WO2023131276A1 - 靶向msln的抗原结合蛋白及其应用 - Google Patents

靶向msln的抗原结合蛋白及其应用 Download PDF

Info

Publication number
WO2023131276A1
WO2023131276A1 PCT/CN2023/070928 CN2023070928W WO2023131276A1 WO 2023131276 A1 WO2023131276 A1 WO 2023131276A1 CN 2023070928 W CN2023070928 W CN 2023070928W WO 2023131276 A1 WO2023131276 A1 WO 2023131276A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
sequence shown
binding protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/070928
Other languages
English (en)
French (fr)
Chinese (zh)
Inventor
王华菁
钟阵威
成超
谢二敏
陈晓锐
何晓文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oricell Therapeutics Co Ltd
Original Assignee
Oricell Therapeutics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oricell Therapeutics Co Ltd filed Critical Oricell Therapeutics Co Ltd
Priority to CN202380015977.8A priority Critical patent/CN118510809A/zh
Priority to JP2024540577A priority patent/JP2025505933A/ja
Priority to EP23737138.0A priority patent/EP4461747A4/en
Priority to US18/726,241 priority patent/US20250213691A1/en
Publication of WO2023131276A1 publication Critical patent/WO2023131276A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein targeting MSLN, a chimeric antigen receptor comprising the antigen-binding protein, and applications thereof.
  • MSLN Mesothelin
  • MSLN is a glycoprotein located on the cell surface, anchored to the cell membrane by glycosylphosphatidylinositol.
  • the mesothelin gene encodes a 69kDa precursor protein, which is hydrolyzed into two chains by furin (paired basic amino acid protease, furin)-like invertase, and the membrane-bound protein of about 40KD at the C-terminal is the mature mesothelial protein
  • furin paired basic amino acid protease, furin
  • MPF megakaryocyte-promoting factor
  • MPF and membrane anchor MSLN are N-glycosylated, MPF can promote the formation of megakaryocyte clones in vitro, membrane anchor MSLN can interact with MUC16, and plays an important role in the process of cell adhesion, so currently targeted therapy is Membrane-anchored MSLN is selected as the target, so currently MSLN specifically refers to the C-terminal 40KD fragment of MSLN, that is, membrane-anchored MSLN.
  • Mesothelin is a glycoprotein present on the cell surface of the mesothelial cell lineage of the peritoneum, pleura, and pericardium. Mesothelin is predominantly expressed (overexpressed) in mesothelioma, ie cancer/tumor cells, ovarian cancer, pancreatic cancer, gastric cancer, lung cancer and endometrial cancer. In contrast, its expression is restricted in normal cells such as mesothelial cells.
  • the present application provides an isolated antigen-binding protein capable of specifically binding to MSLN.
  • the present application also provides a chimeric antigen receptor comprising the antigen-binding protein, and cells comprising and/or expressing the chimeric antigen receptor, and the cells have one or more of the following characteristics: (1) expansion Strong ability; (2) capable of killing target cells expressing MSLN; (3) secreting cytokines under the stimulation of target cells; (4) inhibiting the growth of tumor cells.
  • the application provides an isolated antigen binding protein comprising at least one CDR in the VH of an antibody heavy chain variable region, said VH comprising SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO: 69 and the amino acid sequence shown in any one of SEQ ID NO:70.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:65 and SEQ ID NO:66 amino acid sequence.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24, The amino acid sequence shown in any one of SEQ ID NO:29 and SEQ ID NO:34.
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:67.
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, The amino acid sequence shown in any one of SEQ ID NO:30 and SEQ ID NO:35.
  • the antigen binding protein comprises HCDR1, and the HCDR1 comprises SEQ ID NO: 68 (X 1 X 2 X 3 MG, wherein, X 1 is N, R, S, T or Y, X 2 is N or Y, X 3 is the amino acid sequence shown in A, N or V).
  • the antigen binding protein comprises HCDR1, and the HCDR1 comprises SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, The amino acid sequence shown in any one of SEQ ID NO:31 and SEQ ID NO:36.
  • the antigen binding protein comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise any set of amino acid sequences selected from the group consisting of:
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:14;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the antigen binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises SEQ ID NO: 4, The amino acid sequence shown in any one of 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 antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:5 .
  • the antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:6 .
  • the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO:7 The amino acid sequence shown.
  • the antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69 and SEQ ID NO:70 The amino acid sequence shown in any one.
  • the antigen binding protein comprises an antibody heavy chain variable region VH
  • the VH comprises SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, The amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33 and SEQ ID NO:38.
  • the antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is a VHH
  • the VHH comprises any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69, and SEQ ID NO:70. amino acid sequence.
  • the antigen-binding fragment is a VHH
  • the VHH comprises SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:28, The amino acid sequence shown in any one of SEQ ID NO:33 and SEQ ID NO:38.
  • the antigen-binding protein can compete with a reference antibody for binding to MSLN (Mesothelin, mesothelin) protein, wherein the reference antibody comprises an antibody heavy chain variable region VH, and the reference antibody's VH comprises HCDR1, HCDR2 and HCDR3, and the reference antibody comprises any set of amino acid sequences selected from:
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:14;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgGl.
  • the heavy chain constant region of the antigen binding protein comprises an IgG Fc region.
  • the Fc region comprises the amino acid sequence shown in SEQ ID NO:54.
  • the present application also provides a chimeric antigen receptor comprising a targeting moiety, and the targeting moiety comprises the antigen-binding protein.
  • the chimeric antigen receptor comprises a co-stimulatory domain comprising a co-stimulatory 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, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, Ligands of TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40, and MyD88.
  • a co-stimulatory domain comprising a co-stimulatory 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,
  • said costimulatory domain of said chimeric antigen receptor is an intracellular costimulatory signaling region derived from 4-1BB.
  • the co-stimulatory domain in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:39.
  • the chimeric antigen receptor comprises an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP-12 and a domain containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • said intracellular signaling domain of said chimeric antigen receptor is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:41.
  • the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , 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.
  • proteins selected from the group consisting of: CD8, CD28, 4- 1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5,
  • said transmembrane region of said chimeric antigen receptor is a transmembrane region derived from CD8.
  • the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:40.
  • the chimeric antigen receptor includes 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, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • said hinge region of said chimeric antigen receptor is a hinge region derived from CD8.
  • the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:42.
  • the chimeric antigen receptor further comprises a signal peptide.
  • said signal peptide of said chimeric antigen receptor is derived from a signal peptide of CD8 protein.
  • the signal peptide of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:43.
  • the chimeric antigen receptor further comprises low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • 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.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:44.
  • the chimeric antigen receptor comprises 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 the amino acid sequence shown in any one of SEQ ID NO:53.
  • the present application also provides a polypeptide comprising the antigen-binding protein.
  • the present application also provides one or more isolated nucleic acid molecules encoding said isolated antigen binding protein and/or said chimeric antigen receptor.
  • the nucleic acid molecule comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is the EF1 ⁇ promoter.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the vector comprises a viral vector.
  • the vector comprises a lentiviral vector.
  • the present application also provides a cell comprising the antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule and/or the carrier.
  • the cells are immune effector cells.
  • the cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral Blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • NK cells natural killer cells
  • macrophages NK cells
  • monocytes monocytes
  • dendritic cells granulocytes
  • lymphocytes lymphocytes
  • leukocytes granulocytes
  • peripheral Blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the cells contain and/or express low density lipoprotein receptor-associated protein or fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • 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.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:44.
  • the present application also provides a method for preparing modified immune effector cells, the method comprising culturing the cells under the condition that the antigen-binding protein and/or the chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, which includes introducing the carrier into the immune effector cells.
  • the present application also provides a pharmaceutical composition, which comprises the isolated antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the carrier and/or the cell , and optionally a pharmaceutically acceptable carrier.
  • the present application also provides the isolated antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the carrier, the cell, and/or Or the use of the pharmaceutical composition in the preparation of medicines, the medicines are used to prevent, treat and/or alleviate diseases or diseases related to the abnormal expression of MSLN.
  • the disease or disorder associated with aberrant expression of MSLN comprises tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a tumor expressing a MSLN antigen.
  • the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell cancer, bile duct cancer, triple negative breast cancer, and/or endometrial cancer.
  • the present application also provides a method for preventing, treating and/or alleviating diseases or disorders related to abnormal expression of MSLN, the method comprising administering the isolated antigen-binding protein to a subject in need , the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with aberrant expression of MSLN comprises tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises a tumor expressing a MSLN antigen.
  • the tumor comprises ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell cancer, bile duct cancer, triple negative breast cancer, and/or endometrial cancer.
  • Figure 1 shows the results of the phage Pool ELISA.
  • Figure 2 shows the selection of positive clones.
  • Fig. 3A-Fig. 3D show the detection of the affinity between the antigen binding protein of the present application and MSLN.
  • Figure 4 shows flow cytometric verification of the binding activity of the antigen-binding protein described in this application to 293T overexpressing human MSLN.
  • Figure 5 shows flow cytometric verification of the non-specific binding of the antigen-binding protein described in this application to 293T, KERA, LO2, A549 and HACAT cells.
  • Figure 6 shows the results of repeated stimulation and proliferation experiments of CAR-T cells in vitro.
  • Figure 7 shows the in vitro killing results of CAR-T cells.
  • Figures 8-10 show the results of detecting cytokine release from CAR-T cells in vitro.
  • Figure 11 shows the results of drug efficacy experiments in vivo in SK-OV3 tumor model mice.
  • MSLN also known as mesothelin, or CAK1 antigen or premegakaryocyte enhancer factor
  • MSLN protein is a protein present on normal mesothelial cells and overexpressed in some tumor cells.
  • the term may encompass MSLN protein or a functionally active fragment thereof.
  • the term may also encompass homologues, analogs or variants of the MSLN protein.
  • the MSLN can comprise human MSLN.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • Said “isolated antigen binding protein” may comprise an antigen-binding moiety and optionally a framework or framework portion which permits the antigen-binding moiety to adopt a conformation which facilitates its binding to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative or artificial framework regions with grafted variable regions (CDR) or CDR derivatives.
  • the antigen binding protein may comprise an antibody or an antigen binding fragment thereof.
  • the antigen binding protein can bind MSLN protein.
  • the antigen binding protein can compete with a reference antibody for binding to the MSLN protein.
  • the antigen binding protein may comprise an antibody heavy chain variable region VH.
  • the antigen binding protein may comprise at least one CDR derived from an antibody heavy chain variable region VH.
  • the VH may comprise HCDR3, HCDR2 and/or HCDR1.
  • the VH may include a framework region H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.
  • the VH may comprise a framework region H-FR2 located between the HCDR1 and the HCDR2.
  • the VH may comprise a framework region H-FR3 located between the HCDR2 and the HCDR3.
  • the VH may include a framework region H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.
  • the antigen binding protein can be VHH.
  • the antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from IgG.
  • the antibody heavy chain constant region may be derived from human IgG.
  • the antibody heavy chain constant region may be derived from human IgG1.
  • antibody as used includes whole antibodies and binding fragments thereof. Typically, a fragment competes with the intact antibody from which it is derived for specific binding to the antigen.
  • antibodies or binding fragments thereof can be chemically conjugated to other proteins, or expressed as fusion proteins with other proteins.
  • the antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the binding protein of the antibody or binding fragment thereof can include MSLN.
  • the antibody or binding fragment thereof can be specific for MSLN.
  • antigen-binding fragment refers to a portion of an intact antibody and refers to the antigenically determining variable region of an intact antibody.
  • the antigen-binding fragments may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen-binding fragment can be a VHH.
  • the antigen-binding fragment can bind MSLN.
  • the antigen-binding fragment can be specific for MSLN.
  • VHH generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody.
  • VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies.
  • Nb Nanobodies
  • the VHH can bind MSLN.
  • the VHH can be specific for MSLN.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” 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 can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • 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 the antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors.
  • the term "reference antibody” refers to an antibody that can competitively bind to the same epitope of MSLN as the isolated antigen-binding protein.
  • the reference antibody may comprise a heavy chain variable region VH.
  • the reference antibody can have 3 CDR sequences.
  • the VH of the reference antibody can include HCDR1, HCDR2, and HCDR3.
  • the CDR sequences may be identical to the CDR sequences of the isolated antigen binding protein.
  • IgG refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, and IgG3.
  • chimeric antigen receptor generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds an antigen or a target.
  • a hinge region is included between the extracellular domain and the transmembrane region.
  • the chimeric antigen receptor may also include low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the chimeric antigen receptor can include a signal peptide. Binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell.
  • the extracellular structure may include an antigen binding protein as described above.
  • the extracellular structure can specifically bind MSLN.
  • intracellular domain is meant to include any truncated portion sufficient to transduce an activation signal.
  • the intracellular domain may comprise an intracellular signaling domain and/or a co-stimulatory signaling domain.
  • intracellular signaling region refers to an intracellular region that can generate signals that promote immune effector functions of CAR-containing cells (eg, CART cells or CAR-expressing NK cells).
  • the intracellular signaling region may comprise an intracellular signaling region of one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 , Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • the intracellular signaling region may be a signaling domain derived from CD3 ⁇ .
  • co-stimulatory signaling region refers to a part of the CAR capable of transducing effector signals in the intracellular signaling region.
  • the co-stimulatory signal domain may comprise an intracellular co-stimulatory signal 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, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the co-stimulatory signal domain may be an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the term "transmembrane region” refers to a domain of a peptide, polypeptide or protein capable of spanning the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane.
  • 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, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region
  • the term "hinge region” refers to a part of an antibody heavy chain polypeptide connecting the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 of the EU numbering system according to Kabat.
  • the hinge region is usually a dimeric molecule composed of two polypeptides with the same amino acid sequence.
  • the hinge region generally comprises about 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region.
  • the hinge region can be subdivided into three structural domains: upper, middle, and lower hinge domains.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be derived from the hinge region of CD8.
  • Low-density lipoprotein receptor-related protein refers to a cell surface protein belonging to endocytic receptors, which is widely distributed in organisms and has a large tissue The main function is the uptake of cholesterol into cells for cell proliferation and the synthesis of sterol hormones and bile salts.
  • the low-density lipoprotein receptor-related protein can be from any vertebrate.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the intracellular signaling region.
  • the low-density lipoprotein receptor-associated protein or fragment thereof may comprise one or more selected from the group consisting of low-density lipoprotein receptor-associated protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof.
  • signal peptide refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression upon translation or after translation.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the term can be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of the corresponding natural amino acid, as well as to natural amino acid polymers, those containing modified residues, and non- Natural amino acid polymer.
  • said polypeptide may comprise said antigen binding protein.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • promoter generally refers to a DNA sequence that regulates the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell.
  • the nucleic acid molecule may encode an antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the term "vector” generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the term “cell” refers to a cell into which nucleic acid can be transfected, and the term “cell” includes prokaryotic cells for plasmid propagation, and eukaryotic cells for nucleic acid expression and production of encoded polypeptides.
  • a cell may comprise said antigen binding protein, said nucleic acid molecule and/or said carrier.
  • the cells may be immune effector cells.
  • immune effector cells generally refers to immune cells that participate in the immune response and perform effector functions.
  • the exercising effector functions may include clearing foreign antigens or promoting immune effector responses, etc.
  • immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells , embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • immune effector cells can be T cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian subject.
  • the pharmaceutical composition may comprise the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable Carrier.
  • the pharmaceutical composition can be used to prevent, treat and/or alleviate diseases or conditions related to abnormal expression of MSLN.
  • the disease or condition associated with abnormal expression of MSLN may include tumors.
  • the tumors include solid tumors and/or non-solid tumors.
  • the term "specifically binds" or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, easier, and/or for a longer duration than it binds other targets .
  • an antibody specifically binds an epitope on a protein that is conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, a protein or polypeptide that undergoes substitution, deletion or addition of one or more amino acids in the amino acid sequence of the protein and/or the polypeptide (for example, specifically binding to MSLN) .
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the homologue may have at least about 85% (for example, at least about 85%, about 90%) of the amino acid sequence of the protein and/or the polypeptide (for example, specifically binding to MSLN). , about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or polypeptides.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • Perfectage of sequence homology can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity.
  • Alignment for purposes of determining percent sequence homology can be accomplished in various ways 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 aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA FASTA and BLAST.
  • a description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J.
  • the term "comprises” generally refers to the meanings of including, encompassing, containing or encompassing. In some cases, it also means “for” and “consisting of”.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • said isolated antigen binding protein is defined by the Kabat coding system.
  • the isolated antigen binding protein may comprise at least one CDR in the variable region VH of an antibody heavy chain, for example, the VH may comprise SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO: 69 and the amino acid sequence shown in any one of SEQ ID NO:70.
  • the antigen binding protein may comprise HCDR3, and the HCDR3 comprises the amino acid shown in any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:65 and SEQ ID NO:66 sequence.
  • the HCDR3 can comprise any 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 Amino acid sequence shown in one item.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:67.
  • the HCDR2 can comprise any 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 Amino acid sequence shown in one item.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise SEQ ID NO: 68 (X 1 X 2 X 3 MG, wherein, X 1 is N, R, S, T or Y, X2 is N or Y, X3 is the amino acid sequence shown in A, N or V).
  • the HCDR1 may comprise any 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 Amino acid sequence shown in one item.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO: 68 (X 1 X 2 X 3 MG, wherein X 1 is N, R, S, T or Y, X 2 is N or Y, X 3 is the aminoacid sequence shown in A, N or V), described HCDR2 can comprise the aminoacid sequence shown in SEQ ID NO:67, and described HCDR3 can comprise SEQ ID The amino acid sequence shown in any one of NO:1, SEQ ID NO:9, SEQ ID NO:65 and SEQ ID NO:66.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise any set of amino acid sequences of the following group:
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:1;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:9;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:14;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29;
  • 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
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the antibody framework region FR refers to the part of the antibody variable region that exists between the more divergent (ie hypervariable) CDRs.
  • Such framework regions are typically referred to as Frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide the backbone for representing the six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space, to Forms the antigen-binding surface.
  • the isolated antigen binding protein may comprise H-FR1
  • the H-FR1 may comprise SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 22, SEQ ID NO: 22, SEQ ID NO: The amino acid sequence shown in any one of ID NO:27, SEQ ID NO:32 and SEQ ID NO:37.
  • 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.
  • 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.
  • 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.
  • the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:12, The amino acid sequence shown in any one of 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 SEQ ID NO:5 As shown in the amino acid sequence, the H-FR3 may include the amino acid sequence shown in SEQ ID NO:6, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO:7.
  • the antigen binding protein of described separation can comprise antibody heavy chain variable region VH
  • described VH can comprise SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69 and SEQ ID NO: The amino acid sequence shown in any one of 70.
  • the VH can comprise any 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 Amino acid sequence shown in one item.
  • the antigen-binding fragment may be a VHH
  • the VHH may comprise any one of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:69 and SEQ ID NO:70. amino acid sequence.
  • the VHH can comprise any 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 Amino acid sequence shown in one item.
  • said 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 also include ⁇ and ⁇ .
  • Each heavy chain constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody containing a mu constant region is an IgM antibody
  • an antibody containing an epsilon constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising the ⁇ 1 constant region), IgG2 (comprising the ⁇ 2 constant region), IgG3 (comprising the ⁇ 3 constant region), and IgG4 (comprising the ⁇ 4 constant region) antibodies
  • IgA antibodies include But are not limited to, IgA1 (comprising ⁇ 1 constant region) and IgA2 (comprising ⁇ 2 constant region) antibodies
  • IgM includes but not limited to, IgM1 and IgM2.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from IgG.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from human IgG.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from human IgG1.
  • the heavy chain constant region of the antigen-binding protein may comprise an IgG Fc region.
  • the Fc region may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the present application also 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 is the antigen binding protein described in the application.
  • CAR chimeric antigen receptor
  • the CAR of the present application may comprise a VHH, which may comprise 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 the amino acid sequence shown in any one of SEQ ID NO:38.
  • the CAR includes an extracellular targeting part that binds to the MSLN protein, and may also include an intracellular domain.
  • the CAR may include an intracellular co-stimulatory signal region, which can provide a stimulating signal.
  • the co-stimulatory signal domain may comprise an intracellular co-stimulatory signal domain 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, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the co-stimulatory signal domain may be an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the co-stimulatory signal region may comprise the amino acid sequence shown in SEQ ID NO:39.
  • the CAR may comprise an intracellular signaling region, which may comprise a domain having at least one ITAM motif.
  • the intracellular signaling domain can transmit activation signals to the interior of the cell.
  • the intracellular signaling region may comprise an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia Viral gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and others that contain at least one ITAM domain.
  • EBV Epstein-Barr virus
  • PBj14Nef simian immunodeficiency virus
  • HSKV Kaposi's sarcoma herpesvirus
  • the intracellular signaling region can be a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling region may comprise the amino acid sequence shown in SEQ ID NO:41.
  • the CAR may comprise a transmembrane domain, which is a sequence of a cell surface protein that spans the cell membrane, which may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from any type I transmembrane protein.
  • the transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
  • 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, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region may be a transmembrane region derived from CD8.
  • the transmembrane region may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the CAR may comprise a hinge region, which may be located between the extracellular targeting moiety and the transmembrane domain.
  • 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, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be a hinge region derived from CD8.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:42.
  • the CAR may further include a signal peptide at the N-terminus of the targeting moiety that binds to the MSLN protein.
  • the signal peptide may be a signal peptide derived from CD8 protein.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:43.
  • the CAR may also comprise low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR.
  • the low-density lipoprotein receptor-related protein or fragments thereof may include low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • 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.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may include the amino acid sequence shown in SEQ ID NO:44.
  • the nucleic acid molecule encoding said low-density lipoprotein receptor-associated protein or a fragment thereof may comprise the nucleotide sequence shown in SEQ ID NO:45.
  • the sequence of the low-density lipoprotein receptor-related protein or its fragments in the CAR can be connected to the C-terminal sequence of the CAR through a self-cleaving peptide (for example, 2A peptides such as T2A, P2A, E2A) .
  • a self-cleaving peptide for example, 2A peptides such as T2A, P2A, E2A
  • the low-density lipoprotein receptor-related protein or a fragment thereof can be connected to the C-terminus of the intracellular signaling region through T2A.
  • the cleaved peptide may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the CAR may sequentially comprise a targeting moiety that binds to the MSLN protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the The transmembrane domain, the co-stimulatory signal region and the intracellular signal region.
  • a targeting moiety that binds to the MSLN protein (for example, the antigen-binding protein, and for example, the VHH described in the present application)
  • the hinge region for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the transmembrane domain for example, the co-stimulatory signal region and the intracellular signal region.
  • the CAR may sequentially comprise the VHH, the hinge region derived from CD8, the transmembrane region derived from CD8, the co-stimulatory signal region derived from 4-1BB, and the CD3 ⁇ -derived an intracellular signaling region
  • the VHH may comprise 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 : the amino acid sequence shown in any one of 38.
  • the CAR may sequentially comprise a targeting moiety that binds to the MSLN protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the The transmembrane domain, the co-stimulatory signal region, the intracellular signal region and the low-density lipoprotein receptor-associated protein or fragments thereof.
  • the CAR may sequentially comprise the VHH, the hinge region derived from CD8, the transmembrane region derived from CD8, the co-stimulatory signal region derived from 4-1BB, and the cellular region derived from CD3 ⁇ .
  • Inner signal region, and low-density lipoprotein receptor-associated protein or fragment thereof comprising the amino acid sequence shown in SEQ ID NO:44, and the VHH may comprise SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO :18, the amino acid sequence shown in any one of SEQ ID NO:23, SEQ ID NO:28, SEQ ID NO:33 and SEQ ID NO:38.
  • the CAR may sequentially include a signal peptide, a targeting moiety that binds to the MSLN protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region and the intracellular signal region.
  • a targeting moiety that binds to the MSLN protein for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the hinge region for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the transmembrane domain for example, the co-stimulatory signal region and the intracellular signal region.
  • the CAR may sequentially include a signal peptide, a targeting moiety that binds to the MSLN protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the transmembrane domain, the co-stimulatory signal region, the intracellular signal region and the low-density lipoprotein receptor-associated protein or a fragment thereof.
  • a targeting moiety that binds to the MSLN protein for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the hinge region for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the transmembrane domain for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the co-stimulatory signal region for example, the intracellular signal region and the low-density lipoprotein receptor-associated protein or a fragment thereof.
  • said chimeric antigen receptor can comprise the amino acid sequence shown in SEQ ID NO:47.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:48.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:49.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:50.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:51.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:52.
  • said chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the present application also provides one or more nucleic acid molecules, which can be nucleotides, deoxynucleotides and/ribonucleotides in isolated forms of any length, and can encode said isolated Antigen binding protein and/or said chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the present application also provides one or more nucleic acid molecules, said nucleic acid molecules comprising sequences capable of expressing said chimeric antigen receptor and said low-density lipoprotein receptor-associated protein or fragments thereof in cells .
  • the nucleic acid sequence encoding the chimeric antigenic protein may be connected to the nucleic acid sequence encoding the low-density lipoprotein receptor-related protein or its fragments through a cleavage peptide.
  • the present application also provides a carrier, which may include the nucleic acid molecule.
  • the vector can transform, transduce or transfect host cells, so that the genetic material elements carried by it can be expressed in the host cells.
  • vectors can include promoters, transcripts, enhancers, replicons, selection elements, and reporter genes.
  • a carrier may include components that facilitate entry into cells.
  • the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the present application also provides cells, which may include the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule and/or the carrier.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • the cells may be immune effector cells.
  • 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.
  • the cells may be T cells.
  • the cell may comprise and/or express the CAR.
  • the cell may comprise and/or express the CAR and the low-density lipoprotein receptor-related protein or a fragment thereof.
  • the present application also provides a pharmaceutical composition, which may include the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the cell, and any Optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with drug administration, generally safe, non-toxic , and is neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides a method for preparing the isolated antigen-binding protein and/or the chimeric antigen receptor.
  • the method may comprise culturing said cell under conditions such that said antigen receptor and/or said chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, which may include introducing the carrier into immune cells.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition Use in the preparation of medicaments, which can be used to prevent, alleviate and/or treat diseases and/or conditions.
  • the present application also provides methods for preventing, alleviating and/or treating diseases and/or conditions, which may include administering the isolated antigen-binding protein, the chimeric antigen receptor, Said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition.
  • the present application also provides the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition for use in For the prevention, alleviation and/or treatment of diseases and/or conditions.
  • the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of MSLN.
  • the diseases and/or conditions may include tumors.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the tumor may include hematoma and/or lymphoma.
  • the tumor may include a tumor expressing MSLN antigen.
  • the tumor may include ovarian cancer, pancreatic cancer, gastric cancer, mesothelial cell cancer, cholangiocarcinoma, triple negative breast cancer and/or endometrial cancer.
  • the subject may include humans or non-human animals.
  • the application provides a polypeptide comprising said isolated antigen binding protein.
  • the present application provides a kit or a drug delivery device comprising the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the said pharmaceutical composition.
  • MSLN-D1 (AA 296-390), MSLN-D2 (AA 391-486) and MSLN-D3 (487-598), using IgG-Fc and MSLN -His protein 10 ⁇ g/ml coated plate, 4 °C overnight.
  • the 96-well plate was coated with 2 ⁇ g/ml of MSLN-His, IgG1-Fc and 0.5% BSA, and allowed to stand overnight. Wash three times with 1 ⁇ PBST every other day, and then block with 0.5% BSA for 2 hours.
  • the output reserved for the fourth round was plated on 2YT/carb, and 192 single colonies were randomly picked and placed in 800 ⁇ l of 2YT/carb/M13KO7 the next day, and cultured with shaking overnight to produce phage.
  • the 384-well plate was coated with 2 ⁇ g/ml MSLN-His and IgG1-Fc, and allowed to stand overnight. Wash three times with 1 ⁇ PBST every other day, then block with 0.5% BSA for 2 hours, and centrifuge to collect the phage supernatant.
  • the expression plasmid was transfected into EXPI293 by PEI transfection method, and expressed in a 37°C cell culture incubator for 5 days, after which the cell supernatant was collected, and the antibody was purified with a ProteinA affinity chromatography column. Finally, the antibody protein with a purity of more than 90% is obtained.
  • the OVCAR3 and 293T-MSLN cells were revived and passaged. The cells were harvested on the day of the experiment. After counting, the cell density was adjusted to 1 ⁇ 10 6 /ml, 30ul per well (3 ⁇ 10 4 /well).
  • the representative MSLN nanobody, positive antibody P4, and negative antibody Caplacizumab were used at the highest concentration of 100nM, 3 times ratio, 7 gradients, PBS control, 30ul per well, mixed and incubated at 4°C for 1 hour. Wash twice with PBS containing 0.1% BSA, 500g, 5min, 4°C, and spin dry.
  • Dilute fluorescent secondary antibody Goat pAb to Hu IgG[DyLight 650] (abcam, catalog number: ab98593) at 1:200, 30ul per well, mix well and incubate at 4°C for 30 minutes; wash twice with PBS containing 0.1% BSA, 500g, 5min , 4°C, shake dry, resuspend at 30ul/well, read the value on the high-throughput flow cytometer (IQue), and use Graphpad to make a three-parameter fitting curve for the collected data.
  • the results are shown in Figure 4.
  • the numbers in the legend represent the sequence numbers corresponding to the MSLN VHH.
  • 293T, LO2, KERA, A549, HACAT and K562-MSLN cells were revived and passaged.
  • the cells were harvested on the day of the experiment. After counting, the cell density was adjusted to 1 ⁇ 10 6 /ml, 30ul per well (3 ⁇ 10 4 /well).
  • the representative MSLN nanobody and positive antibody P4 were mixed at a concentration of 20ug/ml, 30ul per well, and incubated at 4°C for 1 hour after mixing. Wash twice with PBS containing 0.1% BSA, 500g, 5min, 4°C, and spin dry.
  • Dilute fluorescent secondary antibody Goat pAb to Hu IgG[DyLight 650] (abcam, catalog number: ab98593) at 1:200, 30ul per well, mix well and incubate at 4°C for 30 minutes; wash twice with PBS containing 0.1% BSA, 500g, 5min , 4°C, shake dry, resuspend at 30ul/well, and read on the high-throughput flow cytometer (IQue).
  • the results are shown in Figure 5. The results showed that the sequences of MSLN 1, MSLN 4, MSLN 5, MSLN 6, MSLN 11, MSLN 36, and MSLN 42 all had non-specific binding activity to 293T, LO2, KERA, A549, and HACAT cells.
  • the vector system used to construct the lentiviral plasmid vector of the present invention belongs to the third-generation lentiviral vector system, and the system has three plasmids in total: the packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein; the encoding envelope protein VSV-G PMD2.G plasmid, containing the CAR core plasmid encoding the target gene.
  • the expression of the CAR gene encoded in the core plasmid based on the BBz/L6 platform is regulated by the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • PBMC cell recovery add 11ml X-VIVO, centrifuge at 500g, 5min; add 12ml X-VIVO, centrifuge at 400g, 5min; add 12ml X-VIVO, centrifuge at 300g, 5min; 1ml X-VIVO suspension count; adjust cell density 1 ⁇ 10 6 cells/ml, add CD3/CD28 (4 ⁇ 10 8 /ml) magnetic beads (Thermo Fisherman, Cat.
  • the number in the legend represents the corresponding sequence number of MSLN VHH, select MSLN3 (VHH sequence as shown in SEQ ID NO:13), MSLN10 (VHH sequence as shown in SEQ ID NO:18 shown), MSLN16 (VHH sequence shown in SEQ ID NO: 8), MSLN27 (VHH sequence shown in SEQ ID NO: 23), MSLN36 (VHH sequence shown in SEQ ID NO: 28), MSLN41 (VHH sequence As shown in SEQ ID NO:33), MSLN42 (VHH sequence as shown in SEQ ID NO:38), wherein MSLN0 is a negative control Caplacizumab, P4 is a positive control (VH sequence as shown in SEQ ID NO:58, VL sequence as shown in Shown in SEQ ID NO:62), T is the blank control without adding target cells. The results showed that all antibodies had a similar proliferation rate as the positive antibodies.
  • LDH is a stable cytoplasmic enzyme, which is released when cells are lysed.
  • the release method is basically the same as that of 51Cr in radioactive analysis.
  • the released LDH is detected in the medium supernatant by coupled enzyme reaction.
  • LDH It can convert a tetrazolium salt (INT) into red formazan, and the amount of red product produced is proportional to the number of lysed cells (Promega, catalog number: G1780).
  • INT tetrazolium salt
  • the positive rate of CAR-T was detected, and the proportion of infected CAR groups was adjusted by uninfected blank T cells.
  • the T cell well was used as the T cell self-release LDH blank control group; the blank T cell well containing only the same effective target ratio as the experimental group was set as the T+ target cell negative control group; Release LDH background group; set the wells containing only the same amount of target cells as the experimental group as the maximum release LDH background group of target cells; set a single well containing only 200uL cell culture medium as the culture medium background LDH control well; set only 200uL cell culture medium alone
  • the wells are volume-corrected control wells; the corresponding effector cells and target cells are co-incubated in 200 ⁇ l of X-VIVO (
  • tumor cells SK-OV3-Luciferase-GFP were co-cultured with example MSLN-CAR-T cells according to different effect-to-target ratios for 18h and 72h, and the killing effect of CAR-T cells on tumor cells was detected.
  • MSLN-CAR-101 wherein, the amino acid sequence of the targeting part of the chimeric antigen receptor is shown in SEQ ID NO: 13, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 40, a total of The amino acid sequence of the stimulating domain is shown in SEQ ID NO:39, the amino acid sequence of the intracellular signaling domain is shown in SEQ ID NO:41, the amino acid sequence of the hinge region is shown in SEQ ID NO:42, and the MSLN-CAR -101 cells also express the exogenous protein fragment shown in SEQ ID NO:44) and the killing effect of the positive sequence P4 is similar to that of the positive sequence P4.
  • Embodiment 7 NSG mouse animal drug efficacy test
  • mice were subcutaneously injected with 3 ⁇ 10 6 SKOV3 tumor cell line, and two weeks later, when the tumor volume was 100-200 mm 3 , they were randomly divided into groups, and CAR-T was infused intravenously the next day, with a cell volume of 5 ⁇ 10 6 . Tumors were measured 3 times a week, and the test was observed for 47 days. Finally, the tumor growth curve was drawn with Graphpad. Among them, 5E6 represents ovarian cancer cells inoculated with 5*10 ⁇ 6 SK-OV3.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/CN2023/070928 2022-01-06 2023-01-06 靶向msln的抗原结合蛋白及其应用 Ceased WO2023131276A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202380015977.8A CN118510809A (zh) 2022-01-06 2023-01-06 靶向msln的抗原结合蛋白及其应用
JP2024540577A JP2025505933A (ja) 2022-01-06 2023-01-06 Ｍｓｌｎを標的とする抗原結合タンパク質及びその使用
EP23737138.0A EP4461747A4 (en) 2022-01-06 2023-01-06 ANTIGEN-BINDING PROTEIN TARGETTING MSLN AND ITS USE
US18/726,241 US20250213691A1 (en) 2022-01-06 2023-01-06 Antigen binding protein targeting msln and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202210011545 2022-01-06
CN202210011545.7 2022-01-06
CN202210018557.2 2022-01-07
CN202210018557 2022-01-07

Publications (1)

Publication Number Publication Date
WO2023131276A1 true WO2023131276A1 (zh) 2023-07-13

Family

ID=87073247

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/070928 Ceased WO2023131276A1 (zh) 2022-01-06 2023-01-06 靶向msln的抗原结合蛋白及其应用

Country Status (5)

Country Link
US (1) US20250213691A1 (https=)
EP (1) EP4461747A4 (https=)
JP (1) JP2025505933A (https=)
CN (1) CN118510809A (https=)
WO (1) WO2023131276A1 (https=)

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103819559A (zh) * 2013-12-10 2014-05-28 中国科学院武汉病毒研究所 一种抗间皮素的纳米抗体及其编码基因和该纳米抗体的用途
US20170267755A1 (en) * 2016-03-16 2017-09-21 Sri International Isolated anti-mesothelin antibodies, conjugates and uses thereof
US20180002439A1 (en) * 2016-06-30 2018-01-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anti-mesothelin antibodies and uses thereof
CN107840891A (zh) * 2016-09-19 2018-03-27 上海吉倍生物技术有限公司 高亲和力的抗msln抗体及其应用
CN108129566A (zh) * 2017-12-31 2018-06-08 中国科学院武汉病毒研究所 靶向间皮素的高亲和力c-型单域抗体及其制备方法与应用
CN109111528A (zh) * 2018-09-20 2019-01-01 杭州普略生物科技有限公司 以msln为靶点的嵌合抗原受体
CN110698562A (zh) * 2019-10-31 2020-01-17 浙江蓝盾药业有限公司 抗人msln单克隆抗体
CN111349165A (zh) * 2020-05-25 2020-06-30 南京蓝盾生物科技有限公司 抗人msln单克隆抗体及其应用
US20200262928A1 (en) * 2017-10-20 2020-08-20 Green Cross Corporation Anti-msln antibody and pharmaceutical composition for cancer treatment comprising same
CN111560072A (zh) * 2020-07-16 2020-08-21 上海恒润达生生物科技有限公司 抗人msln抗体以及靶向msln的免疫效应细胞
CN111848819A (zh) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 一种靶向msln的嵌合抗原受体及其应用
CN112210008A (zh) * 2020-07-16 2021-01-12 上海恒润达生生物科技有限公司 一种新型抗人msln抗体及其用途
CN112225811A (zh) * 2020-07-16 2021-01-15 上海恒润达生生物科技有限公司 新型抗人msln抗体及其用途

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104955845B (zh) * 2012-09-27 2018-11-16 美国政府(由卫生和人类服务部的部长所代表) 间皮素抗体和引起有效的抗肿瘤活性的方法
EP3621994A4 (en) * 2017-05-12 2020-12-30 Harpoon Therapeutics, Inc. MESOTHELIN BINDING PROTEINS
WO2019043026A1 (en) * 2017-08-30 2019-03-07 INSERM (Institut National de la Santé et de la Recherche Médicale) ANTI-MESOTHELIC RADIOMARQUATED SINGLE DOMAIN ANTIBODIES SUITABLE FOR IMAGING AND TREATING CANCERS
WO2019184886A1 (zh) * 2018-03-26 2019-10-03 上海原能细胞医学技术有限公司 促进免疫细胞增殖的方法
EP3806889A4 (en) * 2018-06-18 2022-07-13 Anwita Biosciences, Inc. CYTOKI FUSION PROTEINS AND USES THEREOF
CA3111087A1 (en) * 2018-08-29 2020-03-05 Nanjing Legend Biotech Co., Ltd. Anti-mesothelin chimeric antigen receptor (car) constructs and uses thereof
CA3125484A1 (en) * 2019-01-08 2020-07-16 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Cross-species single domain antibodies targeting mesothelin for treating solid tumors
CN118510890A (zh) * 2022-01-07 2024-08-16 原启生物科技(上海)有限责任公司 靶向cldn18.2和msln的嵌合抗原受体及其用途

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103819559A (zh) * 2013-12-10 2014-05-28 中国科学院武汉病毒研究所 一种抗间皮素的纳米抗体及其编码基因和该纳米抗体的用途
US20170267755A1 (en) * 2016-03-16 2017-09-21 Sri International Isolated anti-mesothelin antibodies, conjugates and uses thereof
US20180002439A1 (en) * 2016-06-30 2018-01-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anti-mesothelin antibodies and uses thereof
CN107840891A (zh) * 2016-09-19 2018-03-27 上海吉倍生物技术有限公司 高亲和力的抗msln抗体及其应用
US20200262928A1 (en) * 2017-10-20 2020-08-20 Green Cross Corporation Anti-msln antibody and pharmaceutical composition for cancer treatment comprising same
CN108129566A (zh) * 2017-12-31 2018-06-08 中国科学院武汉病毒研究所 靶向间皮素的高亲和力c-型单域抗体及其制备方法与应用
CN109111528A (zh) * 2018-09-20 2019-01-01 杭州普略生物科技有限公司 以msln为靶点的嵌合抗原受体
CN110698562A (zh) * 2019-10-31 2020-01-17 浙江蓝盾药业有限公司 抗人msln单克隆抗体
CN111349165A (zh) * 2020-05-25 2020-06-30 南京蓝盾生物科技有限公司 抗人msln单克隆抗体及其应用
CN111560072A (zh) * 2020-07-16 2020-08-21 上海恒润达生生物科技有限公司 抗人msln抗体以及靶向msln的免疫效应细胞
CN112210008A (zh) * 2020-07-16 2021-01-12 上海恒润达生生物科技有限公司 一种新型抗人msln抗体及其用途
CN112225811A (zh) * 2020-07-16 2021-01-15 上海恒润达生生物科技有限公司 新型抗人msln抗体及其用途
CN111848819A (zh) * 2020-07-31 2020-10-30 广东昭泰体内生物医药科技有限公司 一种靶向msln的嵌合抗原受体及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
D. J. LIPMANW. R. PEARSON: "Rapid and Sensitive Protein Similarity Searches", SCIENCE, vol. 227, 1989, pages 1435 - 1441
S. ALTSCHULW. GISHW. MILLERE. W. MYERSD. LIPMAN: "Basic Local Alignment Search Tool", JOURNAL OF MOLECULAR BIOLOGY, vol. 215, 1990, pages 403 - 410
See also references of EP4461747A4
W. R. PEARSOND. J. LIPMAN: "Improved tools for biological sequence comparison", PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 2444 - 2448

Also Published As

Publication number Publication date
CN118510809A (zh) 2024-08-16
EP4461747A4 (en) 2026-04-15
JP2025505933A (ja) 2025-03-05
EP4461747A1 (en) 2024-11-13
US20250213691A1 (en) 2025-07-03

Similar Documents

Publication Publication Date Title
JP7280828B2 (ja) Ｂｃｍａを標的とする抗体およびその使用
CN117229407B (zh) 一种靶向gprc5d的单域抗体、嵌合抗原受体及其应用
US20240384001A1 (en) Anti-gprc5d antigen binding protein and use thereof
US20250177527A1 (en) Chimeric antigen receptor targeting cldn18.2 and msln and use thereof
CN118922451A (zh) 靶向bcma和gprc5d的嵌合抗原受体及其应用
JP2025504803A (ja) Ｇｐｒｃ５ｄを標的とするキメラ抗原受容体及びその使用
US20240182562A1 (en) Cldn18.2 antigen-binding protein and use thereof
CN113698492A (zh) 人间皮素嵌合抗原受体及其用途
CN112661857A (zh) 一种嵌合抗原受体及其用途
TW202317640A (zh) 一種抗人pd-l1人源化抗體或其抗原結合片段及其應用
WO2023024084A1 (zh) 一种嵌合抗原受体及其用途
WO2023131276A1 (zh) 靶向msln的抗原结合蛋白及其应用
CN116284389A (zh) 抗afp/hla02 tcr样抗体及其用途
CN117241824A (zh) 改造的免疫效应细胞及其用途
CN117098782A (zh) 靶向cldn18.2的抗原结合蛋白及其用途
EP4620974A1 (en) Anti-afp/hla02 tcr-like antibody and use thereof
WO2024088371A1 (zh) 靶向msln的抗原结合蛋白
WO2026026960A1 (zh) 一种靶向bcma的人源化纳米抗体及其应用
WO2025067388A1 (zh) 一种靶向cd79b的单域抗体和嵌合抗原受体及其应用
WO2023016554A1 (zh) 靶向cd22的抗原结合蛋白及其用途
TW202521566A (zh) 一種靶向cd38和cll1的雙特異性嵌合抗原受體及其應用
WO2026020799A1 (zh) 靶向b7-h3的抗体、嵌合抗原受体及其应用
WO2026002239A1 (zh) 靶向cd155的抗原结合蛋白及其应用
WO2025167664A1 (zh) Ror1抗原结合蛋白及其用途
WO2026086893A1 (zh) 一种靶向Nectin-4的单域抗体和嵌合抗原受体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23737138

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202380015977.8

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2024540577

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2023737138

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023737138

Country of ref document: EP

Effective date: 20240806

WWP Wipo information: published in national office

Ref document number: 18726241

Country of ref document: US