Antigen-binding proteins targeting MSLN and uses thereof

Abstract

The present application relates to an isolated antigen binding protein capable of targeting MSLN, said isolated antigen binding protein comprising at least one CDR in an antibody heavy chain variable region VH, said VH comprising an amino acid sequence as set forth in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 70. The present application further provides a chimeric antigen receptor comprising said antigen binding protein, and its use in treating tumors.

Description

[Technical field]

[0001] The present application relates to the biomedical field, in particular to an antigen-binding protein that targets MSLN, a chimeric antigen receptor comprising said antigen-binding protein, and uses thereof. [Background technology]

[0002] Mesothelin (MSLN) is a glycoprotein located on the cell surface, anchored to the cell membrane by glycosylphosphatidylinositol. The mesothelin gene encodes a 69 kDa precursor protein, which is hydrolyzed into two chains by furin-like invertase, and the C-terminal approximately 40 KD membrane-bound protein, i.e., mature mesothelin, is shed with an N-terminal approximately 30 KD fragment called megakaryocyte-promoting factor (MPF), which is released outside the cell. Both MPF and membrane-anchored MSLN are N-glycosylated, MPF can promote the formation of megakaryocyte clones in vitro, and membrane-anchored MSLN can interact with MUC16 and play an important role in the cell adhesion process, so that in current targeted therapy, both select membrane-anchored MSLN as the target, and therefore current MSLN technically refers to the C-terminal 40 KD fragment of MSLN, i.e., membrane-anchored MSLN.

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

[0004] The present application provides an isolated antigen binding protein capable of specifically binding to MSLN. The present application further provides a chimeric antigen receptor comprising said antigen binding protein, and a cell comprising and / or expressing said chimeric antigen receptor, said cell having one or more of the following characteristics: (1) high proliferation ability, (2) ability to kill target cells expressing MSLN, (3) secretion of cytokines under stimulation of target cells, and (4) inhibition of tumor cell proliferation.

[0005] In one aspect, the present application provides an isolated antigen binding protein comprising at least one CDR in an antibody heavy chain variable region VH, wherein said VH comprises an amino acid sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:70.

[0006] In some embodiments, the antigen binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:66.

[0007] In some embodiments, the antigen binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:34.

[0008] In some embodiments, the antigen binding protein comprises an HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:67.

[0009] In some embodiments, the antigen binding protein comprises an HCDR2, and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, and SEQ ID NO:35.

[0010] In some embodiments, the antigen binding protein comprises an HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:68 (X1X2X3MG, where X1 is N, R, S, T or Y, X2 is N or Y, and X3 is A, N or V).

[0011] In some embodiments, the antigen binding protein comprises an HCDR1, and the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:36.

[0012] In some embodiments, the antigen binding protein comprises HCDR1, HCDR2 and HCDR3, and said HCDR1, HCDR2 and HCDR3 are (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, 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: 1; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, 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: 9; (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; (6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29; and (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34; The amino acid sequence of the present invention includes any one of the amino acid sequences selected from the group consisting of:

[0013] In some embodiments, the antigen binding protein comprises an H-FR1, wherein the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:37.

[0014] In some embodiments, the antigen binding protein comprises an H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and wherein the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:5.

[0015] In some embodiments, the antigen binding protein comprises an H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0016] In some embodiments, the antigen binding protein comprises an H-FR4, wherein the N-terminus of the H-FR4 is linked directly or indirectly to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7.

[0017] In some embodiments, the antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:70.

[0018] In some embodiments, the antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:38.

[0019] In some embodiments, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0020] In some embodiments, the antigen-binding fragment comprises a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, a VHH, and / or a dAb.

[0021] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0022] In some embodiments, the antigen-binding fragment is a VHH, and the VHH comprises the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:70.

[0023] In some embodiments, the antigen-binding fragment is a VHH, and the VHH comprises the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:38.

[0024] In some embodiments, the antigen binding protein is capable of competing with a reference antibody for binding to MSLN (Mesothelin) protein, wherein the reference antibody comprises an antibody heavy chain variable region VH, the VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the reference antibody comprises (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, 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: 1; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, 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: 9; (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; (6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29; and (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34; The amino acid sequence of the present invention includes any one of the amino acid sequences selected from the group consisting of:

[0025] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from IgG.

[0026] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from human IgG.

[0027] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from human IgG1.

[0028] In some embodiments, the heavy chain constant region of the antigen binding protein comprises an IgG Fc region.

[0029] In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO:54.

[0030] In another aspect, the present application further provides a chimeric antigen receptor comprising a targeting moiety, said targeting moiety comprising said antigen binding protein.

[0031] In some embodiments, the chimeric antigen receptor comprises a costimulatory domain, the 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 for CD83, CD40, and MyD88.

[0032] In some embodiments, the costimulatory domain of the chimeric antigen receptor is an intracellular costimulatory signal region derived from 4-1BB.

[0033] In some embodiments, the costimulatory domain in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:39.

[0034] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling domain, the intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM.

[0035] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor is a signaling domain derived from CD3ζ.

[0036] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:41.

[0037] In some embodiments, the chimeric antigen receptor comprises a transmembrane region, the 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.

[0038] In some embodiments, the transmembrane region of the chimeric antigen receptor is a transmembrane region derived from CD8.

[0039] In some embodiments, the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:40.

[0040] In some embodiments, the chimeric antigen receptor comprises a hinge region between the targeting moiety 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.

[0041] In some embodiments, the hinge region of the chimeric antigen receptor is a hinge region derived from CD8.

[0042] In some embodiments, the hinge region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:42.

[0043] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0044] In some embodiments, the signal peptide of the chimeric antigen receptor is derived from the signal peptide of the CD8 protein.

[0045] In some embodiments, the signal peptide of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:43.

[0046] In some embodiments, the chimeric antigen receptor further comprises a low density lipoprotein receptor-related protein, or a fragment thereof.

[0047] In some embodiments, the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

[0048] In some embodiments, 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.

[0049] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:44.

[0050] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0051] In another aspect, the present application further provides a polypeptide comprising the antigen-binding protein described above.

[0052] In another aspect, the present application further provides one or more isolated nucleic acid molecules encoding said isolated antigen binding protein and / or said chimeric antigen receptor.

[0053] In some embodiments, the nucleic acid molecule comprises a promoter.

[0054] In some embodiments, the promoter is a constitutive promoter.

[0055] In some embodiments, the promoter is an EF1α promoter.

[0056] In another aspect, the present application further provides a vector comprising the above-described nucleic acid molecule.

[0057] In some embodiments, the vector comprises a viral vector.

[0058] In some embodiments, the vector comprises a lentiviral vector.

[0059] In another aspect, the present application further provides a cell comprising said antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule and / or said vector.

[0060] In some embodiments, the cell is an immune effector cell.

[0061] In some embodiments, the cells 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.

[0062] In some embodiments, the cell is a T cell.

[0063] In some embodiments, the cells comprise and / or express a low density lipoprotein receptor-related protein or a fragment thereof.

[0064] In some embodiments, the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

[0065] In some embodiments, 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.

[0066] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:44.

[0067] In another aspect, the present application further provides a method of producing a modified immune effector cell, said method comprising culturing said cell under conditions allowing expression of said antigen binding protein and / or said chimeric antigen receptor.

[0068] In another aspect, the present application further provides a method of producing a modified immune effector cell, comprising introducing the above-described vector into an immune effector cell.

[0069] In another aspect, the present application further provides a pharmaceutical composition comprising said isolated antigen binding protein, said chimeric antigen receptor, said polypeptide, said nucleic acid molecule, said vector and / or said cell, and optionally a pharma- ceutically acceptable vector.

[0070] In another aspect, the present application further provides a 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 manufacture of a medicament, wherein the medicament is used for preventing, treating and / or alleviating a disease or condition associated with abnormal expression of MSLN.

[0071] In some embodiments, the disease or condition associated with aberrant expression of MSLN comprises a tumor.

[0072] In some embodiments, the tumor comprises a solid tumor.

[0073] In some embodiments, the tumor comprises a non-solid tumor.

[0074] In some embodiments, the tumor comprises a tumor that expresses the MSLN antigen.

[0075] In some embodiments, the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial carcinoma, cholangiocarcinoma, triple-negative breast cancer, and / or endometrial cancer.

[0076] In another aspect, the present application further provides a method of preventing, treating and / or alleviating a disease or condition associated with aberrant expression of MSLN, the method comprising administering to a subject in need thereof the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0077] In some embodiments, the disease or condition associated with aberrant expression of MSLN comprises a tumor.

[0078] In some embodiments, the tumor comprises a solid tumor.

[0079] In some embodiments, the tumor comprises a non-solid tumor.

[0080] In some embodiments, the tumor comprises a tumor that expresses the MSLN antigen.

[0081] In some embodiments, the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial carcinoma, cholangiocarcinoma, triple-negative breast cancer, and / or endometrial cancer.

[0082] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the contents of the present application may allow those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention of the present application. Accordingly, the drawings and descriptions in the present application are merely illustrative, not limiting. [Brief description of the drawings]

[0083] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is given below. [Figure 1] The results of phage pool ELISA are shown. [Diagram 2] Selection of positive clones is shown. [Figure 3A-3D] 1 shows detection of affinity between the antigen-binding protein of the present application and MSLN. [Figure 4] 1 shows flow cytometry validation of the binding activity of the antigen-binding protein of the present application with 293T overexpressing human MSLN. [Diagram 5] 1 shows flow cytometry validation of non-specific binding of the antigen-binding protein of the present application to 293T, KERA, LO2, A549 and HACAT cells. [Figure 6] 1 shows the results of an in vitro repeated stimulation proliferation experiment of CAR-T cells. [Figure 7] 1 shows the results of in vitro killing of CAR-T cells. [Figure 8] 1 shows the results of detecting cytokine release from CAR-T cells in vitro. [Figure 9] 1 shows the results of detecting cytokine release from CAR-T cells in vitro. [Figure 10] 1 shows the results of detecting cytokine release from CAR-T cells in vitro. [Figure 11] 1 shows the results of an in vivo efficacy experiment using SK-OV3 tumor model mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Hereinafter, the embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0085] Definition of Terms In the present application, the term "MSLN", also referred to as mesothelin, or CAK1 antigen or prepro-megakaryocyte potentiating factor, is a protein present on normal mesothelial cells and overexpressed in some tumor cells. In the present application, the term may include MSLN protein or a functionally active fragment thereof. In the present application, the term may further include a homolog, analog, or variant of MSLN protein. For example, the MSLN may include human MSLN.

[0086] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability that has been removed from its naturally occurring state. The "isolated antigen-binding protein" may comprise an antigen-binding moiety and, optionally, a framework or framework moiety that allows the antigen-binding moiety to adopt a conformation that promotes its binding to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) from an antibody, or a candidate protein framework region or an artificial framework region with grafted variable regions (CDRs) or CDR derivatives. 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, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1. For example, the VH may comprise a framework region H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR2. For example, the VH may comprise a framework region H-FR3, and the H-FR3 is located between the HCDR2 and the HCDR3. For example, the VH may comprise a framework region H-FR4, and the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3. For example, the antigen binding protein may be a VHH. For example, the antigen binding protein may comprise an antibody heavy chain constant region, and 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.

[0087] The term "antibody" as used includes whole antibodies and binding fragments thereof. Generally, fragments compete with the whole antibody from which they are derived for specific binding to an antigen. Optionally, the antibody or binding fragment thereof may be chemically conjugated to other proteins or expressed in the form of a fusion protein with other proteins. For example, the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. For example, the binding protein of the antibody or binding fragment thereof may include MSLN. For example, the antibody or binding fragment thereof may have specificity for MSLN.

[0088] The term "antigen-binding fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. For example, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments and single chain Fv fragments, tandem Fv fragments, VHH, and bispecific antibodies. For example, the antigen-binding fragment may be a VHH. For example, the antigen-binding fragment may bind to MSLN. For example, the antigen-binding fragment may have specificity for MSLN.

[0089] In this application, the term "VHH" generally refers to an antibody comprising a variable antigen-binding domain of a heavy chain antibody. VHH is also called Nanobody (Nb) and / or single domain antibody. For example, the VHH can bind to MSLN. For example, the VHH can have specificity for MSLN.

[0090] In the present application, the antibody may comprise at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The term "heavy chain constant region" consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The term "light chain constant region" consists of one domain, CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxy 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 the antibodies can mediate the binding of the immunoglobulin to host tissues or factors.

[0091] In the present application, the term "reference antibody" refers to an antibody that can compete with the isolated antigen-binding protein for binding to the same epitope of MSLN. 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 match the CDR sequences of the isolated antigen-binding protein.

[0092] As used herein, the term "IgG" refers to a polypeptide belonging to the antibody class essentially encoded by the known immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.

[0093] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds an antigen or target, a transmembrane region, and an intracellular domain. For example, a hinge region is included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may further comprise a low density lipoprotein receptor-related protein or a fragment thereof. For example, the chimeric antigen receptor may comprise a signal peptide. The extracellular domain of the CAR binds to a target antigen on the surface of a target cell, resulting in CAR clustering and delivering an activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and induce proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate the death of cells expressing the target antigen independently of major histocompatibility (MHC). For example, the extracellular structure may comprise the antigen-binding protein. For example, the extracellular structure may specifically bind to MSLN.

[0094] In the present application, the term "intracellular domain" refers to an intracellular domain including any cleavage 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 capable of producing a signal that promotes immune effector function of a CAR-containing cell (e.g., a CART cell or a CAR-expressing NK cell). For example, the intracellular signal region may include an intracellular signal region of one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM. For example, the intracellular signal region may be a signaling domain derived from CD3zeta. The term "co-stimulatory signal region" refers to a portion of the CAR that can transduce an effector signal within the intracellular signal region. For example, the co-stimulatory signal region may comprise an intracellular co-stimulatory 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, ligand for CD83, CD40, and MyD88. For example, the co-stimulatory signal region may be an intracellular co-stimulatory signal region derived from 4-1BB.

[0095] As used herein, the term "transmembrane domain" refers to a domain of a peptide, polypeptide or protein that is capable of crossing the cytoplasmic membrane. These domains can be used to anchor the extracellular domain to the cell membrane. For example, the transmembrane region may comprise a transmembrane 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 transmembrane region may be derived from the transmembrane region of CD8.

[0096] In the present application, the term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain, for example, about positions 216 to 230 in the EU numbering system according to Kabat. A hinge region is usually a dimeric molecule consisting of two polypeptides having the same amino acid sequence. The hinge region generally contains about 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region. The hinge region may be divided into three domains, the upper, middle, and lower hinge domains. For example, the hinge region may include 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.

[0097] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to endocytosis receptors, which is widely distributed in the body and has a very large tissue differentiation, and its main function is to take up cholesterol into cells for cell proliferation and synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein may be derived from any vertebrate. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the intracellular signal region. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may include one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 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 6 or a fragment thereof.

[0098] In the present application, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of the nascent CAR protein that co- or post-translationally targets the nascent protein to the endoplasmic reticulum and subsequent surface expression. For example, said signal peptide is derived from the signal peptide of the CD8 protein.

[0099] In this application, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms may be used to refer to amino acid polymers in which one or more amino acid residues are artificial synthetic chemical mimics of their corresponding naturally occurring amino acids, and may be used to refer to naturally occurring amino acid polymers, those amino acid polymers containing modified residues, and non-naturally occurring amino acid polymers. For example, the polypeptide may include the antigen-binding protein.

[0100] In this application, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule may 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 operatively 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.

[0101] In the present application, the term "vector" generally refers to a molecule that can be attached to one or more nucleic acid molecules of the present application. For example, the vector may be a viral vector. For example, the vector may be a lentiviral vector.

[0102] In the present application, the term "cell" refers to a cell into 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 coding for polypeptide production. For example, the cell may include the antigen binding protein, the nucleic acid molecule, and / or the vector. For example, the cell may be an immune effector cell. In the present application, the term "immune effector cell" generally refers to an immune cell that participates in an immune response and performs an effector function. For example, performing the effector function may include removing foreign antigens or promoting an immune effector response. For example, the immune effector cell may include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphocyte progenitor cell, and / or a pluripotent stem cell. For example, the immune effector cell may be a T cell.

[0103] In this application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to a mammalian individual. 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 vector. 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.

[0104] In this application, the term "specifically binds" 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 the 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 an antibody that binds to the target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein, the epitope being conserved in proteins of different species. In some embodiments, specific binding may include, but is not required to include, exclusive binding.

[0105] As used herein, the term "subject" refers generally to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.

[0106] In the present application, such proteins, polypeptides and / or amino acid sequences should also be understood to include variants or homologues having at least the same or similar function as the above proteins or polypeptides.

[0107] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., specifically binds to MSLN). For example, the functional variant may include a protein or polypeptide that already has an amino acid modification by substitution, deletion, and / or insertion of at least one, for example 1 to 30, 1 to 20, or 1 to 10, also for example 1, 2, 3, 4, or 5 amino acids. The functional variant can basically retain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0108] In the present application, the homolog may 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 polypeptide (e.g., that specifically binds to MSLN).

[0109] In this application, the above-mentioned homology generally refers to the similarity, similarity or relationship between two or more sequences. The "sequence homology percentage" can be calculated by the following method: comparing two aligned sequences in a comparison window, determining the number of positions in which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exists in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to generate the sequence homology percentage. Alignment for determining sequence homology percentage can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment within the full length sequences being compared or within a region of the target sequence. The homology can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see WR Pearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and DJ Lipman and WR Pearson, "Rapid and Sensitive Protein Similarity Searching," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990.

[0110] In this application, the term "comprise" generally refers to the meaning of including, grouping together, containing or covering. In some cases, it also refers to the meaning of "is" or "consisting of."

[0111] In this application, the term "about" generally refers to a variation within 0.5% to 10% more or less of a specified value, for example, a variation within 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% more or less of a specified value.

[0112] Isolated antigen-binding proteins 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 a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, a VHH, and / or a dAb. In the present application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0113] CDR The CDR of an antibody is also called the complementarity determining region, and is a part of the variable region. The amino acid residues of the region can contact the antigen or antigen epitope. The antibody CDR can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, AbM, and Kabat / Chothia when considered together. These numbering systems are known in the art, and can be specifically referred to, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different numbering systems depending on the sequence and structure of the antibody. Using different numbering systems may result in differences in the CDR region. In the present application, the CDR covers the CDR sequence divided according to any CDR division method, and also covers its variants, and the variants include substitutions, deletions, and / or additions of one or more amino acids in the amino acid sequence of the CDR. For example, 1 to 30, 1 to 20, or 1 to 10, further for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions, and also covers homologues thereof, which may 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 with the amino acid sequence of the CDR. In the present application, the isolated antigen-binding protein is defined by the Kabat numbering system.

[0114] In the present application, the isolated antigen-binding protein may comprise at least one CDR in an antibody heavy chain variable region VH, for example, the VH may comprise an amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:70.

[0115] In the present application, the antigen binding protein may comprise an HCDR3, and the HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:66.

[0116] For example, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:34.

[0117] In the present application, the isolated antigen binding protein may comprise an HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 67. For example, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 30, and SEQ ID NO: 35.

[0118] In the present application, the isolated antigen binding protein may comprise an HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:68 (X1X2X3MG, where X1 is N, R, S, T or Y, X2 is N or Y, and X3 is A, N or V). For example, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:36.

[0119] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:68 (X1X2X3MG, wherein X1 is N, R, S, T or Y, X2 is N or Y, and X3 is A, N or V), the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:67, and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:66.

[0120] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, 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: 1; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, 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: 9; (3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; (4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; (5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; (6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29; and (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34; The amino acid sequence may comprise any one of the following groups:

[0121] FR In the present application, an antibody framework region FR refers to the portion of an antibody variable region that lies 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 scaffold for presenting the six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0122] In the present application, the isolated antigen binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:37.

[0123] In the present application, the isolated antigen binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5.

[0124] In the present application, the isolated antigen binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:6.

[0125] In the present application, the isolated antigen binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:7.

[0126] In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:32, and SEQ ID NO:37, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:5, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:6, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:7.

[0127] VH / VHH In the present application, the isolated antigen binding protein may comprise an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 70. For example, the VH may comprise an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO: 38.

[0128] In the present application, the antigen-binding fragment may be a VHH, and the VHH may comprise an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 70. For example, the VHH may comprise an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO: 38.

[0129] heavy chain constant region In the present application, the isolated antigen-binding protein may comprise 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 gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions further include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Furthermore, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. Some isotypes may be further divided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0130] In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG. In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG. In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG1. In the present application, the heavy chain constant region of the antigen-binding protein may comprise an IgG Fc region. For example, the Fc region may comprise the amino acid sequence shown in SEQ ID NO:54.

[0131] Chimeric Antigen Receptor In another aspect, the present application further provides a chimeric antigen receptor (CAR), which may comprise a targeting moiety that binds to the MSLN protein, for example, the targeting moiety that binds to the MSLN protein may be an antigen binding protein described herein.

[0132] For example, the CAR of the present application may comprise a VHH, and the VHH may comprise an amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:38.

[0133] In the present application, the CAR may not only comprise an extracellular targeting moiety that binds to the MSLN protein, but also may comprise an intracellular domain.

[0134] In the present application, the CAR may include an intracellular costimulatory signal region capable of providing a stimulatory 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, ligand for CD83, CD40, and MyD88.

[0135] For example, the costimulatory signal region may be an intracellular costimulatory signal region derived from 4-1BB. For example, the costimulatory signal region may include the amino acid sequence shown in SEQ ID NO:39.

[0136] In some cases, the CAR may include an intracellular signaling region that may include a domain having at least one ITAM motif. The intracellular signaling domain can transmit an activation signal to the inside of a 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 CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIbeta, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and other domains that include at least one ITAM.

[0137] For example, the intracellular signaling region may be a signaling domain derived from CD3ζ. For example, the intracellular signaling region may include the amino acid sequence shown in SEQ ID NO:41.

[0138] In some cases, the CAR may comprise a transmembrane domain, which may comprise a hydrophobic alpha helix, and is a fragment of a cell membrane-spanning sequence in a cell surface protein. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence that is predicted to form a hydrophobic helix. For example, the transmembrane region may 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.

[0139] For example, the transmembrane region may be a transmembrane region derived from CD8. For example, the transmembrane region may comprise the amino acid sequence set forth in SEQ ID NO:40.

[0140] In some cases, the CAR may comprise a hinge region, which may be located between the extracellular targeting moiety and the transmembrane domain. For example, the hinge region may comprise 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.

[0141] For example, the hinge region may be a hinge region derived from CD8. For example, the hinge region may comprise the amino acid sequence set forth in SEQ ID NO:42.

[0142] 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 CD8 protein. For example, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 43.

[0143] 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 comprise low density lipoprotein receptor-related proteins 1 to 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 comprise the amino acid sequence shown in SEQ ID NO: 44. For example, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof may comprise the nucleotide sequence shown in SEQ ID NO: 45.

[0144] In the present application, the sequence of the low density lipoprotein receptor-related protein or a fragment thereof in the CAR can be linked to the C-terminal sequence of the CAR via a self-cleaving peptide (e.g., a 2A peptide such as T2A, P2A, E2A, etc.). For example, the low density lipoprotein receptor-related protein or a fragment thereof can be linked to the C-terminus of the intracellular signal region via T2A. For example, the cleaved peptide can include the amino acid sequence shown in SEQ ID NO:46.

[0145] In the present application, the CAR may include, in order from the N-terminus to the C-terminus, a targeting moiety that binds to the MSLN protein (e.g., the antigen binding protein, also, for example, the VHH described in the present application), the hinge region, the transmembrane domain, the costimulatory signal region, and the intracellular signal region. For example, from the N-terminus to the C-terminus, the CAR may include, in order, the VHH, a hinge region derived from CD8, a transmembrane domain derived from CD8, a costimulatory signal region derived from 4-1BB, and an intracellular signal region derived from CD3zeta, and the VHH may include an amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:38.

[0146] In the present application, the CAR may include, in order from the N-terminus to the C-terminus, a targeting moiety that binds to an MSLN protein (e.g., the antigen binding protein, also, for example, the VHH described herein), the hinge region, the transmembrane domain, the costimulatory signal region, the intracellular signal region, and the low density lipoprotein receptor-related protein or a fragment thereof. For example, from the N-terminus to the C-terminus, the CAR may include, in order, the VHH, a hinge region derived from CD8, a transmembrane domain 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 set forth in SEQ ID NO:44, and the VHH may include the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33, and SEQ ID NO:38.

[0147] In the present application, from N-terminus to C-terminus, the CAR may include, in order, a signal peptide, a targeting moiety that binds to an MSLN protein (e.g., an antigen binding protein, also e.g., a VHH as described herein), the hinge region, the transmembrane domain, the costimulatory signal region, and the intracellular signal region.

[0148] In the present application, from N-terminus to C-terminus, the CAR may include, in order, a signal peptide, a targeting moiety that binds to an MSLN protein (e.g., an antigen binding protein, also e.g., a VHH as described herein), the hinge region, the transmembrane domain, the costimulatory signal region, the intracellular signal region, and the low density lipoprotein receptor-related protein or a fragment thereof.

[0149] For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 47. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 48. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 49. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 50. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 51. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the chimeric antigen receptor may comprise the amino acid sequence set forth in SEQ ID NO: 53.

[0150] nucleic acid molecule In another aspect, the present application further provides one or more nucleic acid molecules, which may be of any length in isolated form of nucleotides, deoxynucleotides and / or ribonucleotides, and which can encode said isolated antigen binding protein and / or said chimeric antigen receptor.

[0151] 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 the EF1α promoter.

[0152] In another aspect, the present application further 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 a fragment thereof in a cell, wherein 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 a fragment thereof via a truncation peptide.

[0153] vector In another aspect, the present application further provides a vector that may include the nucleic acid molecule. The vector can transform, transduce, or transfect a host cell to express the genetic material elements carried by the vector in the host cell. For example, the vector may include a promoter, a transcript, an enhancer, a replicon, a selection element, and a reporter gene. For example, the vector may include elements that aid in entry into a cell. The 5' and 3' ends of the nucleic acid molecule may further include long terminal repeat sequences to allow the nucleic acid molecule to replicate in the vector.

[0154] For example, the vector may be a viral vector. For example, the vector may be a lentiviral vector.

[0155] cell In another aspect, the present application further provides a cell, which may comprise the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, and / or the vector. The cell may comprise the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny may not necessarily be completely identical (in terms of total DNA complement or genome) to the original parent cell.

[0156] In some embodiments, the cells may be immune effector cells. In some 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, lymphoid progenitor cells, and / or pluripotent stem cells. For example, In some embodiments, the cell may be a T cell.

[0157] 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 fragment thereof.

[0158] Pharmaceutical Compositions In another aspect, the present application further provides a pharmaceutical composition comprising said isolated antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule, said vector and / or said cell, and optionally a pharma- ceutically acceptable adjuvant.

[0159] In some embodiments, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutical effective) carrier agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition are preferably non-toxic to recipients under the dosages and concentrations used. Pharmaceutical compositions of the present invention may include liquid, frozen and lyophilized compositions.

[0160] In some embodiments, such pharma- ceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration, generally safe, non-toxic, and not biologically or otherwise undesirable.

[0161] In some embodiments, the pharmaceutical composition may include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration, or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0162] Manufacturing method In another aspect, the present application further provides a method of producing said isolated antigen binding protein and / or said chimeric antigen receptor, which may comprise culturing said cell under conditions allowing expression of said antigen receptor and / or said chimeric antigen receptor.

[0163] The present application further provides a method of producing a modified immune effector cell, which may comprise introducing the vector into an immune cell.

[0164] use In another aspect, the present application further provides a use of said isolated antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule, said vector, said cell, and / or said pharmaceutical composition in the manufacture of a medicament, wherein said medicament is used to prevent, alleviate and / or treat a disease and / or condition.

[0165] In another aspect, the present application further provides a method of preventing, alleviating and / or treating a disease and / or condition, the method may comprise administering to a subject the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0166] In another aspect, the present application further provides the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition for preventing, alleviating and / or treating a disease and / or condition.

[0167] In the present application, the above diseases and / or conditions may include diseases and / or conditions associated with the abnormal expression of MSLN.

[0168] In the present application, the disease and / or condition may include a tumor.

[0169] In the present application, the tumor may include a solid tumor and / or a non-solid tumor.

[0170] In the present application, the tumor may include a hematological tumor and / or a lymphoma.

[0171] In the present application, the tumor may include a tumor that expresses the MSLN antigen.

[0172] In the present application, the tumor may include ovarian cancer, pancreatic cancer, gastric cancer, mesothelial carcinoma, cholangiocarcinoma, triple-negative breast cancer, and / or endometrial cancer.

[0173] In this application, the subject may include a human or a non-human animal.

[0174] In another aspect, the present application provides a polypeptide comprising the isolated antigen binding protein described above.

[0175] In another aspect, the present application provides a reagent kit or an administration device comprising the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0176] Without intending to be limited by any theory, the following examples are merely intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0177] Example 1 Screening of nanobodies targeting MSLN-His 1.1 Panning against MSLN-His Full-length MSLN and its three domains, MSLN-D1 (AA296-390), MSLN-D2 (AA391-486), and MSLN-D3 (487-598), were constructed and expressed, and plates were coated with IgG-Fc and MSLN-His protein at 10 μg / mL and incubated overnight at 4°C. Every other day, the wells were washed three times with 1x PBST (containing 0.05% Tween 20 in PBS), blocked with 0.5% BSA for 2 hours at room temperature, washed three times with 1x PBST, and negatively selected by adding 100 μL of a phage library (an in-house synthetic nanobody library NanoOri_1.0) to the IgG1-Fc wells. After 1 hour, the phages in IgG1-Fc were transferred to the wells of MSLN-His for positive selection. After 1.5 hours, the wells were washed 10 times with 1x PBST to wash out the phages that did not bind to the antigen, and finally eluted with Glycine-HCl at pH=2.2, 100 μL / well, and then neutralized with Tris-HCl at pH=8.0. Half of the eluted phages were taken and infected into TG1 in the logarithmic growth phase, and after half an hour, they were superinfected into M13KO7 and cultured overnight. Every other day, the phages were precipitated for the next round of screening. A similar screening process was repeated four times, with 20 washes with 1×PBST after the second round of positive selection, 30 washes in the third round, and 40 washes in the fourth round.

[0178] 1.2 Pool ELISA One day before, a 96-well plate was coated with 2 μg / mL MSLN-His, IgG1-Fc and 0.5% BSA and left to stand overnight. Every other day, the plate was washed three times with 1×PBST, and then blocked with 0.5% BSA for 2 hours. After blocking, the plate was washed three times with 1×PBST, 30 μL of each round of phage library was added, and incubated at room temperature for 1 hour with shaking. After washing three times with 1×PBST, secondary antibodies Anti-M13-HRP (Sino Biological, product number: 11973-MM05T-H) and Anti-Flag-HRP (abcam, product number: ab1162) were added, and the plate was incubated at room temperature for 30 to 60 minutes. After washing seven times with 1×PBST, TMB color development solution was added, and after 3 to 5 minutes, 2M phosphoric acid was added to stop the reaction, and the absorbance value at a wavelength of 450 nm was read.

[0179] The results are shown in Figure 1. The results show that specific antibodies targeting MSLN-His began to be enriched from the second round, while the curves of the anti-Flag-HRP secondary antibody showed that the enrichment was more obvious in the third and fourth rounds. Therefore, 96 single clones were collected from the bacterial solutions of the third and fourth rounds, respectively.

[0180] 1.3 Identification of specific positive single clones by phage enzyme-linked immunosorbent assay (ELISA) After 4 rounds of panning, the spare output from the 4th round was plated on 2YT / carb, and every other day 192 single colonies were randomly picked and placed in 800 μL of 2YT / carb / M13KO7, and cultured overnight with shaking to produce phages. One day before, 384-well plates were coated with 2 μg / mL MSLN-His and IgG1-Fc and left to stand overnight. Every other day, the plates were washed three times with 1×PBST, blocked with 0.5% BSA for 2 hours, and centrifuged at the same time to collect the phage supernatant. After blocking, the wells were washed three times with 1x PBST, 30 μL of phage supernatant was added, and incubated at room temperature for 1 hour with shaking. After washing three times with 1x PBST, secondary antibody Anti-M13-HRP was added, and incubated at room temperature for 30-60 minutes. After washing seven times with 1x PBST, TMB color development solution was added, and after 3-5 minutes, 2M phosphoric acid was added to stop the reaction, and the absorbance value at a wavelength of 450 nm was read. If the OD value of the sample well was more than twice as high as that of the control well (0.5% BSA or IgG1-Fc), it was judged to be positive. Finally, the positive phages were reinfected with TG1 and sequenced. The amino acid sequence of each clone was analyzed according to the sequence alignment software BioEdit. Clones with identical CDR1, CDR2, and CDR3 sequences were considered to be the same antibody strain.

[0181] The results are shown in Figure 2, a total of 192 single clones were picked, wells with MSLN / Fc>5 were considered positive, and all 130+ positive clones were subsequently sequenced.

[0182] The sequencing results are shown in Figures 3A to 3D. The sequencing results showed that there were 42 specific nanobody sequences, and after verification by phage ELISA, 21 sequences were retained, all of which were expressed in the form of VHH-Fc fusion proteins and purified before ELISA was performed. 5% Milk was used as a non-specific binding control, P4 was a positive clone (VH of P4 is shown in SEQ ID NO: 58, VL of P4 is shown in SEQ ID NO: 62), and Caplacizumab was the negative control. The results showed that a total of 12 highly specific and high affinity binding MSLNs, MSLN1, MSLN2, MSLN3, MSLN10, MSLN11, MSLN16, MSLN18, MSLN27, MSLN36, MSLN39, MSLN41, and MSLN42, had EC50 similar to that of the positive antibody P4, while a total of six antibodies, MSLN5, MSLN6, MSLN7, MSLN14, MSLN29, and MSLN35, had relatively poor specificity, and antibodies 4 and 15 had very poor specificity, while antibody 30 did not bind at all.

[0183] Example 2 Identification of the binding activity of nanobodies targeting the three MSLN domains 2.1 Expression and purification of VHH-Fc fusion proteins in eukaryotic cells A specific antibody sequence was selected from the results of sequencing and cloned into the vector pcDNA3.4. The recombinant plasmid was transformed into E. coli DH5α (Tiangen Biochemical Technology Co., Ltd., product number: CB101-02). The bacterial suspension was then evenly spread onto 2YT solid medium containing 100 μg / mL ampicillin and left to stand overnight at 37 ° C. A single clone was picked every other day and cultured on a shaker at 37 ° C for about 6 hours, half of the bacterial suspension was taken and sequenced, and the remaining half was stored at 4 ° C. The clone with accurate sequencing was expanded and cultured and extracted from the plasmid. The expression plasmid was transfected into EXPI293 using the method of PEI transfection and expressed in a cell incubator at 37 ° C for 5 days, after which the cell supernatant was collected and the antibody was purified by Protein A affinity chromatography column. Finally, an antibody protein with a purity of more than 90% was obtained.

[0184] 2.2 Detection of specific binding of VHH-Fc to human MSLN protein and its three domains by enzyme-linked immunosorbent assay A high-adsorption 96-well plate was coated with 2 μg / mL of MSLN-His and three MSLN-Domain proteins, left to stand overnight at 4°C, washed three times with 1×PBST, blocked with 0.5% BSA at room temperature for 2 hours, washed three times with 1×PBST, added 100 nM VHH-Fc, incubated with shaking at room temperature for 1 hour, washed three times with 1×PBST, added secondary antibody Anti-human IgG Fc Antibody HRP (Abcam, product number: ab99759), incubated at room temperature for 30 to 60 minutes, washed seven times with 1×PBST, added TMB color development solution, and after 3 to 5 minutes, added 2 M phosphoric acid to stop the reaction, and read the absorbance value at a wavelength of 450 nm.

[0185] 2.3 Identification of the binding activity of bivalent VHH-Fc to human MSLN-transfected 293T and OVCAR3 by flow cytometry OVCAR3 and 293T-MSLN cells were resuscitated and passaged. On the day of the experiment, the cells were harvested and counted, and the cell density was adjusted to 1 × 10 6 Adjust the concentration to 3 × 10 / mL and 30 μL per well. 4The plate was incubated at 4°C for 1 hour after homogenous mixing at 30μL per well; washed twice with PBS containing 0.1% BSA, and dehydrated at 500g for 5min at 4°C. The fluorescent secondary antibody Goat pAb to Hu IgG[DyLight 650] (abcam, product number: ab98593) was diluted 1:200 at 30μL per well; washed twice with PBS containing 0.1% BSA, dehydrated at 500g for 5min at 4°C, and resuspended at 30μL / well. The values ​​were read using a high-throughput flow cytometry detector (IQue), and the collected data was used to create a three-parameter fitting curve using Graphpad. The results are shown in Figure 4, where the numbers indicate the sequence numbers corresponding to the MSLN VHHs. The results showed that MSLN1, MSLN2, MSLN3, MSLN4, MSLN5, MSLN6, MSLN10, MSLN11, MSLN16, MSLN18, MSLN27, MSLN36, MSLN39, MSLN41, and MSLN42 had relatively high flow cytometry binding activity to the MSLN highly expressing ovarian cancer cell line OVCAR3 and the MSLN overexpressing cell line 293-MSLN.

[0186] 2.4 Identification of non-specific binding of bivalent VHH-Fc to tissue cells by flow cytometry 293T, LO2, KERA, A549, HACAT, and K562-MSLN cells were resuscitated and passaged, and the cells were harvested and counted on the day of the experiment, after which the cell density was adjusted to 1 × 10 6 Adjust the concentration to 3 × 10 / mL and 30 μL per well. 4The representative MSLN nanobody and positive antibody P4 were mixed uniformly at a concentration of 20 μg / mL, 30 μL per well, and incubated at 4°C for 1 h. The wells were washed twice with PBS containing 0.1% BSA, and dehydrated at 500 g for 5 min at 4°C. The fluorescent secondary antibody Goat pAb to Hu IgG [DyLight 650] (abcam, product number: ab98593) was diluted 1:200 at 30 μL per well, mixed uniformly and incubated at 4°C for 30 min; the wells were washed twice with PBS containing 0.1% BSA, dehydrated at 500 g for 5 min at 4°C, resuspended at 30 μL / well, and read on a high-throughput flow cytometry detector (IQue). The results are shown in Figure 5 and demonstrated that the sequences MSLN1, MSLN4, MSLN5, MSLN6, MSLN11, MSLN36, and MSLN42 all had non-specific binding activity to 293T, LO2, KERA, A549, and HACAT cells.

[0187] Example 3 Detection of CAR-T lentivirus packaging 3.1 Lentivirus packaging The vector system for constructing the lentiviral plasmid vector of the present invention belongs to the third generation lentiviral vector system, which includes a total of three plasmids: packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein, PMD2.G plasmid encoding envelope protein VSV-G, and core plasmid encoding the target gene CAR. Based on the gene encoding CAR in the BBz / L6 platform core plasmid, the expression was regulated by the elongation factor-1α (EF-1α) promoter. The viral packaging process is as follows: 1×10 6293FT cells were suspended in 2 mL of DMEM (10% FBS) medium and plated on a 6-well plate. The cells were thoroughly and uniformly dispersed and cultured at 5% CO2 and 37°C for approximately 24 h. Plasmids and reagents were equilibrated to room temperature. 100 μL of Opti-MEM medium contained 4.5 μg (psPAX2:PMD2.G:core plasmid = 3:2:4) and 13.5 μL (plasmid:FuGENEHD = 1:3) of transfection reagent (Promega, product number: E2311) and was packaged. The transfection plasmid was thoroughly and homogenously mixed, then the transfection reagent was slowly added drop by drop, and after all were added, they were gently mixed one by one and left at room temperature for 15 min; The original 400 μL of medium DMEM (10% FBS) was aspirated, divided and liposome-cation complex (100 μL of Opti-MEM + PSH1 + PMH2 + core plasmid + FuGENEHD) was added drop by drop, and cultured at 5% CO2 and 37 °C for about 17 hours; The plasmid-containing medium (about 200 μL, One by one) was removed and replaced with 2.5 mL of 5% FBS-containing DMEM medium (preheated at 37 °C for 30 min), and cultured at 5% CO2 and 37 °C for about 24 to 26 hours; The virus supernatant was collected and frozen at 3000 rpm for 10 min, and about 720 μL was dispensed into a cryopreservation tube and frozen at -80 °C, and an appropriate amount was taken for lentivirus titer measurement.

[0188] 3.2 Lentiviral titration 293T cells in good condition were taken, washed with 1x PBS, digested with 1.5 mL of 0.25% pancreatin at 37°C, and stopped in 11 mL of medium. The cell line was cultured at 2x10 5The ratio of cells / well / 2.5mL was adjusted; 10μg / mL polybrene was added to the cell line medium at 1:1000 and mixed thoroughly; 1mL of cell suspension was added to each well of a 12-well plate; 5μL of the obtained virus stock solution was added drop by drop, and 2.5mL was supplemented to each well and mixed thoroughly; The suspension without virus cells was set as a blank control for flow cytometry detection; The cells were cultured at 5% CO2 and 37℃ for about 42~48h; The supernatant was removed, washed with PBS, and digested with 400μL of 0.25% pancreatin (Gibco, product number: 25200072) at 37℃ for about 1min, and digestion was stopped with 2.5mL of complete medium; The cells were gently blown with a 1mL pipette until completely dispersed, transferred to a 1.5mL EP tube, and the cells were counted; 5×10 5 The amount of cells was taken and used for flow cytometry detection.

[0189] Example 4 Infection and expansion of T cells PBMC cells were resuscitated, 11 mL of X-VIVO was added and centrifuged at 500g for 5 min; 12 mL of X-VIVO was added and centrifuged at 400g for 5 min; 12 mL of X-VIVO was added and centrifuged at 300g for 5 min; 1 mL of X-VIVO suspension was counted; cell density was 1 × 10 6 Adjust the volume to 10 cells / mL and mix with CD3 / CD28 (4 × 10 8 Activate the T cells by adding 100 µL of magnetic beads (Thermo Fisher Scientific, product number: 11131D) per well, mix thoroughly and evenly, and plate 700 µL of cell suspension (7 × 10 5cells / well / 700μL); activated PBMC, 37℃, CO2 incubator, 18h; added virus supernatant with MOI of 4-5, polybrene to a final concentration of 5μg / mL (1:2000, based on final volume), supplemented by adding corresponding medium, mixed cells evenly, set up blank control, horizontal centrifuge, 1200rpm, 1h; 37℃, 5% CO2 incubator, 24h; gently Mix evenly, collect cells in 1.5mL EP tubes, and heat at 300g for 5min. Remove the supernatant (approximately 50-100μL of medium remains), resuspend in 1mL of X-VIVO medium in a 12-well plate, add 500μL of medium, and culture in a 37℃, 5% CO2 incubator for 48h; observe the color of the medium, add an appropriate amount of X-VIVO medium (800μL-1mL), and culture in a 37℃, 5% CO2 incubator; count cells every 2 days, and confirm the cell density to 7×10 5 / mL, 6-well-plate / 3mL; total cell number was 2.1 × 10 6 and cultured for 24 h in a 37°C, 5% CO2 incubator; an appropriate amount of medium (1 mL) was added after 24 h observation; gently mixed evenly and counted; based on the counting, the total cell number was divided by the previous total cell number 2.1E+6 to obtain the amplification fold T2; the cell density was 7×10 5 / mL, 6-well plate / 3mL, total cell number was 2.1 × 10 6 The total number of cells was adjusted to 2.1 mL; 6 x 10 5 The number of cells was taken and used for CAR-T positivity rate and CD25 / CD69 CAR detection (magnetic beads were removed for 1 min); cultured for 24 h in a 37°C, 5% CO2 incubator; an appropriate amount of medium (1 mL) was added after 24 h observation; gently mixed evenly and counted; based on the count, the total number of cells was reduced to the previous total number of cells of 2.1 × 10 6 Dividing by 7 x 10 gives the amplification fold T3; the cell density is 7 x 10 5 / mL, 6-well plate / 3mL, total cell number was 2.1 × 10 6and passaged; 9E5 cell count was taken for CAR-T positive rate and PD-L1 / TIM3 / LAG3 CAR detection (magnetic beads were removed for 1 min); appropriate cell count was taken for in vitro tumor killing activity experiment; cultured at 37℃, 5% CO2 incubator for 24 h; appropriate amount of medium (1mL~2.5mL) was added after 48 h observation; gently mixed evenly and counted; based on the count, the total cell count was adjusted to the previous total cell count of 2.1×10 6 Dividing by this, the amplification fold was obtained as T4; the corresponding cells were taken and used for CAR-T repeated stimulation experiment; an appropriate number of cells were taken and used for CAR-T positive rate, CD25 / CD69, PD-1 / TIM3 / LAG3 CAR detection (magnetic beads were removed for 1 min); the amplification fold was: T1*T2*T3*T4.

[0190] The results are shown in Figure 6, where the numbers shown indicate the sequence numbers corresponding to the MSLN VHH, and MSLN3 (VHH sequence is shown in SEQ ID NO: 13), MSLN10 (VHH sequence is shown in SEQ ID NO: 18), MSLN16 (VHH sequence is shown in SEQ ID NO: 8), MSLN27 (VHH sequence is shown in SEQ ID NO: 23), MSLN36 (VHH sequence is shown in SEQ ID NO: 28), MSLN41 (VHH sequence is shown in SEQ ID NO: 33), and MSLN42 (VHH sequence is shown in SEQ ID NO: 38) were selected, among which MSLN0 is the negative control Caplacizumab, P4 is the positive control (VH sequence is shown in SEQ ID NO: 58, VL sequence is shown in SEQ ID NO: 62), and T is the blank control without adding target cells. The results showed that all antibodies had similar proliferation rates to the positive antibodies.

[0191] Example 5 Detection of in vitro tumor-killing activity of CAR-T cells CytoTox96® Non-Radioactive Cytotoxicity Detection LDH is a stable cytoplasmic enzyme that is released when cells lyse. The release method is basically the same as that in the radioactive analysis of 51Cr. The released LDH was detected in the culture supernatant by a coupling enzyme reaction. In the enzyme reaction, LDH can convert tetrazolium salt (INT) into red formazan, and the amount of red product generated was directly proportional to the number of lysed cells (Promega, product number: G1780). The CAR-T positive rate was detected by matching the ratio between the infected CAR groups with uninfected blank T cells and adjusting the total cell number of each group to be consistent; the number of target cells was 2 × 10 4 / 96-well-plate / well, with effector cells:target cells (E:T) = 3:1, 1:1, 1:3, 1:9; wells containing only effector cells in the same amount as the experimental group were set as effector cell self-released LDH background group; wells containing only blank T cells in the same amount as the experimental group were set as T cell self-released LDH blank control group; wells containing only blank T cells with the same effector cell to target cell ratio as the experimental group were set as T + target cell negative control group; wells containing only target cells in the same amount as the experimental group were set as target cell self-released LDH background group; wells containing only target cells in the same amount as the experimental group were set as target cell maximum released LDH background group; single wells containing only 200 μL of cell culture medium were set as culture medium background LDH control wells; single wells containing only 200 μL of cell culture medium were set as volume-corrected control wells; corresponding effector cells and target cells were co-incubated in 200 μL of X-VIVO (without FBS) medium. and cultured at 37℃, 5% CO2 incubator for 20-24h; digested, counted, took the corresponding amount of target cells, resuspended in 100μL of X-VIVO and plated in 96-well plate; took the corresponding amount of effector cells according to the ratio of effector cells to target cells, supplemented the corresponding blank T cells, 500g, 5min; resuspended in 100μL of X-VIVO and mixed evenly with target cells in 96-well plate; 37℃, 5% CO2, 20-24h; about 4 5 min earlier, add 20 μL of lysis solution (10X) to the volume-corrected control wells and target cell maximum released LDH background wells, and incubate at 37°C, 5% CO2 incubator for 45 min; 250xg, 4 min; take 50 μL of the supernatant to the enzyme assay plate, add 50 μL / well of prepared substrate (prepared in detection buffer), and incubate at room temperature in the dark for 20-30 min; add 50 μL / well of stop solution; observe the color (avoid air bubbles); take OD490 reading.

[0192] 1x10 4After co-culture of the tumor cells SK-OV3-Luciferase-GFP and the exemplary MSLN-CAR-T cells with different effector and target cell ratios for 18h and 72h, the killing effect of CAR-T cells on the tumor cells was detected, and the results are shown in FIG. 7. The results show that the killing effect of MSLN-CAR-101 (wherein the amino acid sequence of the targeting portion of the chimeric antigen receptor is shown in SEQ ID NO: 13, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 40, the amino acid sequence of the costimulatory domain is shown in SEQ ID NO: 39, the amino acid sequence of the intracellular signaling domain is shown in SEQ ID NO: 41, and the amino acid sequence of the hinge region is shown in SEQ ID NO: 42, and the above MSLN-CAR-101 cells further express the foreign protein fragment shown in SEQ ID NO: 44) has a similar killing effect to that of the positive sequence P4.

[0193] Example 6 Detection of cytokines CAR-T was produced according to the T cell infection and amplification method in Example 4, and the supernatant was incubated with an appropriate amount of E:T = 1:1 and used to detect cytokines such as IL-2 and IFN-γ. The CAR-T positive rate was detected, and the ratio between the infected CAR groups was matched with uninfected blank T cells, and the total cell number of each group was adjusted to be consistent (the required number of CAR-T cells, the minimum CAR positive ratio is the base number); the total number of T cells was consistent; VT (T + CELL group) = CAR + T; the same amount of blank T cells as the experimental group was placed in the well as the T cell alone group; the same amount of effector cells as the experimental group was placed in the well as the effector cell alone group; the number of target cells was 1 × 10 4 / 96-well / well, effector cells:target cells (E:T)=1:1, corresponding effector cells and target cells were co-incubated according to the ratio of 1:1, effector cells (including CAR-T positive cells 1E4) and target cells (1E4) were mixed and supplemented with serum-free l X-VIVO medium until the final volume was 200μL; gently mixed evenly, 37℃, 5% CO2 incubator, 24h; 400xg, 5min, 50μL of cell supernatant was taken for cytokine detection.

[0194] The results are shown in Figures 8-10, in which KERA is MSLN-negative cells and SK-OV3 is MSLN-positive cells. When CAR-T cells were co-cultured with SK-OV3, the CAR-T cells were activated and produced large amounts of cytokines IL-2, TNF-α, and IFN-γ. Among them, MSLN-CAR-101 has the same amount of cytokine release as the positive sequence P4. Furthermore, when CAR-T cells were co-cultured with KERA, none of the CAR-T cells were activated and there was no cytokine release.

[0195] Example 7 Efficacy test in NSG mice 3x10 on mouse 6 The SKOV3 tumor cell line was subcutaneously injected, and after 2 weeks, the tumor volume was 100-200 mm 3 The subjects were randomly assigned to one group at the time of admission and administered CAR-T intravenous reinfusion every other day. The cell dose was 5 × 10 6 The tumors were measured three times a week for 47 days. Finally, the tumor growth curves were plotted using Graphpad, where 5E6 represents 5*10^6 SK-OV3-inoculated ovarian cancer cells.

[0196] The results are shown in Figure 11, which demonstrated that MSLN-CAR-101 significantly inhibited tumor growth.

[0197] The above detailed description is provided for purposes of explanation and illustration, and is not intended to limit the scope of the appended claims. Various modifications to the present exemplary embodiments will be apparent to those skilled in the art and are intended to remain within the scope of the appended claims and their equivalents.

Claims

1. An isolated antigen-binding protein capable of specifically binding to mesothelin (MSLN), the isolated antigen-binding protein comprising at least one CDR in an antibody heavy chain variable region VH, the VH comprising the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:

70. Antigen-binding proteins.

2. A peptide comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 68 (X1X2X3MG, wherein X1 is N, R, S, T or Y, X2 is N or Y, and X3 is A, N or V), the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 67, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 65, and SEQ ID NO:

66.

2. The antigen-binding protein of claim 1.

3. A method for producing a human ovarian tumor comprising: comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:36; the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, and SEQ ID NO:35; and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:29, and SEQ ID NO:

34.

2. The antigen-binding protein of claim 1.

4. 2. The antigen binding protein of claim 1, comprising HCDR1, HCDR2, and HCDR3, wherein said HCDR1, HCDR2, and HCDR3 are: (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, 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: 1; (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, 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: 9; (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; (4) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; (5) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; (6) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; and (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34; The amino acid sequence of the present invention is selected from the group consisting of:

2. The antigen-binding protein of claim 1.

5. the H-FR1 comprises H-FR1, H-FR2, H-FR3, and H-FR4, the C-terminus of the H-FR1 being directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 4, 12, 17, 22, 27, 32, and 37, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising the amino acid sequence shown in SEQ ID NO: 6, the N-terminus of the H-FR4 being directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprising the amino acid sequence shown in SEQ ID NO: 7; 2. The antigen-binding protein of claim 1.

6. The antibody heavy chain variable region VH comprises an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO:

70.

2. The antigen-binding protein of claim 1.

7. The antibody heavy chain variable region VH comprises an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO:

38.

2. The antigen-binding protein of claim 1.

8. comprising an antibody or antigen-binding fragment thereof, 2. The antigen-binding protein of claim 1.

9. The antigen-binding fragment is a VHH, and the VHH comprises an amino acid sequence set forth in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO:

70.

9. The antigen-binding protein of claim 8.

10. The VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 33, and SEQ ID NO:

38.

10. The antigen-binding protein of claim 9.

11. 2. The antigen binding protein of claim 1, which is capable of competing with a reference antibody for binding to MSLN (Mesothelin) protein, wherein said reference antibody comprises an antibody heavy chain variable region VH, said reference antibody VH comprising HCDR1, HCDR2 and HCDR3, and wherein said reference antibody (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, 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: 1; (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, 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: 9; (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; (4) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 19; (5) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 25, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; (6) The HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; and (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34; The amino acid sequence of the present invention is selected from the group consisting of:

10. The antigen-binding protein of claim 9.

12. an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from IgG; 2. The antigen-binding protein of claim 1.

13. a targeting moiety, said targeting moiety comprising the antigen binding protein of claim 1; Chimeric antigen receptor.

14. a costimulatory domain, an intracellular signaling domain, a transmembrane region, a hinge region between said targeting moiety and said transmembrane region, and / or a signal peptide; the costimulatory domain 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 for CD83, CD40, and MyD88; 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 herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM; the transmembrane region comprises 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; and / or 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; The chimeric antigen receptor of claim 13.

15. the costimulatory domain is an intracellular costimulatory signal region derived from 4-1BB; the intracellular signaling domain is a signaling domain derived from CD3ζ; the transmembrane region is a transmembrane region derived from CD8, the hinge region is a hinge region derived from CD8, and / or The signal peptide is derived from the signal peptide of the CD8 protein. The chimeric antigen receptor of claim 14.

16. Further comprising a low density lipoprotein receptor-related protein or a fragment thereof, The chimeric antigen receptor of claim 13.

17. The low-density lipoprotein receptor-related protein or a fragment thereof includes one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. The chimeric antigen receptor of claim 16.

18. The amino acid sequence of any one of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53 is included. The chimeric antigen receptor of claim 13.

19. 10. The antigen-binding protein of claim 1 , Polypeptide.

20. 10. One or more isolated nucleic acid molecules encoding the antigen-binding protein of claim 1. Nucleic acid molecule.

21. 21. The nucleic acid molecule of claim 20. vector.

22. 10. The antigen-binding protein of claim 1 , cell.

23. further comprising and / or expressing a low density lipoprotein receptor-related protein or a fragment thereof; The cell of claim 22.

24. The low-density lipoprotein receptor-related protein or a fragment thereof includes one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. The cell of claim 23.

25. A method for producing the antigen-binding protein of claim 1.

26. 22. A method for the treatment of immune effector cells comprising introducing the vector of claim 21 into said cells. Methods for producing modified immune effector cells.

27. 10. The isolated antigen binding protein of claim 1, and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions.

28. A pharmaceutical composition comprising the isolated antigen-binding protein of claim 1 for use in the prevention, treatment and / or alleviation of a disease or condition associated with abnormal expression of MSLN.

29. The disease or condition associated with the abnormal expression of MSLN includes tumors; 29. The pharmaceutical composition of claim 28.

Images