln His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 12

<211> 14

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<223> M10 HBiTE CD3 LCDR1

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Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

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Gly Ala Asn Lys Arg Ala Pro

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<211> 9

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Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

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<223> M10 HBiTE CD3 VL

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Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

35 40 45

Gly Leu Ile Gly Gly Ala Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Leu Ser Gly Asp Glu Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 16

<211> 561

<212> PRT

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<223> M10 HBiTE light chain

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Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala

20 25 30

Phe Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Ala Thr Ser

85 90 95

Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Val Thr

115 120 125

Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu

130 135 140

Ser Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

145 150 155 160

Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly

165 170 175

Ala Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu

180 185 190

Ser Gly Asp Glu Ala Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp

195 200 205

Phe Ala Val Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

210 215 220

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

245 250 255

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

260 265 270

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

275 280 285

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

290 295 300

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

305 310 315 320

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

325 330 335

Arg Gly Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

340 345 350

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

355 360 365

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

370 375 380

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

385 390 395 400

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

405 410 415

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

420 425 430

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

435 440 445

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

450 455 460

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Leu

465 470 475 480

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

485 490 495

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

500 505 510

Asp Ser Asp Gly Ser Phe Phe Leu His Ser Lys Leu Thr Val Asp Lys

515 520 525

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

530 535 540

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

545 550 555 560

Lys

<210> 17

<211> 7

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<400> 17

Gly Phe Thr Phe Asn Thr Tyr

1 5

<210> 18

<211> 8

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<223> M10 HBiTE CD3 HCDR2

<400> 18

Arg Ser Lys Tyr Asn Asn Tyr Ala

1 5

<210> 19

<211> 14

<212> PRT

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<220>

<223> M10 HBiTE CD3 HCDR3

<400> 19

His Gly Asn Phe Gly Ser Ser Tyr Val Ser Tyr Phe Ala Tyr

1 5 10

<210> 20

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> M10 HBiTE CD3 VH

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Ser Ser Tyr Val Ser Tyr Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 21

<211> 582

<212> PRT

<213> Artificial sequence

<220>

<223> M10 HBiTE heavy chain

<400> 21

Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly

35 40 45

Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Ser Gly Ser Ser Gly Trp Tyr Gly Tyr Phe Gln His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

130 135 140

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

145 150 155 160

Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys

165 170 175

Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala

180 185 190

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

195 200 205

Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

210 215 220

Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly Ser Ser

225 230 235 240

Tyr Val Ser Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

245 250 255

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

260 265 270

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

275 280 285

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

290 295 300

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

305 310 315 320

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

325 330 335

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

340 345 350

Asp Lys Lys Val Glu Pro Lys Ser Cys Pro Pro Cys Pro Ala Pro Glu

355 360 365

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

370 375 380

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

385 390 395 400

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

405 410 415

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

420 425 430

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

435 440 445

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

450 455 460

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

465 470 475 480

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

485 490 495

Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

500 505 510

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

515 520 525

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu His Ser Lys

530 535 540

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

545 550 555 560

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

565 570 575

Ser Leu Ser Pro Gly Lys

580

<210> 22

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> M10 HBiTE light chain linker

<400> 22

ggggsggggs ggggs 15

<210> 23

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> M10 HBiTE heavy chain linker

<400> 23

gssggggsgg ggs 13

<210> 24

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

gtgtaagctt accatgggtg tgcccactca ggtcctgggg t 41

<210> 25

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

cttacagatg ccagatgttc ctatgtgctg actcag 36

<210> 26

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

cggaattctt atgaacattc tgtaggggcc ac 32

<210> 27

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

tgttctagag ccaccatgga atggagctgg gtctttctct tcttcctgtc agtaactaca 60

ggtgtccact cccagctggt ggaga 85

<210> 28

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

atgtgtgagt tttgtcacaa gatttgggct caact 35

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

gacaaaactc acacatgccc accgt 25

<210> 30

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

ctgagtcgac ttatttaccc ggggacaggg aga 33

<210> 31

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

actacaggtg tccactcctc ctatgtgctg actcagc 37

<210> 32

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

gtaggatcct aggacggtca gcttggtc 28

<210> 33

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

gtgttctaga gccgccacca tggaatggag ctgggtcttt c 41

<210> 34

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

ggcttacaga tgccagatgt cagctggtgg agaccgg 37

<210> 35

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

gatagagctc cctccacctg aggagacggt gacc 34

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds mesothelin, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises amino acid sequences set forth in SEQ ID NOs: 1-3, and the VH comprises amino acid sequences as set forth in SEQ ID NOs: 6-8, and HCDR 1-3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of VL is identical to SEQ ID NO: 4, and the amino acid sequence of said VH has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein

The amino acid sequence of the VL is shown in SEQ ID NO: 4, and the amino acid sequence of the VH is shown as SEQ ID NO: shown at 9.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is of an isotype selected from IgG, IgA, IgM, IgE, and IgD.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG 4.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, whereinThe antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab') ₂ Fv, scFv and ds-scFv.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody is a monoclonal antibody.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain being identical to the amino acid sequence of SEQ ID NO: 5, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody is a bispecific antibody or a multispecific antibody.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the antibody is a bispecific antibody further comprising a second antigen-binding region that binds a second antigen.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the second antigen is a tumor-associated antigen or an immune cell antigen.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the second antigen is a T cell antigen.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the T cell antigen is selected from the group consisting of T Cell Receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG 2D.

14. The antibody or antigen-binding fragment thereof of claim 10, wherein the second antigen is CD3 and the second antigen-binding region comprises a VL and a VH, wherein the VL comprises amino acid sequences set forth in SEQ ID NOs: 12-14, and said VH comprises amino acid sequences set forth in SEQ ID NOs: 17-19, shown as HCDR 1-3.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the second antigen-binding region comprises a VL and a VH, the amino acid sequence of the VL being identical to SEQ ID NO: 15, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the second antigen-binding region comprises a VL and a VH, the amino acid sequence of the VL being as set forth in SEQ ID NO: 15, the amino acid sequence of the VH is shown as SEQ ID NO: shown at 20.

17. The antibody or antigen-binding fragment thereof of any one of claims 14 to 16, wherein the VL of the second antigen-binding region is optionally linked to the C-terminus of the VL of the antibody that specifically binds mesothelin through a first linker, and the VH of the second antigen-binding region is optionally linked to the C-terminus of the VH of the antibody that specifically binds mesothelin through a second linker, wherein the first and second linkers are the same or different.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein the amino acid sequence of the first linker is as set forth in SEQ ID NO:22 and the amino acid sequence of the second linker is as set forth in SEQ ID NO: shown at 23.

19. The antibody or antigen-binding fragment thereof of any one of claims 14 to 18, wherein the bispecific antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain being identical to SEQ ID NO: 16, and the amino acid sequence of said heavy chain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

20. The antibody or antigen-binding fragment thereof of any one of claims 10 to 19, wherein the bispecific antibody is a bispecific T cell engager (BiTE).

21. A bispecific antibody or antigen-binding fragment thereof comprising a first mesothelin-binding antigen-binding region comprising a VL and a VH, and a second CD 3-binding antigen-binding region comprising a VL and a VH, wherein

The VL of the first antigen-binding region comprises amino acid sequences set forth in SEQ ID NOs: 1-3, and the VH of the first antigen-binding region comprises amino acid sequences set forth in SEQ ID NOs: HCDR 1-3 as shown in 6-8; and is

The VL of the second antigen binding region comprises amino acid sequences set forth in SEQ ID NOs: 12-14, and the VH of the second antigen-binding region comprises an amino acid sequence set forth in SEQ ID NOs: 17-19, shown as HCDR 1-3.

22. The bispecific antibody or antigen-binding fragment thereof of claim 21, wherein

The amino acid sequence of the VL of the first antigen-binding region is identical to SEQ ID NO: 4, and the amino acid sequence of the VH of the first antigen-binding region shares at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9 has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity; and is

The amino acid sequence of the VL of the second antigen-binding region is identical to SEQ ID NO: 15, and the amino acid sequence of the VH of the second antigen-binding region shares at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 20 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

23. The bispecific antibody or antigen-binding fragment thereof of claim 22, wherein

The amino acid sequence of the VL of the first antigen-binding region is shown in SEQ ID NO: 4 and the amino acid sequence of the VH of the first antigen-binding region is as set forth in SEQ ID NO: 9 is shown in the figure; and is

The amino acid sequence of the VL of the second antigen-binding region is shown in SEQ ID NO: 15 and the amino acid sequence of the VH of the second antigen-binding region is as shown in SEQ ID NO: shown at 20.

24. The bispecific antibody or antigen-binding fragment thereof of any one of claims 21 to 23, wherein the VL of the second antigen-binding region is optionally linked to the C-terminus of the VL of the first antigen-binding region by a first linker, and the VH of the second antigen-binding region is optionally linked to the C-terminus of the VH of the first antigen-binding region by a second linker, wherein the first linker and the second linker are the same or different.

25. The bispecific antibody or antigen-binding fragment thereof of claim 24, wherein the amino acid sequence of the first linker is as set forth in SEQ ID NO:22 and the amino acid sequence of the second linker is as set forth in SEQ ID NO: shown at 23.

26. The bispecific antibody or antigen-binding fragment thereof of any one of claims 21 to 25, wherein the bispecific antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain being identical to the amino acid sequence of SEQ ID NO: 16, and the amino acid sequence of said heavy chain has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

27. The bispecific antibody or antigen-binding fragment thereof of any one of claims 21 to 26, wherein the bispecific antibody is a bispecific T-cell engager (BiTE).

28. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-20 or the bispecific antibody or antigen-binding fragment thereof of any one of claims 21-27.

29. A vector comprising the nucleic acid of claim 28.

30. A host cell comprising the nucleic acid of claim 28 or the vector of claim 29.

31. A pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, or a bispecific antibody or antigen-binding fragment thereof according to any one of claims 21 to 27; and (ii) a pharmaceutically acceptable carrier or excipient.

32. The pharmaceutical composition of claim 31, further comprising a second therapeutic agent.

33. The pharmaceutical composition of claim 32, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

34. The pharmaceutical composition of claim 32 or 33, wherein the second therapeutic agent is selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid.

35. A conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, or a bispecific antibody or antigen-binding fragment thereof according to any one of claims 21 to 27, and a chemical moiety conjugated thereto.

36. The conjugate of claim 35, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

37. Use of an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 20, the bispecific antibody or antigen-binding fragment thereof of any one of claims 21 to 27, the pharmaceutical composition of any one of claims 31 to 34, or the conjugate of claim 35 or 36 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is mesothelioma, breast, pancreatic, lung, gastric, and ovarian cancer, wherein the cancer is selected from mesothelioma, breast, pancreatic, lung, gastric, and ovarian cancer.

38. The use of claim 37, wherein the cancer is Triple Negative Breast Cancer (TNBC).

39. The use of claim 37 or 38, further comprising administering a second therapeutic agent to the subject.

40. The use of claim 39, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

41. The use of claim 39 or 40, wherein the second therapeutic agent is selected from a Bruton's Tyrosine Kinase (BTK) inhibitor, a PI3K inhibitor, an HDAC inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, an ERK inhibitor, a MAPK inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, and a glucocorticoid.