Antigen binding proteins targeting MSLN

Abstract

An isolated antigen binding protein capable of targeting MSLN, the isolated antigen binding protein comprising at least one CDR in an antibody heavy chain variable region VH, the VH comprising an amino acid sequence represented by any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 45. The invention also relates to a chimeric antigen receptor containing the antigen binding protein and application of the chimeric antigen receptor in the aspect of preventing and / or treating tumors.

Description

Antigen binding proteins targeting MSLN Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting MSLN, a chimeric antigen receptor comprising the antigen binding protein, and applications thereof. Background Art

[0002] Mesothelin (MSLN) is a cell surface glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol complex. The mesothelin gene encodes a 69 kDa precursor protein, which is hydrolyzed by furin-like convertases into two chains: the approximately 40 kDa C-terminal membrane-bound protein, which is the mature mesothelin, and the approximately 30 kDa N-terminal fragment, known as megakaryocyte-promoting factor (MPF), which is shed and released extracellularly. Both MPF and the membrane-anchored MSLN are N-glycosylated. MPF can promote megakaryocyte colony formation in vitro, while the membrane-anchored MSLN can interact with MUC16 and play a key role in cell adhesion. Therefore, the membrane-anchored MSLN is currently the target of targeted therapies. Currently, the term "MSLN" specifically refers to the C-terminal 40 kDa fragment of MSLN, i.e., the membrane-anchored MSLN.

[0003] Mesothelin is a glycoprotein present on the cell surface of mesothelial cells of the peritoneal, pleural and pericardial cavities. Mesothelin is predominantly expressed (overexpressed) in mesotheliomas, i.e., cancer / tumor cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer and endometrial cancer. In contrast, its expression is limited in normal cells such as mesothelial cells.

[0004] Therefore, as a target with development potential, it is urgent to develop more binding proteins that can effectively target MSLN to play a more effective therapeutic role in diseases caused by MSLN overexpression.

[0005] Summary of the Invention

[0006] The present application provides an isolated antigen-binding protein that specifically binds to MSLN. The present application also provides a chimeric antigen receptor comprising the antigen-binding protein, and cells comprising and / or expressing the chimeric antigen receptor, wherein the cells have one or more of the following characteristics: (1) strong proliferation ability; (2) ability to kill target cells expressing MSLN; (3) secretion of cytokines upon stimulation by target cells; and (4) inhibition of tumor cell growth.

[0007] On the one hand, the present application provides an isolated antigen-binding protein comprising at least one CDR in an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.

[0008] In certain embodiments, the isolated antigen binding protein is capable of specifically binding to the mesothelin (MLSN) protein.

[0009] In certain embodiments, the MSLN is a human MSLN.

[0010] In certain embodiments, the isolated antigen-binding protein is capable of competing with a reference antibody for binding to MSLN, wherein the reference antibody comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups:

[0011] 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0012] 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0013] 3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and

[0014] 4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0015] In certain embodiments, the isolated antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.

[0016] In certain embodiments, the isolated antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 19 and SEQ ID NO: 26.

[0017] In certain embodiments, the isolated antigen-binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25.

[0018] In certain embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.

[0019] In certain embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one of the following groups:

[0020] 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0021] 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0022] 3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and

[0023] 4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0024] In certain embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:50.

[0025] In certain embodiments, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.

[0026] In certain embodiments, the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:51.

[0027] In certain embodiments, the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:22 and SEQ ID NO:29.

[0028] In certain embodiments, the isolated antigen-binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:52.

[0029] In certain embodiments, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 23 and SEQ ID NO: 30.

[0030] In certain embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0031] In certain embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any one of the following groups:

[0032] 1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0033] 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0034] 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0035] 4) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 22, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 23, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0036] 5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0037] 6) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 32, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0038] 7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:34, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:13, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7;

[0039] 8) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0040] 9) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0041] 10) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:40, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7;

[0042] 11) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:42, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7; and

[0043] 12) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0044] In certain embodiments, the isolated antigen-binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.

[0045] In certain embodiments, the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0046] In certain embodiments, the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0047] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0048] In certain embodiments, the antigen-binding fragment is a VHH.

[0049] In certain embodiments, the isolated antigen-binding protein is a VHH comprising the amino acid sequence of any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45.

[0050] In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region derived from an IgG heavy chain constant region.

[0051] In certain embodiments, the antibody heavy chain constant region is derived from human IgG.

[0052] In certain embodiments, the antibody heavy chain constant region is derived from the heavy chain constant region of human IgG1.

[0053] In certain embodiments, the antibody heavy chain constant region comprises the Fc region of IgG.

[0054] In certain embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:61.

[0055] On the other hand, the present application also provides a chimeric antigen receptor, which comprises a targeting portion, and the targeting portion comprises the antigen binding protein described in the present application.

[0056] In certain embodiments, the chimeric antigen receptor comprises a costimulatory domain, which comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0057] In certain embodiments, the costimulatory domain is an intracellular costimulatory signaling region derived from 4-1BB.

[0058] In certain embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:53.

[0059] In certain embodiments, the chimeric antigen receptor comprises an intracellular signaling domain, which comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0060] In certain embodiments, the intracellular signaling domain is a signaling domain derived from CD3ζ.

[0061] In certain embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:55.

[0062] In certain embodiments, the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0063] In certain embodiments, the transmembrane region is a transmembrane region derived from CD8.

[0064] In certain embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:54.

[0065] In certain embodiments, the chimeric antigen receptor comprises a hinge region between the targeting portion and the transmembrane region, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0066] In certain embodiments, the hinge region is a hinge region derived from CD8.

[0067] In certain embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:56.

[0068] In certain embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0069] In certain embodiments, the signal peptide is derived from the signal peptide of the CD8 protein.

[0070] In certain embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:57.

[0071] In certain embodiments, the chimeric antigen receptor further comprises a low-density lipoprotein receptor-related protein or a fragment thereof.

[0072] In certain embodiments, the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0073] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

[0074] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:58.

[0075] On the other hand, the present application also provides a polypeptide comprising the antigen-binding protein.

[0076] In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein and / or the chimeric antigen receptor.

[0077] In certain embodiments, the nucleic acid molecule comprises a promoter.

[0078] In certain embodiments, the promoter is a constitutive promoter.

[0079] In certain embodiments, the promoter is the EF1α promoter.

[0080] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0081] In certain embodiments, the vector comprises a viral vector.

[0082] In certain embodiments, the vector comprises a lentiviral vector.

[0083] On the other hand, the present application also provides a cell comprising the antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule and / or the vector.

[0084] In certain embodiments, the cell is an immune effector cell.

[0085] In certain embodiments, the cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and / or pluripotent stem cells.

[0086] In certain embodiments, the cell is a T cell.

[0087] In certain embodiments, the cells contain and / or express low-density lipoprotein receptor-related protein or a fragment thereof.

[0088] In certain embodiments, the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0089] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

[0090] In certain embodiments, the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:58.

[0091] On the other hand, the present application also provides a method for preparing modified immune effector cells, which comprises culturing the cells under conditions such that the antigen binding protein and / or the chimeric antigen receptor are expressed.

[0092] On the other hand, the present application also provides a method for preparing modified immune effector cells, which comprises introducing the vector into immune effector cells.

[0093] On the other hand, the present application also provides a pharmaceutical composition comprising the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0094] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a medicament for preventing, treating and / or alleviating diseases or conditions associated with abnormal expression of MSLN.

[0095] In certain embodiments, the disease or condition associated with abnormal expression of MSLN comprises a tumor.

[0096] In certain embodiments, the tumor comprises a solid tumor.

[0097] In certain embodiments, the tumor comprises a non-solid tumor.

[0098] In certain embodiments, the tumor comprises a tumor expressing the MSLN antigen.

[0099] In certain embodiments, the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct cancer, triple-negative breast cancer, and / or endometrial cancer.

[0100] On the other hand, the present application also provides a method for preventing, treating and / or alleviating diseases or conditions associated with abnormal expression of MSLN, the method comprising administering the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition to a subject in need.

[0101] In certain embodiments, the disease or condition associated with abnormal expression of MSLN comprises a tumor.

[0102] In certain embodiments, the tumor comprises a solid tumor.

[0103] In certain embodiments, the tumor comprises a non-solid tumor.

[0104] In certain embodiments, the tumor comprises a tumor expressing the MSLN antigen.

[0105] In certain embodiments, the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct cancer, triple-negative breast cancer, and / or endometrial cancer.

[0106] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0108] Figure 1 shows the results of phage Pool ELISA.

[0109] Figures 2A-D show the screening results of positive clones.

[0110] FIG3 shows the flow cytometry detection of the specific binding of the antigen binding protein of the present application to MSLN.

[0111] Figure 4 shows the ForteBio test results.

[0112] Figure 5 shows the pCORE-VHH plasmid map.

[0113] Figure 6 shows a schematic diagram of the MSLN-VHH CAR structure.

[0114] FIG7A shows the electrophoresis diagram of the PCR results.

[0115] Figure 7B shows the electrophoresis of vector enzyme digestion

[0116] FIG8 shows the flow cytometry positive rate detection of SK-OV3-MSLN overexpression cell line.

[0117] Figure 9 shows the expansion fold of CAR-T cells after repeated stimulation with target cells.

[0118] FIG10 shows the results of cell killing by CAR-T cells in vitro.

[0119] FIG11A shows the results of IFN-γ cytokine secretion by CAR-T cells in vitro.

[0120] Figure 11B shows the results of IL-2 cytokine secretion by CAR-T cells in vitro.

[0121] FIG12A shows a tumor growth curve after administration of the CAR-T cells described in this application.

[0122] FIG12B shows a graph of animal body weight after administration of the CAR-T cells described in this application. DETAILED DESCRIPTION

[0123] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0124] Definition of terms

[0125] As used herein, the term "MSLN," also known as mesothelin, CAK1 antigen, or promegakaryocytic enhancer factor, is a protein present on normal mesothelial cells and overexpressed in some tumor cells. As used herein, the term may encompass MSLN protein or functionally active fragments thereof. As used herein, the term may also encompass homologs, analogs, or variants of MSLN protein. For example, the MSLN may include human MSLN.

[0126] In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability that has been separated from its naturally occurring state. The "isolated antigen-binding protein" may comprise an antigen-binding portion and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes its antigen binding. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody, or an alternative protein framework region or an artificial framework region having a grafted variable region (CDR) or a CDR derivative. For example, the antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein may bind to the MSLN protein. For example, the antigen-binding protein may compete with a reference antibody for binding to the MSLN protein. For example, the antigen-binding protein may comprise an antibody heavy chain variable region VH. For example, the antigen-binding protein may comprise at least one CDR derived from an antibody heavy chain variable region VH. For example, the VH may comprise HCDR3, HCDR2, and / or HCDR1. For example, the VH may comprise a framework region H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1. For example, the VH may include a framework region H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2. For example, the VH may include a framework region H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3. For example, the VH may include a framework region H-FR4, wherein the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3. For example, the antigen-binding protein may be a VHH. For example, the antigen-binding protein may include an antibody heavy chain constant region, wherein the antibody heavy chain constant region may be derived from IgG. For example, the antibody heavy chain constant region may be derived from human IgG. For example, the antibody heavy chain constant region may be derived from human IgG1.

[0127] The term "antibody" as used herein includes intact antibodies and binding fragments thereof. Generally, fragments compete with the intact antibody from which they are derived for specific antigen binding. Optionally, antibodies or binding fragments thereof can be chemically conjugated to other proteins or expressed as fusion proteins with other proteins. For example, the antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. For example, the binding protein of the antibody or binding fragment thereof can include MSLN. For example, the antibody or binding fragment thereof can be specific for MSLN.

[0128] The term "antigen-binding fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. For example, the antigen-binding fragment can include Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments, tandem Fv fragments, VHH, and bispecific antibodies. For example, the antigen-binding fragment can be a VHH. For example, the antigen-binding fragment can bind to MSLN. For example, the antigen-binding fragment can be specific for MSLN.

[0129] In this application, the term "VHH" generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody. VHHs may also be referred to as nanobodies (Nb) and / or single-domain antibodies. For example, the VHH may bind to MSLN. For example, the VHH may be specific for MSLN.

[0130] In the present application, the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The term "heavy chain constant region" is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The term "light chain constant region" is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors.

[0131] As used herein, the term "reference antibody" refers to an antibody that competitively binds to the same epitope of MSLN as the isolated antigen-binding protein. The reference antibody may include a heavy chain variable region (VH). For example, the reference antibody may have three CDR sequences. For example, the VH of the reference antibody may include HCDR1, HCDR2, and HCDR3. For example, the CDR sequences may be identical to the CDR sequences of the isolated antigen-binding protein.

[0132] In this application, the term "IgG" refers to polypeptides belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.

[0133] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds to an antigen or target. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may further include a low-density lipoprotein receptor-related protein or a fragment thereof. For example, the chimeric antigen receptor may include a signal peptide. The extracellular domain of CAR binds to the target antigen on the surface of the target cell, resulting in CAR clustering and transmitting the activation stimulus to the CAR-containing cell. CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death of cells expressing the target antigen in a manner that is independent of major histocompatibility (MHC). For example, the extracellular structure may include the above-mentioned antigen binding protein. For example, the extracellular structure may specifically bind to MSLN.

[0134] In the present application, the term "intracellular domain" refers to an intracellular domain comprising any truncated portion sufficient to transduce an activation signal. The intracellular domain may include an intracellular signal region and / or a costimulatory signal region. The term "intracellular signal region" refers to an intracellular region that can produce a signal promoting the immune effector function of CAR cells (e.g., CART cells or NK cells expressing CAR). For example, the intracellular signal region may include an intracellular signal region of one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and at least one domain comprising an ITAM. For example, the intracellular signal region may be a signal transduction domain derived from CD3ζ. The term "costimulatory signal region" refers to a part of the CAR that can transduce effector signals in the intracellular signal region. For example, the costimulatory signal region can include an intracellular costimulatory signal region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. For example, the costimulatory signal region can be an intracellular costimulatory signal region derived from 4-1BB.

[0135] In this application, the term "transmembrane region" refers to a domain of a peptide, polypeptide or protein that is capable of crossing the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane. For example, the membrane-spanning region can include the membrane-spanning domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the membrane-spanning region can be derived from the membrane-spanning region of CD8.

[0136] In this application, the term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 according to the EU numbering system of Kabat. The hinge region is typically a dimeric molecule composed of two polypeptides having the same amino acid sequence. The hinge region generally includes about 25 amino acid residues and is flexible, allowing the antigen-binding regions to move independently. The hinge region can be subdivided into three domains: upper, middle, and lower hinge domains. For example, the hinge region can comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be derived from the hinge region of CD8.

[0137] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein that is an endocytic receptor. It is widely distributed in the body and has great differences between tissues. Its main function is to take up cholesterol into cells for cell proliferation and the synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein can be from any vertebrate. For example, the low-density lipoprotein receptor-related protein or its fragment can be located at the C-terminus of the intracellular signaling region. For example, the low-density lipoprotein receptor-related protein or its fragment can include one or more selected from the following group: low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or its fragment can be low-density lipoprotein receptor-related protein 6 or its fragment.

[0138] In this application, the term "signal peptide" refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein that directs the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation. For example, the signal peptide is derived from the signal peptide of a CD8 protein.

[0139] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term may be used to refer to an amino acid polymer in which one or more amino acid residues is a synthetic chemical mimetic of the corresponding naturally occurring amino acid, as well as to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers. For example, the polypeptide may include the antigen-binding protein described herein.

[0140] In this application, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. The term "promoter" generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell. For example, the nucleic acid molecule can encode the antigen-binding protein and / or the chimeric antigen receptor. For example, the nucleic acid molecule can include a promoter. For example, the promoter can be a constitutive promoter. For example, the promoter can be the EF1α promoter.

[0141] In this application, the term "vector" generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached. For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0142] In this application, the term "cell" refers to a cell to which a nucleic acid can be transfected, and the term "cell" includes prokaryotic cells for plasmid propagation, and eukaryotic cells for nucleic acid expression and encoded polypeptide production. For example, a cell can include the antigen binding protein, the nucleic acid molecule and / or the vector. For example, the cell can be an immune effector cell. The term "immune effector cell" generally refers to an immune cell that participates in an immune response and exercises effector functions. For example, the exercise of effector functions can include clearing foreign antigens or promoting immune effector responses, etc. For example, immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells and / or pluripotent stem cells. For example, an immune effector cell can be a T cell.

[0143] As used herein, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to a mammalian subject. For example, the pharmaceutical composition may include the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to prevent, treat, and / or alleviate a disease or condition associated with abnormal expression of MSLN. For example, the disease or condition associated with abnormal expression of MSLN may include a tumor. For example, the tumor may include a solid tumor and / or a non-solid tumor.

[0144] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be one that binds to that target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding may include, but does not require, exclusive binding.

[0145] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0146] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0147] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., specifically binding to MSLN). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example 1-30, 1-20 or 1-10, for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0148] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., specifically binding to MSLN).

[0149] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0150] In this application, the term "comprising" generally means including, encompassing, containing or encompassing. In some cases, it also means "being", "consisting of...".

[0151] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0152] Detailed Description of the Invention

[0153] Isolated antigen binding protein

[0154] In the present application, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb. In the present application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0155] CDR

[0156] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. In the present application, the isolated antigen-binding proteins are defined by the Kabat numbering system.

[0157] In the present application, the isolated antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.

[0158] In the present application, the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 27.

[0159] In the present application, the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 19 and SEQ ID NO: 26.

[0160] In the present application, the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 18 and SEQ ID NO: 25.

[0161] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:25; the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:19 and SEQ ID NO:26; and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:27.

[0162] In the present application, the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise an amino acid sequence selected from any one of the following groups:

[0163] 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0164] 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0165] 3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and

[0166] 4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0167] FR

[0168] In this application, antibody framework region FR refers to the portion of the antibody variable region that is present between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide a skeleton for presenting three CDRs in three-dimensional space to form an antigen-binding surface.

[0169] In the present application, the isolated antigen-binding protein may comprise H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:50.

[0170] In the present application, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.

[0171] In the present application, the isolated antigen-binding protein may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:51.

[0172] In the present application, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 22 and SEQ ID NO: 29.

[0173] In the present application, the isolated antigen-binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:52.

[0174] In the present application, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 23 and SEQ ID NO: 30.

[0175] In the present application, the isolated antigen-binding protein may comprise H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0176] In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.

[0177] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0178] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7.

[0179] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7.

[0180] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:21, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:22, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:23, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0181] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 28, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 29, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7.

[0182] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:32, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0183] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:34, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0184] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:36, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0185] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:38, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0186] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:40, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0187] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:42, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0188] For example, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7.

[0189] VH / VHH

[0190] In the present application, the isolated antigen-binding protein may comprise a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.

[0191] In the present application, the antigen-binding fragment may be a VHH, and the VHH may comprise the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:45.

[0192] Heavy chain constant region

[0193] In the present application, the isolated antigen-binding protein may include a heavy chain constant region. The heavy chain constant region refers to a region comprising at least three heavy chain constant domains: CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. Furthermore, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region); IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region); and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0194] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from IgG. In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG. In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG1. In the present application, the heavy chain constant region of the antigen-binding protein may include an Fc region of IgG. For example, the Fc region may include the amino acid sequence shown in SEQ ID NO:61.

[0195] Chimeric antigen receptor

[0196] On the other hand, the present application also provides a chimeric antigen receptor (CAR), which may comprise a targeting portion that binds to the MSLN protein. For example, the targeting portion that binds to the MSLN protein may be the antigen binding protein described in the present application.

[0197] For example, the CAR of the present application may comprise a VHH, which may comprise an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 45.

[0198] In the present application, the CAR may include an extracellular targeting portion that binds to the MSLN protein and an intracellular domain.

[0199] In the present application, the CAR may include an intracellular costimulatory signal region that may provide a stimulation signal. For example, the costimulatory signal region may include an intracellular costimulatory signal region of one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0200] For example, the costimulatory signal region can be an intracellular costimulatory signal region derived from 4-1BB. For example, the costimulatory signal region can comprise the amino acid sequence shown in SEQ ID NO:53.

[0201] In some cases, the CAR may include an intracellular signaling region, which may include a domain having at least one ITAM motif. The intracellular signaling domain can transmit the activation signal to the interior of the cell. For example, the intracellular signaling region may include an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and other domains comprising at least one ITAM.

[0202] For example, the intracellular signaling region can be a signaling domain derived from CD3ζ. For example, the intracellular signaling region can comprise the amino acid sequence shown in SEQ ID NO:55.

[0203] In some cases, the CAR may include a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane and may include a hydrophobic alpha helix. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane region can comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0204] For example, the transmembrane region may be a transmembrane region derived from CD8. For example, the transmembrane region may comprise the amino acid sequence shown in SEQ ID NO: 54.

[0205] In some cases, the CAR may include a hinge region, which may be located between the extracellular targeting portion and the transmembrane domain. For example, the hinge region may include a hinge region of one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0206] For example, the hinge region may be a hinge region derived from CD8. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 56.

[0207] In the present application, the CAR may further comprise a signal peptide at the N-terminus of the targeting portion that binds to the MSLN protein. For example, the signal peptide may be a signal peptide derived from the CD8 protein. For example, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 57.

[0208] In the present application, the CAR may further comprise a low-density lipoprotein receptor-related protein or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may include low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may include the amino acid sequence shown in SEQ ID NO: 58. For example, the nucleic acid molecule encoding the low-density lipoprotein receptor-related protein or a fragment thereof may include the nucleotide sequence shown in SEQ ID NO: 59.

[0209] In the present application, the sequence of the low-density lipoprotein receptor-related protein or its fragment in the CAR can be connected to the C-terminal sequence of the CAR through a self-cleavage peptide (e.g., 2A peptides such as T2A, P2A, and E2A). For example, the low-density lipoprotein receptor-related protein or its fragment can be connected to the C-terminus of the intracellular signaling region through T2A. For example, the cleavage peptide can comprise the amino acid sequence shown in SEQ ID NO:60.

[0210] In the present application, from N-terminus to C-terminus, the CAR may sequentially include a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described herein), the hinge region, the transmembrane domain, the costimulatory signal region, and the intracellular signal region. For example, from N-terminus to C-terminus, the CAR may sequentially include the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signal region derived from 4-1BB, and an intracellular signal region derived from CD3ζ, and the VHH may include SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: The amino acid sequence shown in any one of SEQ ID NO: 45.

[0211] In the present application, from N-terminus to C-terminus, the CAR may sequentially comprise a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, the intracellular signal region, and the low-density lipoprotein receptor-related protein or a fragment thereof. For example, from N-terminus to C-terminus, the CAR may sequentially comprise the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signal region derived from 4-1BB, an intracellular signal region derived from CD3ζ, and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 58, and the amino acid sequence may comprise any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 45.

[0212] In the present application, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, and the intracellular signal region.

[0213] In the present application, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, a targeting portion that binds to the MSLN protein (e.g., the antigen binding protein, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, the intracellular signal region and the low-density lipoprotein receptor-related protein or a fragment thereof.

[0214] Nucleic acid molecules

[0215] On the other hand, the present application also provides one or more nucleic acid molecules, which can be isolated nucleotides, deoxynucleotides and / or ribonucleotides of any length, and can encode the isolated antigen binding protein and / or the chimeric antigen receptor.

[0216] For example, the nucleic acid molecule may include a promoter. For example, the promoter may be a constitutive promoter. For example, the promoter may be an EF1α promoter.

[0217] On the other hand, the present application also provides one or more nucleic acid molecules comprising sequences capable of expressing the chimeric antigen receptor and the low-density lipoprotein receptor-related protein or its fragment in a cell. In the present application, the nucleic acid sequence encoding the chimeric antigen protein can be linked to the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or its fragment via a cleavage peptide.

[0218] carrier

[0219] On the other hand, the present application also provides a vector, which can include the nucleic acid molecule. The vector can transform, transduce or transfect host cells so that the genetic material elements it carries are expressed in the host cells. For example, the vector can include a promoter, a transcriptor, an enhancer, a replicon, a selection element and a reporter gene. For example, the vector can include components that assist entry into cells. In order to enable the nucleic acid molecule to replicate in the vector, the 5' end and the 3' end of the nucleic acid molecule can also contain long terminal repeats.

[0220] For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0221] cell

[0222] On the other hand, the application also provides cells, which may include the isolated antigen-binding proteins, the chimeric antigen receptor, the nucleic acid molecules and / or the vector. The cells may include offspring of individual cells. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome).

[0223] In certain embodiments, the cells may be immune effector cells. In certain embodiments, the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphocyte progenitor cells and / or pluripotent stem cells. For example,

[0224] In certain embodiments, the cell may be a T cell.

[0225] In the present application, the cell may comprise and / or express the CAR. In the present application, the cell may comprise and / or express the CAR and the low-density lipoprotein receptor-related protein or a fragment thereof.

[0226] Pharmaceutical composition

[0227] On the other hand, the present application also provides a pharmaceutical composition, which may include the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable adjuvant.

[0228] In certain embodiments, the pharmaceutical composition can also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention can include liquid, frozen and lyophilized compositions.

[0229] In certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0230] In certain embodiments, the pharmaceutical composition can comprise parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into a tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0231] Preparation method

[0232] In another aspect, the present application also provides a method for preparing the isolated antigen binding protein and / or the chimeric antigen receptor. The method may include culturing the cell under conditions such that the antigen receptor and / or the chimeric antigen receptor is expressed.

[0233] The present application also provides a method for preparing modified immune effector cells, which may include introducing the vector into immune cells.

[0234] use

[0235] On the other hand, the present application also provides the use of the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug, which can be used to prevent, alleviate and / or treat diseases and / or conditions.

[0236] On the other hand, the present application also provides a method for preventing, alleviating and / or treating a disease and / or condition, which may comprise administering the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject.

[0237] On the other hand, the present application also provides the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition, which are used to prevent, alleviate and / or treat diseases and / or conditions.

[0238] In the present application, the diseases and / or disorders may include diseases and / or disorders associated with abnormal expression of MSLN.

[0239] In the present application, the disease and / or disorder may include a tumor.

[0240] In the present application, the tumor may include solid tumors and / or non-solid tumors.

[0241] In the present application, the tumor may include hematological tumors and / or lymphomas.

[0242] In the present application, the tumor may include a tumor expressing the MSLN antigen.

[0243] In the present application, the tumor may include ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct cancer, triple-negative breast cancer and / or endometrial cancer.

[0244] In the present application, the subject may include a human or a non-human animal.

[0245] In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein.

[0246] On the other hand, the present application provides a kit or an administration device, which comprises the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0247] In another aspect, the present application also provides a method for detecting the presence and / or content of MSLN, comprising administering the isolated antigen-binding protein, polypeptide, chimeric antigen receptor, and / or cell described herein. In certain embodiments, the method may be an in vitro method. In certain embodiments, the method may be for non-diagnostic and non-therapeutic purposes.

[0248] On the other hand, the present application also provides a kit for detecting the presence and / or content of MSLN, which comprises the isolated antigen-binding protein, the polypeptide, the chimeric antigen receptor, and / or the cell described in the present application.

[0249] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0250] Example

[0251] Example 1 Screening of Nanobodies Targeting MSLN-his

[0252] 1.1 Construction of phage display immune nanoantibody library

[0253] First, alpacas expressing recombinant human MSLN and the extracellular domain (ECD) were immunized. This domain was attached to an Fc fragment to facilitate subsequent protein purification. Alpacas were immunized with the MSLN-Fc antigen according to protocols known in the art. Animal immunization was outsourced to Aikonde Biomedical Technology (Suzhou) Co., Ltd., with a single immunization dose of 1-2 mg of protein. After the third and fourth immunizations, 5 mL of peripheral blood was collected, serum was separated, and the immune effect was tested using ELISA. The ELISA immune titer reached above 1:16,000 (coated antigen 5 ug / mL, OD value greater than 2.0). After the shock immunization, 150 mL of peripheral blood was collected. PBMCs were then isolated and total RNA was extracted using an RNA extraction kit provided by QIAGEN. Finally, the extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit.

[0254] The variable regions (VHH) of the heavy chain antibodies were amplified by nested PCR using the following primers: Primers for the first round of PCR:

[0255] CALL001: 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ (SEQ ID NO: 46);

[0256] CALL002: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 47).

[0257] Primers for the second round of PCR:

[0258] VHH-Back: 5′-GATGGTGCAGCTGCAGGAGTCTGGRGGAGG-3′ (SEQ ID NO: 48);

[0259] VHH-For: 5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3' (SEQ ID NO: 49).

[0260] The target fragment was recovered from the gel and cloned into the phage display vector pMES4 (GenBank GQ907248) using restriction enzymes Pst I and Eco91 I (from Thermo). After desalting, the plasmid was electroporated into electrocompetent Escherichia coli TG1 to construct the phage display nanoantibody library NanoMSLN, and the library diversity was evaluated. The library size was calculated to be 1×10 9 , then 24 single clones were selected for sequencing, and a total of 21 sequences were successfully sequenced, of which 2 were useless sequences and 2 were duplicate sequences. Therefore, a total of 85.7% (18 / 21) of the sequences were available, and the total diversity of NanoMSLN was 8.5×10 8 .

[0261] 1.2 Targeting MSLN for panning

[0262] Plates were coated with 10 μg / ml of IgG-Fc and MSLN-His proteins and incubated overnight at 4°C. The following day, the plates were washed three times with 1× PBST (PBS containing 0.05% Tween 20), blocked with 0.5% BSA for 2 hours at room temperature, and washed three times with 1× PBST. 100 μl of the phage library NanoMSLN was added to the IgG1-Fc wells for negative selection. One hour later, phage from the IgG1-Fc wells were transferred to the MSLN-His wells for positive selection. After 1.5 hours, the plates were washed ten times with 1× PBST to remove phage that did not bind to the antigen. Finally, the plates were eluted with 100 μl / well of Glycine-HCl (pH 2.2), followed by neutralization with Tris-HCl (pH 8.0). Half of the eluted phage was used to infect TG1 cells in the logarithmic growth phase. Half an hour later, the cells were superinfected with M13KO7 cells and cultured overnight. The phage were precipitated the following day for the next round of selection. A similar screening process was repeated for 4 rounds. After the second round of positive screening, the cells were washed 20 times with 1× PBST, 30 times in the third round, and 40 times in the fourth round.

[0263] 1.3 Pool ELISA

[0264] One day in advance, a 96-well plate was coated with 2 μg / ml of MSLN-His, IgG1-Fc, and 0.5% BSA and allowed to stand overnight. The next day, the plates were washed three times with 1× PBST and blocked with 0.5% BSA for 2 hours. After blocking, the plates were washed three times with 1× PBST. 100 μl of phage library from each round was added and incubated at room temperature with shaking for 1 hour. After washing three times with 1× PBST, secondary antibodies Anti-M13-HRP (Sino Biological, Catalog No. 11973-MM05T-H) and Anti-Flag-HRP (abcam, Catalog No. ab1162) were added and incubated at room temperature for 30-60 minutes. After washing seven times with 1× PBST, TMB colorimetric solution was added. After 3-5 minutes, 2M phosphoric acid was added to terminate the reaction. The absorbance was read at 450 nm.

[0265] The results are shown in FIG1 , which show that after three rounds of screening using the phage display immune nanoantibody library NanoMSLN, obvious specific enrichment was observed.

[0266] 1.4 Phage enzyme-linked immunosorbent assay (ELISA) to identify specific positive monoclonal

[0267] After three rounds of panning, the reserved output from rounds 1, 2, and 3 was plated on 2YT / carb. Every other day, 96 single colonies were randomly selected from each well and placed in 800 μl of 2YT / carb / M13KO7, incubated with shaking overnight to generate phage. The day before, a 384-well plate was coated with 2 μg / ml of MSLN-His and IgG1-Fc and incubated overnight. The next day, the plate was washed three times with 1× PBST, then blocked with 0.5% BSA for 2 hours. The phage supernatant was collected by centrifugation. After blocking, the plate was washed three times with 1× PBST, 30 μl of phage supernatant was added, and the plate was incubated with shaking at room temperature for 1 hour. After three washes with 1× PBST, the secondary antibody Anti-M13-HRP was added and incubated at room temperature for 30-60 minutes. After seven washes with 1× PBST, TMB colorimetric solution was added. After 3-5 minutes, the reaction was terminated with 2M phosphoric acid, and the absorbance was read at 450 nm. A positive result was determined when the OD value of the sample well was at least 2 times greater than that of the control well (0.5% BSA or IgG1-Fc). Finally, the positive phage was reinfected with TG1 and sequenced. The amino acid sequences of each clone were analyzed using the sequence alignment software BioEdit. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same antibody strain.

[0268] A total of 15 specific sequences were obtained after monoclonal ELISA and positive clone sequencing. Among them, the amino acid sequence of CDR1 of MSLN45 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 8; the amino acid sequence of CDR1 of MSLN46 is shown in SEQ ID NO: 9, the amino acid sequence of CDR2 is shown in SEQ ID NO: 10, the amino acid sequence of CDR3 is shown in SEQ ID NO: 11, and the amino acid sequence of VHH is shown in SEQ ID NO: 15; the amino acid sequence of CDR1 of MSLN47 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 17; the amino acid sequence of CDR1 of MSLN48 is shown in SEQ ID NO: 18, the amino acid sequence of CDR2 is shown in SEQ ID NO: 19, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 20. NO:20, the amino acid sequence of VHH is shown in SEQ ID NO:24; the amino acid sequence of CDR1 of MSLN50 is shown in SEQ ID NO:25, the amino acid sequence of CDR2 is shown in SEQ ID NO:26, the amino acid sequence of CDR3 is shown in SEQ ID NO:27, and the amino acid sequence of VHH is shown in SEQ ID NO:31; the amino acid sequence of CDR1 of MSLN51 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, the amino acid sequence of CDR3 is shown in SEQ ID NO:3, and the amino acid sequence of VHH is shown in SEQ ID NO:33; the amino acid sequence of CDR1 of MSLN52 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO:10, the amino acid sequence of CDR3 is shown in SEQ ID NO:11, and the amino acid sequence of VHH is shown in SEQ ID NO:35; the amino acid sequence of CDR1 of MSLN53 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is shown in SEQ ID NO: NO:10, the amino acid sequence of CDR3 is shown in SEQ ID NO:11, and the amino acid sequence of VHH is shown in SEQ ID NO:37;The amino acid sequence of CDR1 of MSLN55 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 39; the amino acid sequence of CDR1 of MSLN57 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 41; the amino acid sequence of CDR1 of MSLN58 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: 43; the amino acid sequence of CDR1 of MSLN59 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR3 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH is shown in SEQ ID NO: NO:45 shown. ;

[0269] Example 2 Identification of the binding activity of nanobodies targeting MSLN

[0270] 2.1 Expression and purification of VHH-Fc fusion proteins in eukaryotic cells

[0271] Specific antibody sequences were selected from the sequencing results and cloned into the vector pcDNA3.4. The recombinant plasmid was transformed into Escherichia coli DH5α (Tiangen Biochemical Technology Co., Ltd., catalog number: CB101-02). The bacterial solution was then evenly spread on 2YT solid culture medium containing 100 μg / ml ampicillin and allowed to stand at 37°C overnight. The next day, a single clone was picked and cultured on a 37°C shaker for about 6 hours. Half of the bacterial solution was sent for sequencing, and the other half was stored at 4°C. The clones that were sequenced correctly were amplified and cultured, and the plasmid was extracted. The expression plasmid was transfected into EXPI293 using the PEI transfection method and expressed in a 37°C cell culture incubator for 5 days. Afterwards, the cell supernatant was collected and the antibody was purified using a Protein A affinity chromatography column. Finally, an antibody protein with a purity of over 90% was obtained.

[0272] 2.2 ELISA assay to detect the specific binding of VHH-Fc to human MSLN protein

[0273] High-absorption 384-well plates were coated with 2 μg / ml MSLN-His and 5% skim milk, incubated overnight at 4°C, washed three times with 1× PBST, and blocked with 5% skim milk for 2 hours at room temperature. During the blocking period, the MSLN-VHH antibody was diluted starting at 200 nM and then diluted 2.5-fold in a series of 11 dilutions. After blocking, the plates were washed three times with 1× PT, and the corresponding diluted primary antibody was added at 30 μl / well. The plates were incubated with shaking at room temperature for 1 hour. After washing three times with 1× PBST, the secondary antibody, Anti-human IgG Fc Antibody HRP (abcam, Cat. No. ab99759), was added and incubated at room temperature for 30-60 minutes. After washing seven times with 1× PBST, TMB colorimetric solution was added. After 3-5 minutes, 2M phosphoric acid was added to terminate the reaction. The absorbance was read at 450 nm.

[0274] The results are shown in Figure 2A-D. Thirteen sequences with high monoclonal ELISA signals were selected for ELISA detection. Except for MSLN49, all other sequences could bind to MSLN-his protein.

[0275] 2.3 Identification of the Binding Activity of Bivalent VHH-Fc to SK-VO3 by Flow Cytometry

[0276] SK-OV3 cells were revived and passaged. On the day of the experiment, cells were harvested and counted, and the cell density was adjusted to 1×10 6 / ml, 30ul per well (3×10 4 Representative MSLN nanobodies 45-59, the positive antibody P4, and the negative antibody caplacizumab were added to each well in a 3-fold gradient, starting at 200 nM, with a PBS control. The cells were mixed and incubated at 4°C for 1 hour. The wells were washed twice with PBS containing 0.1% BSA, centrifuged at 500g for 5 minutes at 4°C, and then dried. A 1:200 diluted fluorescent secondary antibody, Goat anti-Human IgG (H+L) Secondary Antibody [DyLight 650], was added to each well in 30ul of solution, mixed, and incubated at 4°C for 30 minutes. The cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500g for 5 minutes at 4°C, and then dried. The cells were resuspended at 30ul / well and read on a high-throughput flow cytometer (IQue). The collected data were fitted with a three-parameter curve using GraphPad.

[0277] The results are shown in FIG3 , which show that MSLN46, 52 and 53 can specifically bind to MSLN on the cell surface.

[0278] 2.4 ForteBio assays for affinity of blocking antibodies

[0279] Dilute the MSLN nanobody with flow cytometric binding activity to 100 nM and add the antibody to a 384-well plate. Dilute the human MSLN-His antigen to 100 nM in a 2-fold dilution series of seven steps, then add the antibody to the 384-well plate. Use an AHC probe (Sartorius, Cat. No. 18-5060) with an association time of 180 seconds, a dissociation time of 360 seconds, a baseline of 60 seconds, and three regeneration cycles of 5 seconds each. Then, use software to fit the association-dissociation curves and calculate the antibody affinity.

[0280] The results are shown in Figure 4. The ForteBio test results showed that the affinities of MSLN46, 52 and 53 were all at the nM level, significantly higher than the positive control antibody P4, with KD values ​​of 2.15E-09M, 2.58E-09M and 1.41E-09M, respectively.

[0281] Example 3 VHH sequence CAR-T vector construction, lentiviral packaging and CAR-T cell preparation

[0282] 3.1 Construction of VHH Sequence CAR-T Vector

[0283] The Genesis platform plasmid (Genesis self-constructed) was double-digested with SphI and NotI, and the linearized vector fragment was recovered and mixed with the target antibody sequence VHH at a 1:3 molar ratio. After homologous recombination, the fragment was transformed into E. coli DH5α competent cells. After heat shock for 90 seconds, the cells were plated on LB solid medium containing ampicillin and cultured overnight at 37°C. Single colonies were picked, and the plasmid was sequenced to verify its correctness. The plasmid was then shaken and aspirated for future use. The core plasmid is shown in Figure 5, and the CAR-T structure is shown in Figure 6. 41BB (SEQ ID NO: 53) and CD3ζ (SEQ ID NO: 55) were selected as costimulatory domains.

[0284] 3.2 Lentiviral packaging and titer determination

[0285] The vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third generation lentiviral vector system. The system has three plasmids: the packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein; the PMD2.G plasmid encoding the envelope protein VSV-G; and the constructed core plasmid containing the target gene CAR. The CAR gene in the core plasmid based on the BBz platform plasmid is expressed by the elongation factor-1α (EF-1α) promoter. The packaging process of the lentivirus is as follows: 1x10 6293T cells were suspended in 2 ml of 10% FBS DMEM medium and plated in a single well of a 6-well plate and cultured overnight; 1 ml of medium was aspirated, and 1 ml of 2.88 ug of packaging plasmid (psPAX2: PMD2.G: core plasmid = 3:2:4) and 8.64 ul of FuGENEHD transfection reagent were mixed, and 144 ul of Opti-MEM medium (50 times the mass of the plasmid) was added. After gently mixing, the cells were cultured at 37°C in a CO2 incubator for 12 hours; the medium containing the plasmid was removed, the cells were washed once with PBS, and then replaced with 2 ml of DMEM medium containing 5% FBS and cultured for 48 hours; 2.5 ml of viral supernatant was collected, centrifuged at 3000 rpm for 5 minutes, aliquoted and frozen at -80°C for use, and the titer was detected.

[0286] 3.3 VHH sequence CAR-T cell preparation

[0287] The method for producing CAR-T cells containing VHH sequences is as follows: human peripheral blood mononuclear cells are obtained by density gradient centrifugation; the peripheral blood mononuclear cells are resuspended in medium containing 200 U / ml interleukin-2 to a cell density of 2x10 6 / ml, CD3 / CD28 magnetic beads were added at a ratio of 1:3 (cells: magnetic beads) to activate T cells; the activated peripheral blood mononuclear cells were cultured in a 37°C CO2 incubator for 24 hours; the above-obtained lentiviral supernatant was added at a virus infection multiplicity (MOI) of 3, polybrene was added to a final concentration of 5ug / ml, the cell suspension was placed in a well plate and centrifuged at 1200rpm for 1 hour in a horizontal centrifuge; the well plate was returned to a 37°C CO2 incubator and cultured for 24 hours; centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells were resuspended in fresh X-VIVO medium containing 500U / ml interleukin-2 to a cell density of 0.6x106 / ml, and placed in a 37°C CO2 incubator for culture; the cells were counted every 2 days, and fresh X-VIVO medium containing 500U / ml interleukin-2 was added to adjust the cell density back to 0.6x10 6 / ml; CAR-T cells cultured for 9-14 days detect cell positivity: The lentivirus used to infect cells carries GFP. After the lentivirus infects the cells, the GFP positivity rate is detected by flow cytometry to obtain the CAR expression positivity rate. Cells with a positivity rate >20% can be used for tumor killing experiments.

[0288] Example 4 Construction and detection of overexpression cell line SK-OV3-MSLN

[0289] The full-length MSLN gene was introduced into SK-OV3 cells using lentiviral packaging and lentiviral infection to generate a SK-OV3-MSLN overexpressing cell line. pLV-C-GFPS was used as a vector to insert the full-length MSLN sequence and construct the recombinant plasmid pLV-C-GFPS-MSLN. PCR and enzyme digestion results are shown in Figure 7 . The PCR results are as follows (A): from left to right, Tiangen Marker IV, 1 and 2 are full-length MSLN bands (approximately 1900 bp); the vector was double-digested with Not I and Xba I as follows (B): Tiangen Marker IV, A and B vectors (approximately 8200 bp). After lentiviral infection, the recombinant plasmid was introduced into SK-OV3 cells, and SK-OV3-MSLN-positive cell lines were obtained by flow cytometry sorting and expanded into culture as target cells. The positive rate of SK-OV3-MSLN overexpressing cell lines was detected by flow cytometry. The results are shown in FIG8 . The positive rate of SK-OV3-MSLN overexpressing cells reached 97.98%.

[0290] Example 5 Cell Killing and Factor Detection of VHH Sequence CAR-T

[0291] 5.1 CAR-T repeated stimulation experiment in vitro

[0292] CAR-T cells were prepared according to Example 3. On the 9th to 12th day of amplification, the positive rate was detected and the CAR-T cells were resuspended in serum-free X-VIVO medium (manufacturer: LONZA) to 4×10 5 / ml as effector cells; OV-CAR-3 cells were resuspended in serum-free X-VIVO medium to a density of 4×10 5 The target cells were mixed at a density of 1000 / ml at a ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator. The culture medium was observed every 2 days and 1 volume of culture medium was added when the culture medium turned from orange to yellow. The cells were counted on the 4th to 5th day to calculate the expansion times. After counting, 5×10 5 CAR-T cells were expanded again for the second and third rounds using the same method as described above. The total expansion fold was calculated by multiplying the first round expansion fold by the second round expansion fold and then by the third round expansion fold.

[0293] The results of repeated stimulation are shown in Figure 9. The results show that after three rounds of repeated stimulation, the cumulative expansion of CAR-T cells (MSLN-52) reached 543.75 times, which is comparable to the control antibody P4 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 63, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 64).

[0294] 5.2 CAR-T cell killing assay in vitro

[0295] SK-OV3-MSLN cells were used as target cells, cells infected with CAR-T lentivirus were used as effector cells, and uninfected T cells were used as control effector cells. The specific experimental process was as follows: the infection efficiency of CAR was detected, and the infection ratio of CAR was adjusted to the same level in each group using uninfected T cells; the effector cell: target cell (effector-target ratio) was 3:1, 1:1, 0.3:1, and 0.1:1 in 200 μl X-VIVO medium, and the number of target cells was 1x10 4 / well, as the experimental group; wells containing only effector cells equal to the experimental group were used as the effector cell self-release background group; wells containing only target cells equal to the experimental group were used as the target cell self-release background group; the obtained cells were cultured in a 37°C CO2 incubator for 18 hours; 20μl 10x lysis buffer was added to some wells containing only target cells and reacted for 45 minutes as the maximum release of target cells. The obtained cell culture well plates were centrifuged at 300g for 5 minutes, and 50μl supernatant was collected for detection of the release of lactate dehydrogenase LDH. The detection method was referred to the instructions of the CytoTox96 non-radioactive cytotoxicity kit (manufacturer: Promega). The released LDH in the culture medium supernatant can be detected by a coupled enzyme reaction. The cell killing activity calculation formula is:

[0296] Toxicity % = 100 x (experimental group - effector cell self-release - target cell self-release + culture base value) / (maximum target cell release - target cell self-release).

[0297] According to the cell killing formula, the killing effect of each antibody sequence on the target cells was analyzed, and antibodies with obvious killing effects were selected for in vivo functional evaluation.

[0298] The cell killing results are shown in Figure 10. The results show that under the condition of an effector-target ratio of 1:1, MSLN-46 has the lowest cell killing effect, and there is little difference in the cell killing effect between MSLN-52 and MLSN-53, which is equivalent to the positive control P4.

[0299] 5.3 CAR-T in vitro cytokine secretion experiment

[0300] SK-OV-3-MSLN cells were used as target cells, cells infected with CAR-T lentivirus were used as effector cells, and uninfected T cells were used as control effector cells. The specific experimental process is as follows: to detect the infection efficiency of CAR, uninfected T cells were used to adjust the infection ratio of CAR to the same level in each group; the effector cell: target cell ratio (effector-target ratio) was 1:1, and the number of target cells was 2x10 in 200μl X-VIVO medium. 4 / well, as the experimental group; wells containing only effector cells equal to those in the experimental group served as the background group; the obtained cells were cultured in a 37°C CO2 incubator for 18 hours; the obtained cell culture well plates were centrifuged at 300g for 5 minutes, and 50μl of supernatant was collected for detection of IL2 and IFN-γ expression levels. The detection method was referred to the instructions of the R&D DuoSet ELISA kit (manufacturer: R&D SYSTEM).

[0301] The IFN-γ detection results are shown in Figure 11 (A), and the IL2 detection results are shown in Figure 12 (B). MSLN-46 secreted more IFN-γ, but had higher self-activation. Among the IL-2 results, MSLN-53 had the highest secretion, followed by MSLN-52, both of which were better than the positive control P4.

[0302] Example 6: In vivo efficacy verification of VHH sequence CAR-T

[0303] SK-OV-3-MSLN (3E6 / mouse) were subcutaneously implanted in the dorsal midsection of the right upper limb. When tumors reached 78 mm³, the NDG mice were divided into three groups: T cell, P4, and MSLN-VHH, with 5 mice in each group. Each group received 1E7 cryopreserved CAR-T cells via the tail vein. Post-administration body weight and tumor size were recorded, and various biological responses were observed. As shown in Figure 12A, mice showed no significant weight changes and were in good condition, with no unexplained deaths. As shown in Figure 12B, MSLN-VHH mice showed significant tumor suppression, and four mice achieved complete tumor elimination by Day 40, confirming their safety and anti-tumor efficacy.

[0304] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. An isolated antigen-binding protein comprising at least one CDR of an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO:

45.

2. The isolated antigen-binding protein according to claim 1, which is capable of specifically binding to the mesothelin (MLSN) protein.

3. The isolated antigen binding protein of claim 2, wherein the MSLN is human MSLN.

4. The isolated antigen-binding protein according to any one of claims 1 to 3, which is capable of competing with a reference antibody for binding to MSLN, wherein the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are selected from any one of the following groups: 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; 3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and 4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

27.

5. The isolated antigen-binding protein according to any one of claims 1 to 4, comprising a HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO:

27.

6. The isolated antigen-binding protein according to any one of claims 1 to 5, comprising a HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 19 and SEQ ID NO:

26.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, comprising HCDR1, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 18 and SEQ ID NO:

25.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 18 and SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 2, SEQ ID NO: 19 and SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO:

27.

9. The isolated antigen-binding protein according to any one of claims 1 to 8, comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups: 1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; 2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; 3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and 4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

27.

10. The isolated antigen-binding protein according to any one of claims 1 to 9, comprising H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:

50.

11. The isolated antigen-binding protein of claim 10, wherein the H-FR1 comprises the amino acid sequence of any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, and SEQ ID NO:

44.

12. The isolated antigen-binding protein according to any one of claims 1 to 11, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:

51.

13. The isolated antigen-binding protein of claim 12, wherein the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 22 and SEQ ID NO:

29.

14. The isolated antigen-binding protein according to any one of claims 1 to 13, comprising an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:

52.

15. The isolated antigen-binding protein of claim 14, wherein the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 23 and SEQ ID NO:

30.

16. The isolated antigen-binding protein according to any one of claims 1 to 15, comprising H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

7.

17. The isolated antigen-binding protein according to any one of claims 1 to 16, comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any one of the following groups: 1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 2) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 3) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 4) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 22, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 23, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7; 6) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 32, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 7) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:34, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:13, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:14, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7; 8) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7; 9) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 38, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; 10) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:40, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7; 11) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:42, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:7; and 12) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:44, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

7.

18. The isolated antigen-binding protein according to any one of claims 1 to 17, comprising a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO:

45.

19. The isolated antigen binding protein of any one of claims 1-18, which comprises an antibody or antigen binding fragment thereof.

20. The isolated antigen binding protein of claim 19, wherein the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

21. The isolated antigen binding protein of any one of claims 19-20, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

22. The isolated antigen binding protein of any one of claims 19-21, wherein the antigen binding fragment is a VHH.

23. The isolated antigen-binding protein of claim 22, wherein the VHH comprises the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO:

45.

24. The isolated antigen binding protein of any one of claims 1-23, comprising an antibody heavy chain constant region derived from the heavy chain constant region of IgG.

25. The isolated antigen binding protein of claim 24, wherein the antibody heavy chain constant region is derived from human IgG.

26. The isolated antigen binding protein of any one of claims 24-25, wherein the antibody heavy chain constant region is derived from the heavy chain constant region of human IgGl.

27. The isolated antigen binding protein of any one of claims 24-26, wherein the antibody heavy chain constant region comprises the Fc region of IgG.

28. The isolated antigen-binding protein of claim 27, wherein the Fc region comprises the amino acid sequence shown in SEQ ID NO:

61.

29. A chimeric antigen receptor comprising a targeting moiety comprising the antigen binding protein of any one of claims 1-28.

30. The chimeric antigen receptor of claim 29, comprising a costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

31. The chimeric antigen receptor of any one of claims 29-30, wherein the costimulatory domain is an intracellular costimulatory signaling region derived from 4-1BB.

32. The chimeric antigen receptor of any one of claims 30-31, wherein the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

53.

33. The chimeric antigen receptor of any one of claims 29-32, comprising an intracellular signaling domain, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

34. The chimeric antigen receptor of claim 33, wherein the intracellular signaling domain is a signaling domain derived from CD3ζ.

35. The chimeric antigen receptor of any one of claims 33-34, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:

55.

36. The chimeric antigen receptor of any one of claims 29-35, comprising a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. The chimeric antigen receptor according to claim 36 , wherein the transmembrane region is a transmembrane region derived from CD8.

38. The chimeric antigen receptor of claims 36-37, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

54.

39. The chimeric antigen receptor of any one of claims 29-38, comprising a hinge region between the targeting portion and the transmembrane region, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

40. The chimeric antigen receptor of claim 39, wherein the hinge region is a hinge region derived from CD8.

41. The chimeric antigen receptor of any one of claims 39-40, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

56.

42. The chimeric antigen receptor of any one of claims 29-41, further comprising a signal peptide.

43. The chimeric antigen receptor of claim 42, wherein the signal peptide is derived from the signal peptide of the CD8 protein.

44. The chimeric antigen receptor according to any one of claims 42-43, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:

57.

45. The chimeric antigen receptor of any one of claims 29-44, further comprising a low-density lipoprotein receptor-related protein or a fragment thereof.

46. ​​The chimeric antigen receptor of claim 45, wherein the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

47. The chimeric antigen receptor according to any one of claims 45-46, wherein the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

48. The chimeric antigen receptor of any one of claims 45-47, wherein the low-density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

58.

49. A polypeptide comprising the antigen binding protein of any one of claims 1-28.

50. One or more isolated nucleic acid molecules encoding the antigen binding protein of any one of claims 1-28 and / or the chimeric antigen receptor of any one of claims 29-48.

51. The nucleic acid molecule of claim 50, further comprising a promoter.

52. The nucleic acid molecule of claim 51, wherein the promoter is a constitutive promoter.

53. The nucleic acid molecule according to any one of claims 51-52, wherein the promoter is the EF1α promoter.

54. A vector comprising the nucleic acid molecule of any one of claims 50-53.

55. The vector of claim 54, comprising a viral vector.

56. The vector of any one of claims 54-55, comprising a lentiviral vector.

57. A cell comprising the antigen binding protein of any one of claims 1-28, the chimeric antigen receptor of any one of claims 29-48, the polypeptide of claim 49, the nucleic acid molecule of any one of claims 50-53, and / or the vector of any one of claims 54-56.

58. The cell of claim 57, which is an immune effector cell.

59. The cell of any one of claims 57-58, comprising a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell.

60. The cell according to any one of claims 57-59, which is a T cell.

61. The cell according to any one of claims 57-60, further comprising and / or expressing low-density lipoprotein receptor-related protein or a fragment thereof.

62. The cell according to claim 61, wherein the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

63. The cell according to any one of claims 61-62, wherein the low-density lipoprotein receptor-related protein or fragment thereof is low-density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

64. The cell according to any one of claims 61-63, wherein the low-density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

58.

65. The cell of any one of claims 61-64, wherein the low-density lipoprotein receptor-related protein or fragment thereof is an exogenous low-density lipoprotein receptor-related protein or fragment thereof.

66. A method of producing the antigen binding protein of any one of claims 1-28 and / or the chimeric antigen receptor of any one of claims 29-48, the method comprising culturing the cell of any one of claims 57-65 under conditions such that the antigen binding protein of any one of claims 1-28 and / or the chimeric antigen receptor of any one of claims 29-48 is expressed.

67. A method for preparing a modified immune effector cell, comprising introducing the vector of any one of claims 54-56 into the immune effector cell.

68. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-28, the chimeric antigen receptor of any one of claims 29-48, the polypeptide of claim 49, the nucleic acid molecule of any one of claims 50-53, the vector of any one of claims 54-56, and / or the cell of any one of claims 57-65, and optionally a pharmaceutically acceptable carrier.

69. Use of the isolated antigen-binding protein of any one of claims 1-28, the chimeric antigen receptor of any one of claims 29-48, the polypeptide of claim 49, the nucleic acid molecule of any one of claims 50-53, the vector of any one of claims 54-56, and / or the cell of any one of claims 57-65, and / or the pharmaceutical composition of claim 68 in the preparation of a medicament for preventing, treating and / or alleviating a disease or condition associated with abnormal expression of MSLN.

70. The use according to claim 69, wherein the disease or disorder associated with abnormal expression of MSLN comprises a tumor.

71. The use of claim 70, wherein the tumor comprises a solid tumor.

72. The use of claim 70, wherein the tumor comprises a non-solid tumor.

73. The use of any one of claims 70-72, wherein the tumor comprises a tumor expressing the MSLN antigen.

74. The use according to any one of claims 70-73, wherein the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell carcinoma, bile duct cancer, triple-negative breast cancer and / or endometrial cancer.

75. A method for preventing, treating and / or alleviating a disease or condition associated with abnormal expression of MSLN, the method comprising administering to a subject in need thereof the isolated antigen-binding protein of any one of claims 1-28, the chimeric antigen receptor of any one of claims 29-48, the polypeptide of claim 49, the nucleic acid molecule of any one of claims 50-53, the vector of any one of claims 54-56, and / or the cell of any one of claims 57-65, and / or the pharmaceutical composition of claim 68.

76. The method of claim 75, wherein the disease or condition associated with aberrant expression of MSLN comprises a tumor.

77. The method of claim 76, wherein the tumor comprises a solid tumor.

78. The method of claim 76, wherein the tumor comprises a non-solid tumor.

79. The method of any one of claims 76-78, wherein the tumor comprises a tumor expressing the MSLN antigen.

80. The method of any one of claims 76-79, wherein the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell cancer, bile duct cancer, triple-negative breast cancer, and / or endometrial cancer.

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