WO2019104562A1 - 一种嵌合抗原受体及其应用 - Google Patents

一种嵌合抗原受体及其应用 Download PDF

Info

Publication number
WO2019104562A1
WO2019104562A1 PCT/CN2017/113661 CN2017113661W WO2019104562A1 WO 2019104562 A1 WO2019104562 A1 WO 2019104562A1 CN 2017113661 W CN2017113661 W CN 2017113661W WO 2019104562 A1 WO2019104562 A1 WO 2019104562A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
seq
amino acid
cells
acid sequence
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/CN2017/113661
Other languages
English (en)
French (fr)
Inventor
胡文
姚永超
郭文中
江银波
黄烁洲
江亭
李姣姣
陶铸
古艳丽
张会会
秦莉
陈小平
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.)
Guangzhou Cas Lamvac Biotech Co Ltd
Original Assignee
Guangzhou Cas Lamvac Biotech 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 Guangzhou Cas Lamvac Biotech Co Ltd filed Critical Guangzhou Cas Lamvac Biotech Co Ltd
Priority to US17/051,747 priority Critical patent/US12221475B2/en
Priority to GB2015416.7A priority patent/GB2585607B/en
Priority to EP17933216.8A priority patent/EP3744738A4/en
Priority to AU2017441551A priority patent/AU2017441551B2/en
Priority to CA3098330A priority patent/CA3098330C/en
Priority to CN201780001820.4A priority patent/CN110325551B/zh
Priority to PCT/CN2017/113661 priority patent/WO2019104562A1/zh
Publication of WO2019104562A1 publication Critical patent/WO2019104562A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C07K16/20—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • 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
    • A61K40/43—Protozoan antigens
    • 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
    • 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
    • C07K14/70517—CD8
    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/24—Dimerizable CARs; CARs with adapter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of cellular immunotherapy of tumors, in particular to a chimeric antigen receptor and application thereof, in particular to a chimeric antigen receptor with VAR2CSA protein as a binding domain and its application in tumor therapy.
  • CAR Chimeric Antigen Receptor
  • CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy
  • the treatment has achieved great success (Porter et al., N Engl J Med. 2011, 365(8): 725-33; Grupp et al., N Engl J Med.
  • TIL tumor infiltrating T lymphocytes
  • TCR-T engineered T cell antigen receptor T cell
  • CAR-T technology can recombine CAR molecules into CD3-positive T cells, and the binding to target cells is independent of MHC molecules, avoiding the natural state.
  • the many intermediate links that the immune cells rely on for cancer cell clearance enable CAR-T cells to accurately recognize the antigens of cancer cells and directly target killing cancer cells (Fesnak et al., Nat Rev Cancer. 2016, 16(9): 566 -81; Lim and June, Cell. 2017, 168(4): 724-740.).
  • Immunological checkpoint blockers typified by antibodies specific for CTLA-4 and PD-1/PD-L1. Treatment (Pauken et al., Science. 2016, 354 (6316): 1160-1165.), CAR-T cells are able to proliferate and maintain their anti-tumor activity in cancer patients, On the treatment of immune CAR-T than the checkpoint blocker therapy more durable.
  • CAR-T Improve the safety of clinical application of CAR-T, such as integration of "suicide genes” (Jensen et al., Biol Blood Marrow Transplant. 2010, 16(9): 1245-56. ;Gargett and Brown, Front. Pharmacol. 2014, 5(235): 1-7.) or "Molecular Switch” (Rodgers et al., Proc Natl Acad Sci USA. 2016, 113(4): E459-68; Wu Et al., Science. 2015, 350 (6258): aab4077 ; Morsut et al., Cell.
  • VAR2CSA is a protein expressed by human Plasmodium falciparum and transported to the surface of infected red blood cells (PfEMP1, PlasmoDB: PF3D7_1200600), which is a protein modified by targeting placenta-like chondroitin sulfate (pl-CSA).
  • PfEMP1 Plasmodium falciparum and transported to the surface of infected red blood cells
  • pl-CSA a protein modified by targeting placenta-like chondroitin sulfate
  • Polysaccharides mediate the adhesion of erythrocytes infected by Plasmodium to the extracellular matrix and plasma membrane of placental syncytia (Salanti et al., Mol Microbiol.
  • Plasmodium falciparum and humans have at least millions of years of co-evolutionary history (Rich and Ayala, In Krishna R.Dronamraju, Paolo Arese (Ed). Emerging Infectious Diseases of the 21st Century: Malaria-Genetic and Evolutionary Aspects. Springer US 2006.pp.125-146.), its life history mainly includes three stages: mosquito stage, liver stage and red stage (Bousema et al., Nat Rev Microbiol.
  • human erythrocytes are the host cells of their erythropoiesis, and their clearance from the immune system is mainly through the erythrocyte membrane protein 1 (PfEMP1) expressed by Plasmodium falciparum, which is infected with P. falciparum.
  • PfEMP1 erythrocyte membrane protein 1
  • VAR2CSA protein in patients with placental malaria may also be the result of co-evolution with human Plasmodium falciparum and humans.
  • pl-CSA is mainly expressed on the surface of different types of tumor cells and its extracellular matrix (Salanti et al., Cancer Cell. 2015, 28(4): 500-14; Ayres Pereira et al., PLoS Pathog. 2016, 12 (8): e1005831; Seiler et al., Eur Urol. 2017, 72(1): 142-150.), we speculate that the targeted binding of VAR2CSA protein to pl-CSA may help to break through the immunosuppression of solid tumors. Microenvironment.
  • Salanti and co-workers showed that the rVAR2 protein was coupled to the hemiasterlin toxin analog KT886 from the sponge Hemiasterella minor to obtain the drug VDC886 (rVAR2 drug conjugated KT886), which showed a contradiction in a non-pregnant tumor-bearing mouse model.
  • VDC886 Another study of VDC886 also found that in vitro, VDC886 was able to effectively remove cultured invasive bladder cancer (MIBC) cell lines with IC50 at low nanomolar levels; in vivo, weekly Intravenous injections (four times in total) VDC886 effectively prevented chemoresistance (resistance to cisplatin-based neoadjuvant chemotherapeutic drugs) growth of orthotopic bladder cancer xenografts and prolonged survival of tumor-bearing mice (Seiler et al., Eur Urol. 2017, 72(1): 142-150.). The above studies show that rVAR2 protein has a certain stability in tumor-bearing mice, and its protein itself has no adverse effects on the immunogenicity of mice.
  • MIBC invasive bladder cancer
  • CAR-T cells mainly stimulate the cytokine release and cell proliferation of CAR-T cells through phosphorylation signaling through the interaction of CAR with tumor cell surface antigens, ultimately killing or eliminating tumor cells (Chmielewski et Al., Immunol Rev. 2014, 257(1): 83-90.).
  • CN 105753991A discloses a chimeric antigen receptor against placental-like chondroitin sulfate and its use, and it has been found that replacing a single-chain antibody in a general CAR-T with a VAR2CSA protein domain that specifically interacts with pl-CSA (single- The CART-rVAR2 obtained from the fragment fragment variable (ScFv) fragment has in vitro killing activity against a variety of different types of tumor cells, but subsequent studies have found that it does not secrete cytokines.
  • ScFv fragment fragment variable
  • CAR-T cells in clinical trials target only specific tumor types and/or specific protein targets (clinicaltrials.gov), such as CAR-T for CD19 and CD22 targeting B-cell leukemia and B-cell lymphoma ( ClinicalTrials.gov Identifier: NCT00450944); CAR-T against the target of liver cancer GPC3 (ClinicalTrials.gov Identifier: NCT02723942); CAR-T expressing anti-PD1 antibody against lung cancer, liver cancer and gastric cancer (ClinicalTrials.gov Identifier: NCT02862028 CAR-T (ClinicalTrials.gov Identifier: NCT03252171) for GD2-positive glioma patients; CAR-T (ClinicalTrials.gov Identifier: NCT02575261) for EphA2-positive glioblastoma patients; lung cancer treatment for MUC1 target CAR-T (ClinicalTrials.gov Identifier: NCT03198052); multiple myelom
  • Gene insertion mutation occurred in the process of constructing CAR-T cells due to the use of lentiviral vector or other retroviral vector causes the possibility of cell carcinogenesis, for example, in a clinical trial using retroviral therapy for severe combined immunodeficiency disease caused by cytokine receptor gamma chain deletion, CD34 receiving retroviral transduction + bone marrow progenitor cells in patients treated with 9 4 develop acute T-cell leukemia, and the emergence of this condition is considered to be inserted LMO2 oncogene mutations result in CD34 + bone marrow progenitors transduced retrovirus The amount of proliferation is related (Hacein-Bey-Abina et al., N Engl J Med.
  • a "suicide gene switch" type CAR-T cell based on an inducible Caspase 9 (iCasp9) protein regulatory system is said to be able to effectively limit "on-target, on-tumor toxicities” by clearing transduced CAR-T cells.
  • iCasp9 inducible Caspase 9
  • anti-CD20 chimeric antibodies may be difficult to apply to CAR-T against B lymphoma, while the Myc-tag strategy lacks clinical grade antibodies (Paszkiewicz et al., J Clin Invest. 2016, 126(11): 4262-4272.).
  • cetuximab (Erbitux TM)
  • cetuximab (Erbitux TM)
  • the human epidermal growth factor receptor polypeptide tEGFR is co-expressed on CAR-T cells via a 2A sequence as a cell surface marker for enrichment and tracking identification of CAR+-T cells in vivo, and can be used clinically.
  • the grade of cetuximab mAb controls the activity of infused CAR+-T cells in vivo by antibody-dependent cytotoxicity (Wang et al., Blood. 2011, 118(5): 1255-63.). However, in the case of severe toxicity, this apoptosis by antibody-dependent cytotoxicity can be initiated rapidly and needs to be verified in clinical trials.
  • CAR-T technology applied to the treatment of solid tumors remains difficult (Newick et al., Annu Rev Med. 2017, 68: 139-152.).
  • CAR-T cells must enter the solid tumor from the blood system, infiltrating and passing through the tumor's stroma to produce tumor-associated antigen-specific cytotoxicity and play a tumor killing function.
  • the function of CAR-T cells is rapidly lost, including: 1. inhibition of the tumor microenvironment, oxidative stress response of the tumor microenvironment, Nutritional deprivation, acidic pH environment, hypoxia, etc. can inhibit the activity of T cells; 2.
  • Negative effects of soluble T cell inhibitory factors and cytokines 3. Inhibitory immune cells, such as regulatory T cells (Tregs), Inhibition of myeloid-derived suppressor cells (MDSCs), as well as tumor-associated macrophages (TAMs) or neutrophils (TANs); 4. Endogenous negative regulatory mechanisms of T cells themselves, such as intracellular and cellular Inhibition of upregulation of expression of surface inhibitory receptors.
  • Tregs regulatory T cells
  • MDSCs Inhibition of myeloid-derived suppressor cells
  • TAMs tumor-associated macrophages
  • TANs neutrophils
  • Endogenous negative regulatory mechanisms of T cells themselves such as intracellular and cellular Inhibition of upregulation of expression of surface inhibitory receptors.
  • CAR-T cell technology is basically an individualized treatment technology, often accompanied by serious toxic side effects and even threatening the life of the patient, and CAR-T cell therapy for solid tumors. Still difficult. How to develop a broad-spectrum CAR-T cell capable of killing activity against many different types of tumors, and to reduce its toxic side effects is an urgent problem to be solved.
  • the present invention provides a chimeric antigen receptor and the use thereof, the chimeric antigen receptor having multiple targets The ability of different types of tumor cells, with ordinary The broad spectrum of CAR-T cells is not available and can be regulated.
  • the invention provides a chimeric antigen receptor comprising any one of a compound that recognizes Plasmodium VAR2CSA, a protein tag on Plasmodium VAR2CSA, or a protein capable of labeling Plasmodium VAR2CSA Or a combination of at least two domains.
  • the chimeric antigen receptor comprises a binding domain capable of recognizing the Plasmodium protein VAR2CSA, which can recognize the VAR2CSA protein, that is, can recognize any combination of the VAR2CSA protein and the placenta-like chondroitin sulfate (pl-CSA).
  • One or at least two domain recombinant proteins can also recognize a marker molecule on the Plasmodium protein VAR2CSA, a fusion protein tag capable of recombinant expression with the Plasmodium protein VAR2CSA or a marker capable of labeling the Plasmodium VAR2CSA
  • Compounds that is, chimeric antigen receptors capable of recognizing VAR2CSA protein in any way are within the scope of the present application, either by recognizing the VAR2CSA protein itself or by recognizing other proteins capable of fusion with the VAR2CSA protein or Compounds capable of labeling VAR2CSA proteins are within the scope of the invention.
  • the VAR2CSA protein has the ability to target a plurality of different types of tumor cells by binding to placental-like chondroitin sulfate pl-CSA on the surface of tumor cells, and almost 95% (106/111) of cancer-derived patients, Human cancer cell lines including hematopoietic, epithelial, and mesenchymal sources can be targeted.
  • the inventors by specifically binding a chimeric antigen receptor to a VAR2CSA protein, the inventors have found that the chimeric antigen receptor can be further regulated by regulating the content of the VAR2CSA protein, and when there is no VAR2CSA protein, the chimeric antigen The receptor does not work and does not cause toxic side effects on healthy cells.
  • a fusion protein is formed by fusion expression of a protein tag with a VAR2CSA protein, so that the chimeric antigen receptor domain capable of recognizing the protein tag can also indirectly recognize the VAR2CSA fusion protein containing the protein tag; likewise, by the compound tag VAR2CSA
  • the protein is then used to construct a chimeric antigen receptor using a single-chain antibody capable of specifically recognizing the compound, and the purpose of indirectly recognizing the VAR2CSA protein containing the compound can also be achieved.
  • the identification VAR2CSA Plasmodium protein domain comprises a heavy chain variable region (V H) and an anti-VAR2CSA antibody light chain variable region (V L).
  • the antigen of the VAR2CSA protein antibody is any one or a combination of at least two of DBL1X, ID1, DBL2X, ID2a, ID2b, DBL3X, ID3, DBL4 ⁇ , ID4, DBL5 ⁇ , ID5 or DBL6 ⁇ , preferably ID1 , a combination of DBL2X and ID2a.
  • the VAR2CSA protein can be recognized by recognizing any one of the VAR2CSA proteins, particularly the combination of ID1, DBL2X and ID2a, that is, the VAR2CSA protein can be combined with placental-like chondroitin sulfate (pl-CSA). Binding of any one or at least two domains of recombinant protein (rVAR2) to enable accurate recognition of VAR2CSA protein, and rVAR2 can bind to placental-like chondroitin sulfate (pl-CSA) on the surface of tumor cells, and can also be used in the present invention
  • the chimeric antigen receptor targets an intermediate of the tumor cell surface pl-CSA antigen.
  • the DBL1X comprises an amino acid sequence as shown in SEQ ID NO. 1, and the amino acid sequence shown in SEQ ID NO. 1 is as follows:
  • the ID1 comprises an amino acid sequence as shown in SEQ ID NO. 2, and the amino acid sequence shown in SEQ ID NO. 2 is as follows:
  • the DBL2X comprises an amino acid sequence as shown in SEQ ID NO. 3, and the amino acid sequence shown in SEQ ID NO. 3 is as follows:
  • DBL2X (SEQ ID NO. 3):
  • the ID2a comprises an amino acid sequence as shown in SEQ ID NO. 4, and the amino acid sequence shown in SEQ ID NO. 4 is as follows:
  • the ID2b comprises an amino acid sequence as shown in SEQ ID NO. 5, and the amino acid sequence shown in SEQ ID NO. 5 is as follows:
  • the DBL3X comprises an amino acid sequence as shown in SEQ ID NO. 6, the amino acid sequence shown in SEQ ID NO. 6 is as follows:
  • DBL3X (SEQ ID NO. 6):
  • the ID3 comprises an amino acid sequence as shown in SEQ ID NO. 7, and the amino acid sequence shown in SEQ ID NO. 7 is as follows:
  • the DBL4 ⁇ comprises an amino acid sequence as shown in SEQ ID NO. 8, the amino acid sequence shown in SEQ ID NO. 8 is as follows:
  • DBL4 ⁇ (SEQ ID NO. 8):
  • the ID4 comprises an amino acid sequence as shown in SEQ ID NO. 9, and the amino acid sequence shown in SEQ ID NO. 9 is as follows:
  • the DBL5 ⁇ comprises an amino acid sequence as shown in SEQ ID NO. 10, and the amino acid sequence shown in SEQ ID NO. 10 is as follows:
  • DBL5 ⁇ (SEQ ID NO. 10):
  • the ID5 comprises an amino acid sequence as shown in SEQ ID NO. 11, and the amino acid sequence shown in SEQ ID NO. 11 is as follows:
  • the DBL6 ⁇ comprises an amino acid sequence as shown in SEQ ID NO. 12, and the amino acid sequence shown in SEQ ID NO. 12 is as follows:
  • the anti-VAR2CSA protein antibody is a single-chain antibody linked by a short peptide of 15-20 amino acids.
  • the binding domain of the Plasmodium protein VAR2 CSA comprises a complementarity determining region (CDR) sequence of a heavy chain variable region and a light chain variable region of an antibody having an anti-VAR2 CSA protein.
  • CDR complementarity determining region
  • the CDR of the heavy chain variable region having a single-chain antibody has the following sequence:
  • CDR1 is the amino acid sequence shown in SEQ ID NO. 13;
  • CDR2 is the amino acid sequence shown in SEQ ID NO.
  • the amino acid sequence shown in SEQ ID NO. 15 is as follows:
  • CDR1 (SEQ ID NO. 13): GFTFSNYA;
  • CDR2 (SEQ ID NO. 14): ISITGRYT;
  • CDR3 (SEQ ID NO. 15): TREGYDYAPSWFAY.
  • the CDR of the light chain variable region having a single-chain antibody has the following sequence:
  • CDR1 is represented by the amino acid sequence of SEQ ID NO. 16;
  • CDR2 is represented by the amino acid sequence of SEQ ID NO. 17;
  • the amino acid sequence shown in SEQ ID NO. 18 is as follows:
  • CDR1 (SEQ ID NO. 16): QTLVHRNGITY;
  • CDR2 (SEQ ID NO. 17): KVS;
  • CDR3 (SEQ ID NO. 18): FQGSHVPRT.
  • the heavy chain variable region of the single-chain antibody of the binding domain of VAR2CSA comprises or has at least 70% amino acid sequence identity, preferably 90% or more, as set forth in SEQ ID NO. A variant of amino acid identity.
  • the variant still has activity in binding to a VAR2CSA protein, and the amino acid sequence shown in SEQ ID NO. 19 is as follows:
  • the light chain variable region of the single-chain antibody of the binding domain of VAR2CSA comprises an ammonia as shown in SEQ ID NO.
  • the variant still has activity in binding to a VAR2CSA protein, and the amino acid sequence shown in SEQ ID NO. 20 is as follows:
  • the heavy chain variable region of the single-chain antibody of the binding domain of VAR2CSA comprises or has at least 60% nucleotide sequence identity with the nucleotide sequence as shown in SEQ ID NO. 21, preferably A variant that is more than 80% nucleotide identity.
  • the variant still has an amino acid capable of expressing a binding to a VAR2 CSA protein, and the nucleotide sequence shown in SEQ ID NO. 21 is as follows:
  • the light chain variable region of the single-chain antibody of the binding domain of VAR2CSA comprises or has at least 60% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID NO. A variant that is more than 80% nucleotide identity.
  • the variant still has an amino acid capable of expressing a binding to a VAR2 CSA protein, and the nucleotide sequence shown in SEQ ID NO. 22 is as follows:
  • the protein tag on the Plasmodium protein VAR2CSA is selected from, but not limited to, PNE-tag, human myc-tag CaptureSelect C-tag, FLAG-tag, 3 ⁇ FLAG-tag, Strep-tag, 6 ⁇ His-tag Any one or combination of at least two of V5tag, S-tag, HA-tag, VSV-G-tag, GST-tag, HaloTag, XTEN-tag or huEGFRt-tag protein tag.
  • the PNE-tag (peptide neo-epitopes) is a 14 amino acid comprising the transcription factor GCN4 derived from yeast, having the amino acid sequence shown in SEQ ID NO. 23, and the amino acid sequence shown in SEQ ID NO. as follows:
  • PNE-tag (SEQ ID NO. 23): NYHLENEVARLKKL.
  • the human myc-tag comprises 10 amino acids from a human c-myc protein having the amino acid sequence set forth in SEQ ID NO. 24, and the amino acid sequence set forth in SEQ ID NO. 24 is as follows:
  • Myc-tag (SEQ ID NO. 24): EQKLISEEDL.
  • CaptureSelect C-tag has the amino acid sequence set forth in SEQ ID NO. 25, and the amino acid sequence shown in SEQ ID NO. 25 is as follows:
  • CaptureSelect C-tag (SEQ ID NO. 25): EPEA.
  • the FLAG-tag has the amino acid sequence shown as SEQ ID NO. 26, and the amino acid sequence shown in SEQ ID NO. 26 is as follows:
  • FLAG-tag (SEQ ID NO. 26): DYKDDDDK.
  • the 3 ⁇ FLAG-tag has the amino acid sequence shown as SEQ ID NO. 27, and the amino acid sequence shown in SEQ ID NO. 27 is as follows:
  • 3 ⁇ FLAG-tag (SEQ ID NO. 27): DYKDHDGDYKDHDIDYKDDDDK.
  • the Strep-tag has an amino acid sequence as shown in SEQ ID NO. 28, and the amino acid sequence shown in SEQ ID NO. 28 is as follows:
  • Strep-tag (SEQ ID NO. 28): WSHPQFEK.
  • the 6 ⁇ His-tag has the amino acid sequence shown as SEQ ID NO. 29, and the amino acid sequence shown in SEQ ID NO. 29 is as follows:
  • the V5-tag has the amino acid sequence shown as SEQ ID NO. 30, and the amino acid sequence shown in SEQ ID NO. 30 is as follows:
  • V5-tag (SEQ ID NO. 30): GKPIPNPLLGLDST.
  • the S-tag has the amino acid sequence set forth in SEQ ID NO. 31, and the amino acid sequence shown in SEQ ID NO. 31 is as follows:
  • S-tag SEQ ID NO. 31: KETAAAKFERQHMDS.
  • the HA-tag has the amino acid sequence set forth in SEQ ID NO. 32, and the amino acid sequence set forth in SEQ ID NO. 32 is as follows:
  • HA-tag (SEQ ID NO. 32): YPYDVPDYA.
  • the VSV-G-tag has an amino acid sequence as shown in SEQ ID NO. 33, and the amino acid sequence shown in SEQ ID NO. 33 is as follows:
  • VSV-G-tag (SEQ ID NO. 33): YTDIEMNRLGK.
  • the GST-tag has the amino acid sequence set forth in SEQ ID NO. 34, and the amino acid sequence set forth in SEQ ID NO. 34 is as follows:
  • the HaloTag has the amino acid sequence set forth in SEQ ID NO. 35, and the amino acid sequence set forth in SEQ ID NO. 35 is as follows:
  • HaloTag SEQ ID NO. 35:
  • the XTEN-tag has the amino acid sequence set forth in SEQ ID NO. 36, and the amino acid sequence shown in SEQ ID NO. 36 is as follows:
  • the huEGFRt-tag having a truncated to retain only complete cetuximab (cetuximab (Erbitux TM)) and does not contain binding sites for N- terminal extracellular ligand-binding domain and an intracellular receptor complex
  • cetuximab cetuximab (Erbitux TM)
  • the human epidermal growth factor receptor polypeptide tEGFR of the kinase kinase domain has the amino acid sequence set forth in SEQ ID NO. 37, and the amino acid sequence set forth in SEQ ID NO. 37 is as follows:
  • huEGFRt-tag (SEQ ID NO. 37):
  • the compound capable of labeling the Plasmodium protein VAR2CSA means that the Plasmodium protein VAR2CSA can be recognized by the compound, and those skilled in the art can select according to the Plasmodium protein VAR2CSA, and the compound of the present invention may be a cyclooctyne group-containing group.
  • the chimeric antigen receptor further comprises any one or a combination of at least two of a hinge region, a transmembrane region and an intracellular signal region.
  • the hinge region is a conventional hinge region, and those skilled in the art can select according to needs, and are not particularly limited herein.
  • the present invention adopts a human CD8 ⁇ hinge region.
  • the transmembrane region is a conventional transmembrane region, and a person skilled in the art can select according to needs, and is not particularly limited herein.
  • the present invention employs a human CD28 transmembrane region.
  • the intracellular signal region is a conventional intracellular signal region, and can be selected by a person skilled in the art as needed, and is not particularly limited herein.
  • the present invention adopts a human CD27 intracellular signal region, a human CD134 intracellular signal region, Human CD28 intracellular signal region or human 4-1BB (CD137) Any one or combination of at least two of the intracellular signal regions.
  • the amino terminal of the chimeric antigen receptor contains a CD8 ⁇ signal peptide; the carboxy terminus of the chimeric antigen receptor further comprises a human CD3 intracellular signal region.
  • the invention provides a nucleic acid encoding a chimeric antigen receptor as described in the first aspect or a nucleic acid having at least 60% identity, preferably at least 80% identity thereto.
  • the nucleic acid has an amino acid capable of expressing a binding to a VAR2 CSA protein.
  • the invention provides a chimeric antigen receptor-expressing cell, the expression cell comprising the nucleic acid of the second aspect.
  • the cell is an immune effector cell, further preferably any one or a combination of at least two of T cells, B cells, NK cells, NKT cells, dendritic cells or macrophages.
  • the present invention provides a recombinant vector comprising the nucleic acid of the second aspect.
  • the vector is any one or a combination of at least two of a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant lentiviral vector, preferably a recombinant lentiviral vector.
  • the recombinant cloning vector is selected from, but not limited to, any one of pUC18, pUC19, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 or a combination of at least two.
  • the eukaryotic expression plasmid is selected from the group consisting of, but not limited to, a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pCI-neo series vector, a pEGFP series vector, a pSPT series vector, a pFLAG-CMV series vector, Any one of pRL series vectors, pUC57 vectors, pMAX or pDC315 or a combination of at least two.
  • the recombinant lentiviral vector is selected from, but not limited to, any one or at least two of a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retroviral vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector. The combination.
  • the recombinant vector by recombining the nucleic acid construct of the chimeric antigen receptor with the vector, the recombinant vector can be transfected into an immune cell to obtain an immunity expressing the chimeric antigen receptor. Cells that function as chimeric antigen receptors.
  • the present invention provides a recombinant virus comprising the recombinant virus obtained by co-transfecting a mammalian cell with the recombinant vector and the packaging helper plasmid according to the fourth aspect;
  • the present invention provides a chimeric antigen receptor T cell (CAR-T cell) which is expressed by transfecting a recombinant virus according to the fifth aspect into a T cell.
  • CAR-T cell chimeric antigen receptor T cell
  • the invention provides a chimeric antigen receptor according to the first aspect, a nucleic acid according to the second aspect, a recombinant vector according to the fourth aspect or a recombinant virus according to the fifth aspect, Transfect and amplify CAR-T cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the chimeric antigen of the third aspect A receptor expressing cell or an expression vector as described in the fourth aspect, and optionally a pharmaceutically acceptable excipient.
  • the ninth aspect provides the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the chimeric antigen receptor expression cell of the third aspect, or the method of the fourth aspect Use of an expression vector for the preparation of a medicament for the treatment and/or prevention of an autoimmune disease or tumor.
  • the tumor is a solid tumor and/or a hematoma
  • the tumor may be any one or at least two domains capable of binding to placenta-like chondroitin sulfate (pl-CSA) in VAR2CSA protein or VAR2CSA protein.
  • the recombinant protein (rVAR2) specifically recognizes and binds to any tumor tissue and cells, and may be, for example, a human lung cancer cell line, including NCI-H460 (large cell lung cancer cell line, ATCC #HTB177), NCI-H520 (squamous cell lung cancer).
  • the cell line, ATCC#HTB182) and A549 are all able to specifically bind to rVAR2 protein to varying degrees; rVAR2 protein can also target human placental choriocarcinoma cell line BeWo (ATCC#) CCL98);
  • rVAR2 protein can also target human placental choriocarcinoma cell line BeWo (ATCC#) CCL98);
  • the B cell lymphoma cell line Raji (ATCC#CCL86) and the acute myeloid leukemia cell line KG-1a (ATCC#CCL246.1) can also be specifically bound by rVAR2 protein; however, rVAR2 protein and healthy people
  • the PBMC was negatively bound to normal human umbilical vein endothelial cells (HUVEC, ATCC #PCS-100-010).
  • the present invention provides a method of treating a subject having an autoimmune disease and/or a disease associated with expression of a tumor antigen, comprising administering to the subject an effective amount comprising the ninth aspect The drug of the pharmaceutical composition.
  • the tumor is a solid tumor and/or a hematoma
  • the tumor may be any one or at least two domains capable of binding to placenta-like chondroitin sulfate (pl-CSA) in VAR2CSA protein or VAR2CSA protein.
  • pl-CSA placenta-like chondroitin sulfate
  • the recombinant protein specifically recognizes and binds to any tumor tissue and cells, and may be, for example, a human lung cancer cell line, including NCI-H460 (large cell lung cancer cell line, ATCC#HTB177), NCI-H520 (squamous cell lung cancer cell line, ATCC#HTB182) and A549 (lung adenocarcinoma cell line, ATCC#CCL185) can all be specifically bound by rVAR2 protein to varying degrees; rVAR2 protein can also target Binding to human placental choriocarcinoma cell line BeWo (ATCC#CCL98); in addition, B cell lymphoma cell line Raji (ATCC#CCL86) and acute myeloid leukemia cell line KG-1a (ATCC#CCL246.1) can also be The rVAR2 protein specifically binds; however, the rVAR2 protein is negatively bound to healthy human PBMC and normal human umbilical vein endothelial cells (
  • variant refers to any variant comprising a substitution, deletion or addition of one or several to more amino acids, provided that the variant substantially retains the same function as the original sequence.
  • the present invention has the following beneficial effects:
  • the chimeric antigen receptor of the present invention can recognize a recombinant protein (rVAR2) of any one or at least two domains capable of binding to placental-like chondroitin sulfate (pl-CSA) in a VAR2CSA protein or a VAR2CSA protein,
  • rVAR2 recombinant protein
  • the VAR2CSA or rVAR2 protein has the ability to target a variety of different types of tumor cells by binding to placental-like chondroitin sulfate pl-CSA on the surface of tumor cells, and almost 95% (106/111) of human cancer cells derived from cancer patients The system can be targeted;
  • the present invention allows a chimeric antigen receptor to specifically bind to a recombinant protein (rVAR2) of any one or at least two domains capable of binding to placental-like chondroitin sulfate (pl-CSA) in a VAR2CSA protein or a VAR2CSA protein.
  • rVAR2 recombinant protein
  • pl-CSA placental-like chondroitin sulfate
  • Indirect recognition and killing of tumor cells expressing surface antigens containing placenta-like chondroitin sulfate pl-CSA can further regulate the action of chimeric antigen receptors by regulating the content of VAR2CSA protein.
  • VAR2CSA protein is absent, chimerism
  • the antigen receptor does not work and does not cause toxic side effects on human healthy cells;
  • the extracellular recognition domain portion of the chimeric antigen receptor of the present invention that is, the single-chain antibody (ScFv) portion can be competitively bound to the VAR2CSA protein or rVAR2 thereof by which the monoclonal antibody to which it is homologously binds.
  • the binding domain on the protein can regulate the function of the chimeric antigen receptor cell by indirectly limiting the content of the monoclonal antibody of the VAR2CSA protein, and is beneficial for reducing immune cells such as T cells expressing the chimeric antigen receptor. Toxic side effects;
  • the present invention allows a chimeric antigen receptor to specifically bind to a recombinant protein (rVAR2) of any one or at least two domains capable of binding to placental-like chondroitin sulfate (pl-CSA) in a VAR2CSA protein or a VAR2CSA protein.
  • rVAR2 recombinant protein
  • pl-CSA placental-like chondroitin sulfate
  • the chimeric antigen receptor of the present invention can recognize a recombinant protein (rVAR2) of any one or at least two domains capable of binding to placental-like chondroitin sulfate (pl-CSA) in a VAR2CSA protein or a VAR2CSA protein,
  • the VAR2CSA or rVAR2 protein specifically targets the combined placenta-like chondroitin sulfate pl-CSA, which is widely and abundantly present in the cell surface and extracellular matrix of tumor tissues, such that the chimeric antigen receptor is expressed
  • the synergistic action of the immune cells with the VAR2CSA protein has the potential to target the tumor microenvironment and may help the immune cells expressing the chimeric antigen receptor to break through the immunosuppressive microenvironment of the solid tumor and ultimately achieve targeting.
  • the chimeric antigen receptor of the present invention can be used as a drug for treating cancer, and its action can be regulated, opening up a new idea for cancer treatment.
  • Figure 1 (A) shows the results of 10% SDS-PAGE protein gel electrophoresis analysis of prokaryotic expression purified recombinant plasmid containing rrep2 with Strep-tag protein tag
  • Figure 1 (B) shows prokaryotic expression purified with Strep-tag protein.
  • Figure 2 shows the function of murine anti-rVAR2 protein antibody by enzyme-linked immunosorbent assay (ELISA).
  • NC was diluted 1:500 in serum of unimmunized mice and used as a negative control for ELISA.
  • Other antigen dilutions (rVAR2 recombinant protein) in the control group and the experimental group were 1 ⁇ g/ml;
  • Figure 3 is a flow cytometric specificity of tumor cell targeting of rVAR2 protein
  • Figure 4 shows the binding of monoclonal antibody 5H4 to rVAR2 protein by Western blot.
  • A is prokaryotic expression of purified rVAR2 protein
  • B is eukaryotic expression and purified rVAR2 protein.
  • Figure 5 is a 12% SDS-PAGE protein gel electrophoresis assay for prokaryotic expression of purified ID1, DBL2X and ID2a polypeptides with Strep-tag protein tag, M, protein molecular weight standard; NC, negative control, empty vector transfection E. coli lysate; ID2a, purified ID2a protein with Strep-tag tag, DBL2X, purified DBL2X protein with Strep-tag tag, ID1, purified ID1 protein with Strep-tag tag;
  • Figure 6 shows the epitope mapping of anti-rVAR2 monoclonal antibody 5H4, in which Mock is a blank control.
  • the detection system is identical to the experimental group except that no monoclonal antibody 5H4 is added;
  • HCS is hybridoma cell supernatant, mouse B cell The supernatant of hybridoma expression;
  • Figure 7 (A) is a T cell expressing the chimeric antigen receptor of the present invention, a VAR2 CSA full-length protein (or its pl-CSA binding domain recombinant protein rVAR2), and a tumor cell expressing a pl-CSA epitope.
  • Figure 7 (B) shows several different types of T cells constructed using the chimeric antigen receptor of the present invention and their VAR2CSA full-length protein (or pl-CSA binding domain recombinant protein rVAR2) Schematic diagram of the binding mode between the tumor cells and the tumor cells expressing the pl-CSA epitope;
  • Figure 8 is a plasmid map of lentiviral expression vector of pLentiCART-anti-rVAR2 based on 5H4 single-chain antibody;
  • Figure 9 (A) shows the stability of rVAR2 recombinant protein in human serum by 10% SDS-PAGE protein gel electrophoresis
  • Figure 9 (B) shows the stability of rVAR2 recombinant protein in human serum by Western blot;
  • FIG 10 shows the flow cytometry of CART-anti-rVAR2 (5H4 ScFv) cells and the expression of the extracellular antigen recognition region (5H4 ScFv).
  • the Mock is a single T cell group without Alexa Fluor. 647-labeled goat anti-mouse F(ab') 2 IgG antibody treatment; Negative Control as a negative control group, T cells plus Alexa Fluor 647-labeled goat anti-mouse F(ab') 2 IgG antibody treatment;
  • CART-anti-rVAR2 ( 5H4 ScFv) is a CAR-T cell expression group obtained by transfecting T cells with a lentivirus expressing CART-anti-rVAR2 (5H4 ScFv);
  • Figure 11 (A) shows the secretion level of IL-2 during co-incubation of sCART-anti-rVAR2 (5H4 ScFv) with Raji cells;
  • Figure 11 (B) shows that sCART-anti-rVAR2 (5H4 ScFv) is shared with Raji cells. Detection of the secretion level of IFN- ⁇ during incubation;
  • Figure 12 shows the in vitro killing ability of sCART-anti-rVAR2 (5H4 ScFv) on tumor cell Raji by flow cytometry.
  • the signal in Far Red + gate indicates the ratio of Raji cells, while the signal in GFP + gate indicates CART-anti.
  • Figure 13 shows the in vitro real-time dynamic monitoring of the killing effect of sCART-anti-rVAR2 (5H4 ScFv) on Raji cells, wherein the white arrow-labeled cells are Far Red-labeled tumor cells Raji, while the unlabeled cells are co-expressed GFP reporter genes.
  • CART-anti-rVAR2 (5H4 ScFv) cells are used to determine the killing effect of sCART-anti-rVAR2 (5H4 ScFv) on Raji cells, wherein the white arrow-labeled cells are Far Red-labeled tumor cells Raji, while the unlabeled cells are co-expressed GFP reporter genes.
  • CART-anti-rVAR2 (5H4 ScFv) cells are co-expressed GFP reporter genes.
  • Example 1 Expression and purification of related recombinant proteins of pl2CSA protein pl-CSA binding domain
  • the DNA molecule encoding the amino acid sequence of the pl-CSA binding domain of the VAR2CSA protein was cloned into a prokaryotic or eukaryotic expression vector, and the constructed prokaryotic expression vector was separately transferred into E. coli (for example, in BL21 (DE3), the constructed eukaryotic expression vector is transferred into eukaryotic cells (for example, human embryonic kidney cell line HEK293T containing large T antigen) for protein expression, and protein expression cells or medium supernatant are collected. Protein purification was performed using the corresponding protein tag affinity purification medium. The steps are as follows:
  • Escherichia coli expressing the Strep-tag-tagged rVAR2 recombinant protein was first collected by centrifugation, and E. coli was resuspended per gram of cells by adding 10 ml of ice-cold Buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA).
  • the cells were lysed by an AH-1500 ultrahigh pressure homogenizer (ATS Engineering Inc.) under a pressure of 100 MPa at a low temperature of 2-3 times to obtain a cell lysate.
  • the cell lysate supernatant was collected by centrifugation at 14,000 rpm, 10 min, 4 °C.
  • the resin was washed and equilibrated with 2 times Strep-Tactin resin volume of Buffer W (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA).
  • Buffer W 100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA.
  • the collected cell lysate supernatant was incubated with the equilibrated Strep-Tactin resin for 30 min-1 h, and after the penetrating solution was completely discharged, the ice pre-cooled Buffer W of at least 5 times Strep-Tactin resin volume was added in multiple times.
  • the resin was washed (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) and 3 times Strep-Tactin resin volume of ice-cold Buffer E (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin) was added.
  • the rrep2 recombinant protein with Strep-tag tag was eluted from Strep-Tactin resin in 3 portions, and a certain amount of recombinant plasmid containing rVAR2 with Strep-tag protein tag was purified for SDS-PAGE protein gel.
  • the predicted average molecular weight of the rVAR2 recombinant protein with the Strep-tag protein tag was about 74 kDa.
  • the amino acid sequence of the rVAR2 recombinant protein carrying the Strep-tag protein tag is as follows (SEQ ID NO. 38):
  • the black bold amino acid sequence is the amino acid sequence introduced from the vector backbone DNA introduced during gene cloning, and the lower row
  • the amino acid sequence of the line marker is the Strep-tag sequence (SEQ ID NO. 28), and the rVAR2 recombinant protein with the Strep-tag protein tag is purified by its affinity with the Strep-Tactin resin.
  • An eukaryotic (mammalian cell) protein expression vector carrying a secreted signal peptide or a non-secretory signal peptide is used to construct an expression vector for the rVAR2 recombinant protein with a 3 ⁇ FLAG-tag protein tag, and the expression vector is subjected to electroporation or PEI.
  • a 5% pH adjustment buffer (1 M Tris-HCl, 3 M NaCl, pH 7.4) was added to the cell culture supernatant for use.
  • Cell lysate Buffer L [50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, 1% TRITON X-100, and 1% protease inhibition was added to the collected cells at a ratio of 10 6 -10 7 cells/ml.
  • the agent (Sigma, Cat. #P8340) was incubated at room temperature for 30 min. 12,000 x g, and centrifuged at 4 ° C for 10 min to collect the supernatant of the cell lysate, and set aside.
  • the prepared rVAR2 recombinant protein expression supernatant containing the 3 ⁇ FLAG-tag protein tag or the magnetic affinity of the cell lysate supernatant coupled with the well-balanced ANTI-FLAG M2 antibody or the ANTI-FLAG M2 antibody Beads were incubated for 2 h on ice, and the resin or magnetic beads bound with rVAR2 recombinant protein with 3 ⁇ FLAG-tag protein tag were collected by magnetic column or magnetic frame, using 20 times resin or magnetic bead volume of TBS buffer ( 50 mM Tris HCl, 150 mM NaCl, pH 7.4) Wash the collected resin or magnetic beads in three portions, using 5x resin or magnetic bead volume of 3 ⁇ FLAG polypeptide eluate (prepared with TBS buffer, 3 ⁇ FLAG polypeptide concentration is 150 ng/ ⁇ l) eluted and collected rVAR2 recombinant protein with 3 ⁇ FLAG-tag protein tag, and then a certain amount of
  • the predicted average molecular weight of the rVAR2 recombinant protein containing the 3 ⁇ FLAG-tag protein tag is about 76 kDa
  • the molecular weight of 10% SDS-PAGE gel electrophoresis is about 115 kDa, indicating There may be post-translational modifications of the protein.
  • amino acid sequence of the rVAR2 recombinant protein carrying the 3xFLAG-tag tag is as follows (SEQ ID NO. 39):
  • the black bold amino acid sequence is the amino acid sequence introduced from the vector backbone DNA introduced during gene cloning, and the underlined 3X FLAG-tag amino acid sequence (SEQ ID NO. 27), through which it is associated with ANTI-
  • the affinity of the FLAG M2 antibody-conjugated resin or magnetic beads was used to purify the rVAR2 recombinant protein with the 3X FLAG-tag protein tag.
  • the prokaryotic expression purified rVAR2 recombinant protein was immunized with 3 4-6 week old BALB/c mice respectively.
  • the specific steps were as follows: First, the purified rVAR2 recombinant protein was buffered with phosphate buffer (PBS, pH 7.4). The solution was dialyzed 3 times, and the concentration of protein was ultrafiltered to 1 ⁇ g/ ⁇ l; 100 ⁇ l of rVAR2 recombinant protein solution (100 ⁇ g) was emulsified with 100 ⁇ l of Freund's complete adjuvant, and then the mice were injected with multiple injections on the back for primary immunization, and then 100 ⁇ l were used respectively.
  • PBS phosphate buffer
  • mice After rMF2 recombinant protein solution (100 ⁇ g) was emulsified with 100 ⁇ l of Freund's incomplete adjuvant, the mice were subjected to multi-point injection of the back for 2-3 times of boosting, and the serum of the immunized mice was detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the function and potency of the polyclonal antibody are shown in Figure 2.
  • the OD450 value is about 1.0
  • the detected mouse antiserum (polyclonal antibody) titer is about 1:25000.
  • mice Prepare appropriate amount of feeder cells from mouse peritoneal cavity and mouse myeloma cells SP2/0 as needed; take the immunized BALB/c mice, remove the eyeballs with anesthesia with isoflurane, and separate the serum for antibody detection. Positive control serum at the time.
  • the BALB/c mice that had been immunized were sacrificed by cervical dislocation, and the spleen was surgically removed and placed in a dish containing 10 ml of incomplete medium, gently washed, and the surrounding connective tissue was carefully peeled off.
  • the isolated mouse spleens were transferred to another stainless steel mesh plate containing 10 mL of incomplete medium plates, and ground into a cell suspension with a syringe needle to allow the spleen cells to enter the incomplete medium in the plate. Sip several times with a pipette to make a single cell suspension and count. Usually 1 x 10 8 - 2.5 x 10 8 splenocytes per mouse.
  • Hybridomas were prepared by fusing 1 ⁇ 10 8 splenocytes with 1 ⁇ 10 7 myeloma cells SP2/0 by PEG treatment, and plated into 96-well plates containing feeder cells using HAT medium [containing hypoxantin (hypoxantin) , cell culture medium of aminopterin and thymidin] culture and screening of hybridoma cells, microscopic observation of hybridoma cell growth, until it reaches the bottom area of the pore When the cells are above 10, the cell culture supernatant is taken for antibody detection, and the hybridoma cells of the positive antibody detection well are monoclonalized by the limiting dilution method, and the cell culture supernatant is taken up again after the cell bottom area is more than 1/10.
  • HAT medium containing hypoxantin (hypoxantin) , cell culture medium of aminopterin and thymidin
  • a monoclonal antibody hybridoma cell strain with high affinity for the recombinant antibody and rVAR2 recombinant protein was selected, and a part of the cells were frozen for storage; the remaining cells were further cultured to collect the supernatant containing the rVAR2 recombinant protein monoclonal antibody.
  • a portion of the RNA was extracted and the variable region coding sequences of the heavy and light chains of the murine IgG antibody were analyzed by RT-PCR using specific primers. From this, a cell line secreting monoclonal antibody 5H4 was selected, and the variable region coding sequences of the heavy and light chains are shown in Table 1.
  • the variable region amino acid sequences of the heavy and light chains of 5H4 are shown in Table 2.
  • the sequence of the complementary determining region (CDR) of the heavy and light chains of 5H4 is shown in Table 3.
  • V H monoclonal antibody heavy chain variable region
  • V L light chain variable region
  • V H monoclonal antibody heavy chain variable region
  • V L light chain variable region
  • Example 3 Flow cytometry detection of tumor cell targeting by rVAR2 protein
  • mice 5 ⁇ 10 5 different types of cells isolated and cultured in vitro (including normal cell control group and tumor cell group), purified prokaryotic or eukaryotic rVAR2 protein, and anti-rVAR2 obtained by immunizing mice
  • the mouse polyclonal antibody of the protein and the purchased FITC dye-labeled goat anti-mouse secondary antibody (IgG H&L, Abcam, cat#ab6785) were separately incubated for 45 min, pre-cooled at 4 °C before incubation of the next reagent or after incubation of the reagent.
  • the PBS solution containing 0.02% NaN3 and 2% FBS was washed 3 times with 2 min intervals.
  • flow cytometry was used to detect the targeting of rVAR2 protein to many different types of tumor cells.
  • the experimental grouping is shown in Table 4 below:
  • human lung cancer cell lines include NCI-H460 (large cell lung cancer cell line, ATCC#HTB177), NCI-H520 (squamous cell lung cancer cell line, ATCC#HTB182), and A549 (lung adenocarcinoma cell line, ATCC#CCL185).
  • rVAR2 protein can also target human placental choriocarcinoma cell line BeWo (ATCC#CCL98); in addition, B cell lymphoma cell line Raji (ATCC#CCL86) and acute bone marrow
  • the leukemia cell line KG-1a ATCC#CCL246.1
  • rVAR2 protein is associated with healthy human PBMC and normal human umbilical vein endothelial cells (HUVEC, ATCC#PCS-100-010) ) The combination is negative.
  • the structural and functional properties of the core domain rVAR2 which binds VAR2CSA to pl-CSA (placenta-like chondroitin sulfate), can be subdivided into three major domain components, including ID1, DBL2X and ID2a (Clausen et al. , 2012).
  • the amino acid sequence of the rVAR2 protein and its domain components is as follows:
  • amino acid sequence of the rVAR2 protein is as follows (SEQ ID NO. 39):
  • amino acid sequence of the ID1 polypeptide is as follows (SEQ ID NO. 2):
  • the amino acid sequence of the DBL2X polypeptide is as follows (SEQ ID NO. 3):
  • the amino acid sequence of the ID2a polypeptide is as follows (SEQ ID NO. 4):
  • the DNA coding sequences of ID1, DBL2X and ID2a polypeptides were cloned into E. coli expression vector, and the proteins of the three were obtained after protein expression purification. The results of SDS-PAGE protein gel electrophoresis are shown in Figure 5, and the purified bands are shown.
  • the Strep-tag-tagged ID2a protein has a predicted average molecular weight of approximately 15 kDa; the purified Strep-tag-tagged DBL2X protein has a predicted average molecular weight of 40 kDa; a purified Strep-tag-tagged ID1 protein, The predicted average molecular weight was 17 kDa.
  • anti-rVAR2 monoclonal antibody 5H4 was preliminarily confirmed by antigen-specific ELISA: the purified ID1, DBL2X and ID2a antigen peptides were diluted with Na 2 CO 3 -NaHCO 3 buffer (pH 9.6) to the final concentration.
  • the ELISA results are shown in Figure 6. Only the detection results of the coated wells of the ID2a antigen peptide are consistent with the detection results of the rVAR2 protein coated wells. The epitope of the anti-rVAR2 monoclonal antibody 5H4 was shown to be above the epitope of the ID2a polypeptide.
  • VAR2CSA-based broad-spectrum CAR-T cell In order to further enhance the killing activity of this VAR2CSA-based broad-spectrum CAR-T cell and enhance its application safety.
  • Construction of a CAR-T CART-anti-VAR2CSA or CART-anti
  • VAR2CSA or its pl-CSA binding domain recombinant protein rVAR2
  • rVAR2CSA ScFv or anti-rVAR2 ScFv single-chain antibody
  • Cell-regulated CAR-T cells the tumor cell targeting using full-length VAR2CSA protein or rVAR2 protein to bind and label tumor cells, and then use the full-length VAR2CSA protein or rVAR2 protein we developed.
  • the regulatable CAR-T cell system consisting of the recombinant protein VAR2CSA full-length protein or its pl-CSA binding domain and its single-chain antibody-constituted CAR-T cells was named sCART-anti- VAR2CSA (system containing full-length protein of VAR2CSA) or sCART-anti-rVAR2 cells (system containing pl-CSA binding domain recombinant protein of VAR2CSA protein).
  • the single-chain antibody ScFv sequence responsible for recognizing the ID2a epitope on rVAR2 includes the VH chain of the anti-rVAR2 monoclonal antibody 5H4 and the V of 5H4.
  • the L chain and the linker sequence L (Linker) therebetween, the linker sequence includes, but is not limited to, an amino acid sequence such as GGGGSGGGGSGGGGS.
  • Example 7 Stability of rVAR2 protein in human serum
  • rVAR2 protein In order to confirm whether the rVAR2 protein can serve as a medium for CART-anti-rVAR2 cells to target tumor cells, it is first necessary to determine whether the rVAR2 protein is stably present in human blood.
  • ⁇ VAR protein low-adsorption filter was used to filter the sterilized rVAR2 recombinant protein to a final concentration of 0.2 ⁇ g/ ⁇ l of rVAR2 recombinant protein, which was plated and placed in a 37 ° C, 5% CO 2 incubator.
  • Samples were taken according to the number of days of the samples placed in the incubator (Day).
  • Day 0 indicates that on day 0, 30 ⁇ l of the just-mixed sample was taken, and Day 1 indicated that 30 ⁇ l was taken in a 37 ° C, 5% CO 2 incubator for one day ( Samples of 24h), and so on, take samples of Day 2, Day 3, Day 4, Day 5, Day 7, Day 9, Day 11, Day 14, Day 17, and Day 22, respectively.
  • Samples of 24h Samples of 24h
  • Samples of 24h take samples of Day 2, Day 3, Day 4, Day 5, Day 7, Day 9, Day 11, Day 14, Day 17, and Day 22, respectively.
  • 570 ⁇ l SDS-PAGE protein loading buffer (2 ⁇ ) boiled for 3 min, cooled and stored at -20 ° C until use.
  • NC negative control group
  • PC purified rVAR2 recombinant protein
  • the rVAR2 recombinant protein with Strep-tag protein tag has an average molecular weight of about 74 kDa, human serum albumin, and an average molecular weight of about 66.5 kDa, indicating 37 ° C, 5%. Under the conditions of CO 2 incubator treatment, rVAR2 recombinant protein can be stably stored in the serum of lung cancer patients for more than 3 weeks.
  • CART-anti-rVAR2 cells were constructed.
  • the vector was ligated with the CD8 signal peptide, 5H4 ScFv and CD8a hinge region downstream of the EF1 ⁇ promoter.
  • CD8Hinge CD28 transmembrane sequence
  • TM CD28 transmembrane sequence
  • CD28 costimulatory factor CD137 (4-1BB) costimulatory factor
  • CD3 ⁇ domain sequence as shown in Figure 8.
  • CART-anti-rVAR2 (5H4 ScFv) expression plasmid was compared with the third generation lentiviral packaging plasmid pMDLg-pRRE:pRSV-Rev:pMD2.G by mass ratio of 3:1:1:1 using PEI transfection method.
  • HEK293T cells were co-transfected after mixing, and the cell culture supernatant was collected 72 h after transfection.
  • the collected culture supernatant was filtered through a 0.45 ⁇ m membrane and concentrated by ultrafiltration to a lentivirus titer of 1.6 ⁇ 10 8 TU/ml- 1 ⁇ 10 10 TU/ml, kept in a refrigerator at -80 °C for use.
  • produce CART-anti-rVAR2 (5H4 ScFv) cells as follows:
  • centrifuge at 800g for 20min acceleration set to 6, deceleration set to 1
  • gently suck out the grayish mononuclear cells with a Pasteur pipette add another 10ml containing RF-10 (containing 10% off)
  • RF-10 containing 10% off
  • a centrifuge tube of live FBS RPMI 1640 medium mix; centrifuge at 500g for 5min, discard the supernatant; add 10mL RF-10 to resuspend the cells, trypan blue staining and counting, centrifugation at 350g for 10min, discard the supernatant.
  • CD4 Positive Isolation Kit Invitrogen, Cat. #11331D
  • CD8 Positive Isolation Kit Invitrogen, Cat. #11333D
  • the basic operation steps are as follows: vortex the magnetic beads, take 25 ⁇ l magnetic beads (Dynabeads) in a test tube, add 1 ml Buffer 1 and mix, place the test tube on a magnetic stand for 1 min, and collect the supernatant.
  • Buffer 1 Add 25 ⁇ l Buffer 1 and resuspend the magnetic beads for use; resuspend PBMC cells with Buffer 1 to a density of 1 ⁇ 10 7 cells/ml; add 25 ⁇ l of washed magnetic beads to 1 ml of PBMC cells, and incubate at 2-8 ° C for 20 min.
  • CD4 + T and CD8 + T cells after magnetic bead sorting were centrifuged for 10 min at 350 g; RF-10 (RPMI 1640 medium containing 10% inactivated FBS) was resuspended; CD4 + T was added in a ratio of 1:1.
  • RF-10 RPMI 1640 medium containing 10% inactivated FBS
  • CD4 + T was added in a ratio of 1:1.
  • Cultured in CD8 + T cell culture plate the cell density is 5 ⁇ 10 5 /ml; add CD3/CD28 antibody magnetic beads in T cell-specific medium, and add the amount of magnetic beads to the ratio of cells to 1:1; rhIL-2 was made to a final concentration of 10 ng/ml; 2-3 times per week, and cell proliferation was recorded.
  • the CD4 + T and CD8 + T cells after magnetic bead sorting were centrifuged at 350g for 10min, and the whole medium was resuspended; the CD4 + T and CD8 + T cells were added in a ratio of 1:1 in 96-well plates.
  • the density is 5 ⁇ 10 5 /ml, and the number of cells per well is 1 ⁇ 10 5 ; the magnetic beads are washed, and the magnetic beads with the ratio of 1:1 to the cells are added to the culture dish; rhI-2 is added to make the final concentration 10 ⁇ g.
  • the infection efficiency of flow detection is about 45%, and CAR-T cells can be produced more efficiently.
  • Example 9 Detection of cytokine secretion during co-incubation of sCART-anti-rVAR2, a regulatable CAR-T, with tumor cells
  • sCART-anti-rVAR2 a regulatable CAR-T cell, has potential killing function on tumor cells.
  • cytokine detection kit R&D Systems, Human IFN- ⁇ ELISA Kit (Cat.#) Dif50) and Human IL-2 ELISA Kit (Cat.#d2050)
  • the cytokine secretion levels during sCART-anti-rVAR2 system co-incubation with tumor cells were tested as follows:
  • CAR-T cells CART-anti-rVAR2 (5H4 ScFv)
  • target cells cancer cells
  • Effector cells [CART-anti-rVAR2 (5H4 ScFv), CARTV2] and target cells (Raji, R) were evenly spread in a 48-well plate at a ratio of 1:1, and each cell was set to 1.25 ⁇ 10 5 . /well / 250 ⁇ l, the total volume is 500 ⁇ l; in which the effector cells (or target cells) and rVAR2 recombinant protein (rVAR2, V) are co-incubated in advance, the rVAR2 protein and effector cells (or target cells) are first co-incubated at 37 ° C After the incubation for 1 h, the subsequent operations were continued.
  • Control group Normal T cells (T) plus target cells, ie T+R group, each cell was set to 1.25 ⁇ 10 5 /well/250 ⁇ l, and the insufficient fraction was 250 ⁇ l RPMI-1640 medium. Filled up, the total volume was kept at 500 ⁇ l.
  • the rVAR2 recombinant protein was co-incubated with the target cells, and then added to the normal T cell group, namely [V+R]+T group;
  • the cells in each group were incubated for 24 h in a 37 ° C incubator, and the supernatant was collected for testing.
  • Example 10 Far Red method to verify the killing function of sCART-anti-rVAR2, a regulatable CAR-T cell on tumor cells
  • Kit labeled target cells (1) tumor cells take appropriate, 3min centrifugation after 300 ⁇ g to remove the supernatant; (2 Resuspend the cells in PBS, centrifuge at 300 ⁇ g for 3 min, remove the supernatant, repeat this step; (3) resuspend in PBS to a cell density of 1 ⁇ 106 cells/ml; (4) per 1 ml of system cells Add 1 ⁇ l of Far-Red at a concentration of 200 ⁇ M and incubate for 20 min in a 37 ° C water bath; (6) After the incubation, add 5 volumes of RF-10 medium (RPMI 1640 medium containing 10% inactivated FBS) to stop the reaction (5 min) (7) 300 g was centrifuged for 3 min, and the supernatant was removed; (8) Resuspended to 5 ⁇ 105 / ml with T
  • the CAR-T cells were centrifuged at 300 ⁇ g for 3 min, and the supernatant was removed. After resuspending in PBS, centrifuged at 300 ⁇ g for 3 min, the supernatant was removed, and the medium was repeated; T cell expansion medium (Gibco, Cat.#A10485) Resuspended to 5 ⁇ 105 / ml;
  • CAR-T cells CART-anti-rVAR2 (5H4 ScFv)] to target cells (cancer cell Raji) mediated by rVAR2 recombinant protein
  • the experiments were divided into the following groups, such as As shown in Table 5, in each group, the rVAR2 recombinant protein was co-incubated with which cells, and the molar concentration of the protein was 500 times that of the corresponding co-incubated cells; and the effector cells and target cells were effective. The ratio is 2:1.
  • the cells were collected by centrifugation at 300 g for 3 min; the centrifuged cells were washed twice with PBS buffer containing 2% inactivated FBS and 0.02% NaN 3 , and then used. The cells were resuspended in PBS buffer; the signal of Far-Red (excitation wavelength 630 nm, emission wavelength 661 nm) was detected by an upflow cytometer.
  • Example 11 Real-time dynamic monitoring of the killing effect of sCART-anti-rVAR2, a regulatable CAR-T cell, on tumor cells in vitro
  • the target cell Raji was stained and labeled with the same Far-Red method as in Example 10, and then resuspended in the corresponding T cell medium, and then Raji cells were plated on a 96-well culture plate at 5000/100 ⁇ l holes.
  • the prepared CART-anti-rVAR2 (5H4 ScFv) cells were added at a ratio of effect ratio of 4:1. Since the lentiviral transfection efficiency was about 45% or more, the actual target ratio was between about 2:1 to 4:1.
  • rVAR2 recombinant protein and CART-anti-rVAR2 cells (rVAR2 recombinant protein final concentration was 18 nM, CART-anti-rVAR2 cells were After incubated at 37 °C for 1 h, it was added to tumor cells and named as [CART-anti-rVAR2(5H4 ScFv)+rVAR2]+Raji group; or rVAR2 recombinant protein (rVAR2 recombinant protein) After incubation with Raji cells for 1 h at 37 °C, 20,000 cells/100 ⁇ l of CART-anti-rVAR2 cells were added and named [Raji+rVAR2]+CART-anti-rVAR2 (5H4 ScFv).
  • Raji is a B cell lymphoma cell line with high cell surface expression of CD19
  • CAR19 cells targeting CD19 CART-CD19 + cells, Porter et al., N Engl J Med. 2011, 365(8): 725-33; Grup et al., N Engl J Med. 2013; 368(16): 1509-18.
  • sCART-anti-rVAR2 5H4 ScFv

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种嵌合抗原受体及其应用，该嵌合抗原受体包含识别疟原虫蛋白VAR2CSA、疟原虫蛋白VAR2CSA上的蛋白标签或能够标记疟原虫蛋白VAR2CSA的化合物中的任意一种或至少两种的组合的结构域。该嵌合抗原受体可以识别VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)，该VAR2CSA蛋白或者rVAR2蛋白通过结合肿瘤细胞表面的pl-CSA从而具有靶向多种不同类型肿瘤细胞的能力。

Description

一种嵌合抗原受体及其应用 技术领域
本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种嵌合抗原受体及其应用，具体为以VAR2CSA蛋白为结合域的嵌合抗原受体及其在肿瘤治疗中的应用。
背景技术
癌症是仅次于心血管疾病的世界第二大致死性疾病，仅2015年全世界就有约880万人因癌症死亡(GBD 2015 Mortality and Causes of Death Collaborators，Lancet.2016，388(10053)：1459-1544.)。免疫治疗被认为是继手术、放疗和化疗之后的第四种癌症治疗方法。其中，嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor(CAR)T-Cell Immunotherapy，CAR-T)是近年来最热门也是最成功的细胞免疫疗法，其在B细胞白血病和B细胞淋巴瘤的治疗上取得了巨大的成功(Porter et al.，N Engl J Med.2011，365(8)：725-33；Grupp et al.，N Engl J Med.2013，368(16)：1509-18；Gill et al.，Blood Rev.2016，30(3)：157-67.)，其在实体瘤治疗领域的潜力也正在被挖掘(Newick et al.，Annu Rev Med.2017，68：139-152.)。
与肿瘤疫苗在临床试验中的低客观缓解率相比，细胞免疫疗法更值得期待(Rosenberg et al.，Nat Med.2004，10(9)：909-15.)。但不同于传统的过继性细胞免疫治疗(例如肿瘤浸润性T淋巴细胞(TIL)的分离和体外扩增回输疗法(Rosenberg and Restifo，Science.2015，348(6230)：62-8.)、DC-CIK疗法(Mesiano et al.，Expert Opin Biol Ther.2012，12(6)：673-84.)和工程化的T细胞抗原受体T细胞(TCR-T)疗法(Klebanoff et al.，Nat Med.2016，22(1)：26-36.)，CAR-T技术能够将CAR分子重组导入CD3阳性的T细胞，与靶细胞的结合不依赖MHC分子的介导，避开了自然状态下免疫细胞对癌细胞清除所依赖的诸多中间环节，使得CAR-T细胞能够精准识别癌细胞的抗原直接靶向杀伤癌细胞(Fesnak et al.，Nat Rev Cancer.2016，16(9)：566-81；Lim and June，Cell.2017，168(4)：724-740.)。相对于以特异性针对CTLA-4与PD-1/PD-L1的抗体为代表的免疫检查点阻断剂治疗(Pauken et al.，Science.2016，354(6316)：1160-1165.)，CAR-T细胞能够在癌症病人体内增殖并维持其抗肿瘤活性，理论上CAR-T的治疗将比免疫检查点阻断剂治疗更持久。
当前，国际上对CAR-T细胞的优化研究主要还是集中在以下几个方面(Fesnak et al.，Nat Rev Cancer.2016，16(9)：566-81.)：一、发掘特异的实体瘤表面抗原，避免CAR-T细胞的脱靶效应；二、在实体瘤中重定向T细胞靶向性，例如通用细胞因子杀伤重定向T细胞(T cells redirected for universal cytokine killing，TRUCKs)的应用；三、优化CAR-T细胞的基因编辑和细胞转染技术(Eyquem et al.，2017)；四、增强CAR-T细胞的向肿瘤组织的运输和归巢肿瘤细胞的能力；五、避免肿瘤微环境的免疫抑制和肿瘤细胞的免疫逃逸；六、提高CAR-T的临床应用安全性，例如整合“自杀基因”(Jensen et al.，Biol Blood Marrow Transplant.2010，16(9)：1245-56.；Gargett and Brown，Front.Pharmacol.2014，5(235)：1-7.)或者“分子开关”(Rodgers et al.，Proc Natl Acad Sci USA.2016，113(4)：E459-68；Wu et al.，Science.2015，350(6258)：aab4077； Morsut et al.，Cell.2016，164(4)：780-91.)以及敲除内源性TCRs预防移植物抗宿主病(Graft versus Host Disease，GvHD，Galetto et al.，Blood，2014，124(21)：1116.)等。
VAR2CSA是人恶性疟原虫(Plasmodium falciparum)表达并输送到其感染的红细胞表面的一种蛋白质(PfEMP1，PlasmoDB：PF3D7_1200600)，它可通过靶向结合胎盘样硫酸软骨素(pl-CSA)修饰的蛋白多糖来介导疟原虫感染的红细胞与胎盘合胞体的细胞外基质和细胞质膜的黏附(Salanti et al.，Mol Microbiol.2003，49(1)：179-91；Salanti et al.，J Exp Med.2004，200(9)：1197-203.)，从而导致疟原虫感染孕妇的胎盘，临床上称作胎盘疟疾，可引发流产和死胎。研究表明，肿瘤组织和胎盘组织表达相似类型的硫酸软骨素，VAR2CSA能够通过与pl-CSA的相互作用来特异性地靶向多种不同种类的肿瘤组织和细胞而非正常的组织和细胞(Salanti et al.，Cancer Cell.2015，28(4)：500-14.)。
人恶性疟原虫和人类至少有几百万年的共进化历史(Rich and Ayala，In Krishna R.Dronamraju，Paolo Arese(Ed).Emerging Infectious Diseases of the 21st Century：Malaria-Genetic and Evolutionary Aspects.Springer US 2006.pp.125-146.)，其生活史主要包括蚊期、肝期和红内期三个阶段(Bousema et al.，Nat Rev Microbiol.2014，12(12)：833-40.)，在共进化过程中，人红细胞是其红内期宿主细胞，其逃避人免疫系统的清除主要是通过人恶性疟原虫表达的红细胞膜蛋白1(PfEMP1)介导的人恶性疟原虫感染红细胞与宿主血管内皮细胞的黏附以及约60个编码PfEMP1蛋白的var基因(包括var2CSA)的抗原变异来实现的(Pasternak and Dzikowski，Int J Biochem Cell Biol.2009，41(7)：1463-6.)。因此，VAR2CSA蛋白在患胎盘疟疾患者体内的特异性表达也可能是与人恶性疟原虫与人类共进化的结果。由于pl-CSA主要表达在不同类型肿瘤细胞的表面及其细胞外基质(Salanti et al.，Cancer Cell.2015，28(4)：500-14；Ayres Pereira et al.，PLoS Pathog.2016，12(8)：e1005831；Seiler et al.，Eur Urol.2017，72(1)：142-150.)，我们推测VAR2CSA蛋白对pl-CSA的靶向结合作用可能有助于突破实体瘤的免疫抑制微环境。Salanti及其合作者的研究表明，将rVAR2蛋白与来自海绵Hemiasterella minor的hemiasterlin毒素类似物KT886偶联得到药物VDC886(rVAR2 drug conjugated KT886)，在非怀孕的荷瘤小鼠模型中显示出了对非霍奇金淋巴瘤，前列腺癌和转移性乳腺癌的抗肿瘤活性，并且没有观察到任何不良反应(Salanti et al.，Cancer Cell.2015，28(4)：500-14.)。针对VDC886的另一项研究还发现，在体外实验中，VDC886能够有效清除培养的肌肉浸润性膀胱癌(MIBC)细胞系，并且IC50为低纳摩尔级浓度水平；而在体内实验中，每周静脉注射两次(共四次)VDC886有效地阻止了化学耐药性(对基于顺铂的新辅助化疗药耐药)原位膀胱癌异种移植物的生长并且延长了荷瘤小鼠的生存期(Seiler et al.，Eur Urol.2017，72(1)：142-150.)。以上研究表明，rVAR2蛋白在荷瘤小鼠体内具有一定的稳定性，并且其蛋白本身免疫原性对小鼠没有不良的影响。
普通的CAR-T细胞主要是通过CAR与肿瘤细胞表面抗原的相互作用，通过磷酸化信号传导刺激CAR-T细胞的细胞因子释放和细胞增殖，最终起到杀伤或清除肿瘤细胞的效果(Chmielewski et al.， ImmunolRev.2014，257(1)：83-90.)。CN 105753991A公开了一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用，发现以与pl-CSA特异性相互作用的VAR2CSA蛋白结构域替换一般CAR-T中的单链抗体(single-chain fragment variable，ScFv)片段获得的CART-rVAR2对多种不同类型的肿瘤细胞具有体外杀伤活性，但后续研究发现其并不分泌细胞因子。
现有的CAR-T细胞技术的不足之处在于：
一、临床试验中多数CAR-T细胞只针对特定肿瘤类型和/或特定的蛋白靶点(clinicaltrials.gov)，例如针对B细胞白血病和B细胞淋巴瘤的靶点CD19和CD22的CAR-T(ClinicalTrials.gov Identifier：NCT00450944)；针对肝癌的靶点GPC3的CAR-T(ClinicalTrials.gov Identifier：NCT02723942)；针对肺癌、肝癌和胃癌的表达anti-PD1抗体的CAR-T(ClinicalTrials.gov Identifier：NCT02862028)；针对GD2阳性胶质瘤患者的CAR-T(ClinicalTrials.gov Identifier：NCT03252171)；针对EphA2阳性恶性胶质瘤患者的CAR-T(ClinicalTrials.gov Identifier：NCT02575261)；针对MUC1靶点的肺癌治疗性CAR-T(ClinicalTrials.gov Identifier：NCT03198052)；针对BCMA靶点的多发性骨髓瘤治疗性CAR-T(ClinicalTrials.gov Identifier：NCT03070327)；针对CD123靶点的急性骨髓性白血病治疗性CAR-T(ClinicalTrials.gov Identifier：NCT03190278)；针对EGFRvIII靶点的胶质母细胞瘤治疗性CAR-T(NCT02209376，NCT02664363)；针对CD138和BCMA靶点的多发性骨髓瘤治疗性CAR-T(ClinicalTrials.gov Identifier：NCT03196414)等。
二、严重的毒副作用，包括：1、对低表达CAR-T细胞靶蛋白(肿瘤相关抗原)的正常组织细胞的毒性，即所谓的“on-target，off-tumor toxicities”效应，例如针对CD19靶点的CAR-T治疗急性B淋巴细胞白血病，无论是在小鼠模型上(Davila et al.，PLoS One.2013，8(4)：e61338.)还是临床治疗中(Brentjens et al.，Blood.2011，118(18)：4817-28.)都观测到B细胞增殖障碍；2、在构建CAR-T细胞的过程中，因慢病毒载体或其他逆转录病毒载体的使用而存在基因插入突变(insertional mutagenesis)引起细胞癌变的可能性，例如在一项利用逆转录病毒治疗因细胞因子受体共同γ链缺失导致的重症联合免疫缺陷疾病的临床试验中发现，接受逆转录病毒转导的CD34+骨髓祖细胞治疗的9个患者中的4个发展为急性T细胞白血病，而这一病症的出现被认为与LMO2原癌基因的插入突变导致逆转录病毒转导的CD34+骨髓祖细胞的大量增殖有关(Hacein-Bey-Abina et al.，N Engl J Med.2003，348(3)：255-6；Hacein-Bey-Abina et al.，N Engl J Med.2010，363(4)：355-64.)；3、CAR-T细胞对肿瘤细胞的强力杀伤效应引起的严重不良反应，即所谓的“on-target，on-tumor toxicities”效应，例如肿瘤裂解综合症(the tumor lysis syndrome，TLS；Kochenderfer et al.，Blood.2013，122(25)：4129-39.)，细胞因子释放综合症/细胞因子风暴(cytokine release syndrome，CRS；Maude et al.，N Engl J Med.2014，371(16)：1507-17.)以及与之相关的巨噬细胞激活综合症(macrophage activation syndrome，MAS；Grupp et al.，ASH Annu Meet Abstr.2012，120(21)：2604.)等。一种基于可诱导的Caspase 9 (iCasp9)蛋白调控系统构建的“自杀基因开关”型CAR-T细胞据称能够通过清除转导的CAR-T细胞，有效限制“on-target，on-tumor toxicities”效应的发生(Gargett and Brown，Front.Pharmacol.2014，5(235)：1-7；Tey，Clin Transl Immunology.2014，3(6)：e17.)，但是iCasp9在CAR-T细胞治疗中的有效性和必要性还需要进一步验证(Ledford，Nature.2016，538(7624)：150-151；Paszkiewicz et al.，J Clin Invest.2016，126(11)：4262-4272.)。通过在CAR-T细胞上共表达另一个蛋白分子，再利用该蛋白特异性的中和抗体来清除带有该蛋白标签的CAR-T细胞也是一种有效的策略，例如利用抗CD20的嵌合抗体rituximab来清除表达CD20的T细胞(Vogler et al，Mol Ther.2010，18(7)：1330-1338；Philip et al.Blood.2014，124(8)：1277-1287.)，利用抗Myc-tag的抗体来清除表达Myc-tag的T细胞(Kieback et al.，Proc Natl Acad Sci USA.2008，105(2)：623-628.)。但是，抗CD20的嵌合抗体可能很难应用在针对B淋巴细胞瘤的CAR-T上，而Myc-tag策略缺乏临床级抗体(Paszkiewicz et al.，J Clin Invest.2016，126(11)：4262-4272.)。将一个截短的仅保留完整的西妥昔单抗[cetuximab(ErbituxTM)]结合位点而不包含其胞外N-末端配体结合结构域和胞内的受体络氨酸激酶结构域的人类表皮生长因子受体多肽tEGFR通过2A序列相连接共表达在CAR-T细胞上以作为细胞表面标志物用于体内输注的CAR+-T细胞的富集和跟踪鉴定，并且可以通过使用临床级的cetuximab单抗通过抗体依赖的细胞毒性来控制输注的CAR+-T细胞在体内的活性(Wang et al.，Blood.2011，118(5)：1255-63.)。然而，在发生严重毒性的情况下，这种通过抗体依赖性细胞毒性的细胞凋亡是否可以迅速起始，需要在临床试验中验证。
三、应用于实体瘤治疗的CAR-T技术依然是难点(Newick et al.，Annu Rev Med.2017，68：139-152.)。与血液性恶性肿瘤不同，CAR-T细胞必须从血液系统进入到实体瘤部位，浸润并透过肿瘤的间质才能产生肿瘤相关抗原特异性的细胞毒性并发挥肿瘤杀伤的功能。然而，尽管CAR-T细胞成功的运输并浸润到实体肿瘤部位，CAR-T细胞的功能也会很快丧失，其原因包括：1、肿瘤微环境的抑制，肿瘤微环境的氧化应激反应，营养匮乏，酸性pH环境，缺氧等都会抑制T细胞的活性；2、可溶的T细胞抑制性因子和细胞因子的负面影响；3、抑制性免疫细胞，例如调节性T细胞(Tregs)，髓性来源的抑制细胞(MDSCs)，以及肿瘤相关的巨噬细胞(TAMs)或中性白细胞(TANs)等的抑制作用；4、T细胞自身的内源性负调控机制，例如胞内和细胞表面抑制性受体的表达上调产生的抑制作用。
因此，普通的CAR-T细胞技术基本上都属于个体化的治疗技术，往往在治疗过程中伴随着发生严重的毒副作用乃至威胁病人的生命的可能性，并且针对实体瘤的CAR-T细胞治疗依然是难点。如何开发一种能够针对多种不同类型肿瘤都具有杀伤活性的广谱性CAR-T细胞，并且能够降低其毒副作用是一个亟待解决的问题。
发明内容
针对目前CAR-T技术治疗肿瘤中伴随着发生严重的毒副作用乃至威胁病人的生命的可能性，本发明提供一种嵌合抗原受体及其应用，所述嵌合抗原受体具有靶向多种不同类型肿瘤细胞的能力，具有普通 CAR-T细胞所不具备的广谱性，并且具有可调控性。
为达此目的，本发明采用以下技术方案：
一方面，本发明提供一种嵌合抗原受体，所述嵌合抗原受体包含识别疟原虫蛋白VAR2CSA、疟原虫蛋白VAR2CSA上的蛋白标签或能够标记疟原虫蛋白VAR2CSA的化合物中的任意一种或至少两种的组合结构域。
本发明中，所述嵌合抗原受体包含能够识别疟原虫蛋白VAR2CSA的结合域，其可以识别VAR2CSA蛋白，即可以通过识别VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)，还可以识别疟原虫蛋白VAR2CSA上的标记分子，即能够与疟原虫蛋白VAR2CSA重组表达的融合蛋白标签(tag)或者能够标记疟原虫蛋白VAR2CSA的化合物，也就是说，能够以任何方式识别到VAR2CSA蛋白的嵌合抗原受体都在本申请的保护范围内，无论是识别VAR2CSA蛋白本身，或是通过识别其他能够与VAR2CSA蛋白发生融合的蛋白或者能够标记VAR2CSA蛋白的化合物都在本发明的保护范围内。
本发明中，所述VAR2CSA蛋白通过结合肿瘤细胞表面的胎盘样硫酸软骨素pl-CSA从而具有靶向多种不同类型肿瘤细胞的能力，几乎95％(106/111)的来源于癌症患者的，包括造血系统、上皮组织和间质来源的人类癌细胞系都能被靶向。
本发明中，通过将嵌合抗原受体特异性结合VAR2CSA蛋白，发明人发现，可以通过调控VAR2CSA蛋白的含量，从而进一步调控嵌合抗原受体的作用，当不存在VAR2CSA蛋白时，嵌合抗原受体也就不起作用，不会对人体健康细胞产生毒副作用。
本发明中，通过将蛋白标签与VAR2CSA蛋白融合表达形成融合蛋白，这样能够识别该蛋白标签的嵌合抗原受体结构域也能够间接识别含有该蛋白标签的VAR2CSA融合蛋白；同样，通过化合物标记VAR2CSA蛋白，然后利用能够特异性识别该化合物的单链抗体来构建嵌合抗原受体，也可以达到间接识别含有该化合物的VAR2CSA蛋白的目的。
根据本发明，所述识别疟原虫蛋白VAR2CSA的结构域包含具有抗VAR2CSA蛋白抗体的重链可变区(VH)和轻链可变区(VL)。
根据本发明，所述VAR2CSA蛋白抗体的抗原为DBL1X、ID1、DBL2X、ID2a、ID2b、DBL3X、ID3、DBL4ε、ID4、DBL5ε、ID5或DBL6ε中的任意一种或至少两种的组合，优选为ID1、DBL2X和ID2a的组合。
本发明中，发明人发现，可以通过识别VAR2CSA蛋白中的任意一个片段来识别VAR2CSA蛋白，特别是识别ID1、DBL2X和ID2a的组合，即VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)，从而能够准确识别VAR2CSA蛋白，并且rVAR2能够与肿瘤细胞表面的胎盘样硫酸软骨素(pl-CSA)结合，也能够作为本发明中所述嵌合抗原受体靶向肿瘤细胞表面pl-CSA抗原的中间体。
优选地，所述DBL1X包含具有如SEQ ID NO.1所示的氨基酸序列，所述SEQ ID NO.1所示的氨基酸序列如下：
DBL1X(SEQ ID NO.1)：
Figure PCTCN2017113661-appb-000001
优选地，所述ID1包含具有如SEQ ID NO.2所示的氨基酸序列，所述SEQ ID NO.2所示的氨基酸序列如下：
ID1(SEQ ID NO.2)：
Figure PCTCN2017113661-appb-000002
Figure PCTCN2017113661-appb-000003
优选地，所述DBL2X包含具有如SEQ ID NO.3所示的氨基酸序列，所述SEQ ID NO.3所示的氨基酸序列如下：
DBL2X(SEQ ID NO.3)：
Figure PCTCN2017113661-appb-000004
优选地，所述ID2a包含具有如SEQ ID NO.4所示的氨基酸序列，所述SEQ ID NO.4所示的氨基酸序列如下：
ID2a(SEQ ID NO.4)：
Figure PCTCN2017113661-appb-000005
优选地，所述ID2b包含具有如SEQ ID NO.5所示的氨基酸序列，所述SEQ ID NO.5所示的氨基酸序列如下：
ID2b(SEQ ID NO.5)：
Figure PCTCN2017113661-appb-000006
优选地，所述DBL3X包含具有如SEQ ID NO.6所示的氨基酸序列，所述SEQ ID NO.6所示的氨基酸序列如下：
DBL3X(SEQ ID NO.6)：
Figure PCTCN2017113661-appb-000007
优选地，所述ID3包含具有如SEQ ID NO.7所示的氨基酸序列，所述SEQ ID NO.7所示的氨基酸序列如下：
ID3(SEQ ID NO.7)：
Figure PCTCN2017113661-appb-000008
优选地，所述DBL4ε包含具有如SEQ ID NO.8所示的氨基酸序列，所述SEQ ID NO.8所示的氨基酸序列如下：
DBL4ε(SEQ ID NO.8)：
Figure PCTCN2017113661-appb-000009
优选地，所述ID4包含具有如SEQ ID NO.9所示的氨基酸序列，所述SEQ ID NO.9所示的氨基酸序列如下：
ID4(SEQ ID NO.9)：
Figure PCTCN2017113661-appb-000010
优选地，所述DBL5ε包含具有如SEQ ID NO.10所示的氨基酸序列，所述SEQ ID NO.10所示的氨基酸序列如下：
DBL5ε(SEQ ID NO.10)：
Figure PCTCN2017113661-appb-000011
优选地，所述ID5包含具有如SEQ ID NO.11所示的氨基酸序列，所述SEQ ID NO.11所示的氨基酸序列如下：
ID5(SEQ ID NO.11)：
Figure PCTCN2017113661-appb-000012
优选地，所述DBL6ε包含具有如SEQ ID NO.12所示的氨基酸序列，所述SEQ ID NO.12所示的氨基酸序列如下：
DBL6ε(SEQ ID NO.12)：
Figure PCTCN2017113661-appb-000013
根据本发明，所述抗VAR2CSA蛋白抗体为通过15-20个氨基酸的短肽连接而成的单链抗体。
根据本发明，所述疟原虫蛋白VAR2CSA的结合域包含具有抗VAR2CSA蛋白的抗体的重链可变区和轻链可变区的互补决定区(CDR)序列。
根据本发明，所述具有单链抗体的重链可变区的CDR具有以下序列：CDR1如SEQ ID NO.13所示的氨基酸序列；CDR2如SEQ ID NO.14所示的氨基酸序列；CDR3如SEQ ID NO.15所示的氨基酸序列，具体如下：
CDR1(SEQ ID NO.13)：GFTFSNYA；
CDR2(SEQ ID NO.14)：ISITGRYT；
CDR3(SEQ ID NO.15)：TREGYDYAPSWFAY。
根据本发明，所述具有单链抗体的轻链可变区的CDR具有以下序列：CDR1如SEQ ID NO.16所示的氨基酸序列；CDR2如SEQ ID NO.17所示的氨基酸序列；CDR3如SEQ ID NO.18所示的氨基酸序列，具体如下：
CDR1(SEQ ID NO.16)：QTLVHRNGITY；
CDR2(SEQ ID NO.17)：KVS；
CDR3(SEQ ID NO.18)：FQGSHVPRT。
根据本发明，所述VAR2CSA的结合域的单链抗体的重链可变区包含具有如SEQ ID NO.19所示的氨基酸序列或与其具有至少70％以上氨基酸序列同一性，优选为90％以上氨基酸同一性的变体。
本发明中，所述变体仍然具有结合VAR2CSA蛋白的活性，所述SEQ ID NO.19所示的氨基酸序列如下：
Figure PCTCN2017113661-appb-000014
根据本发明，所述VAR2CSA的结合域的单链抗体的轻链可变区包含具有如SEQ ID NO.20所示的氨 基酸序列或与其具有至少70％以上氨基酸序列同一性，优选为90％以上氨基酸同一性的变体。
本发明中，所述变体仍然具有结合VAR2CSA蛋白的活性，所述SEQ ID NO.20所示的氨基酸序列如下：
Figure PCTCN2017113661-appb-000015
根据本发明，所述VAR2CSA的结合域的单链抗体的重链可变区包含具有如SEQ ID NO.21所示的核苷酸序列或与其具有至少60％以上核苷酸序列同一性，优选为80％以上核苷酸同一性的变体。
本发明中，所述变体仍然具有能够表达与VAR2CSA蛋白结合的氨基酸，所述SEQ ID NO.21所示的核苷酸序列如下：
Figure PCTCN2017113661-appb-000016
根据本发明，所述VAR2CSA的结合域的单链抗体的轻链可变区包含具有如SEQ ID NO.22所示的核苷酸序列或与其具有至少60％以上核苷酸序列同一性，优选为80％以上核苷酸同一性的变体。
本发明中，所述变体仍然具有能够表达与VAR2CSA蛋白结合的氨基酸，所述SEQ ID NO.22所示的核苷酸序列如下：
Figure PCTCN2017113661-appb-000017
优选地，所述疟原虫蛋白VAR2CSA上的蛋白标签选自但不限于PNE-tag、人myc-tag CaptureSelect C-tag、FLAG-tag、3×FLAG-tag、Strep-tag、6×His-tag、V5tag、S-tag、HA-tag、VSV-G-tag、GST-tag、HaloTag、XTEN-tag或huEGFRt-tag蛋白标签中的任意一个或至少两个的组合。
所述PNE-tag(peptide neo-epitopes)为包含来自酵母的转录因子GCN4上的14个氨基酸，具有如SEQ ID NO.23所示的氨基酸序列，所述SEQ ID NO.23所示的氨基酸序列如下：
PNE-tag(SEQ ID NO.23)：NYHLENEVARLKKL。
所述人myc-tag包含来自人c-myc蛋白的10个氨基酸，具有如SEQ ID NO.24所示的氨基酸序列，所述SEQ ID NO.24所示的氨基酸序列如下：
Myc-tag(SEQ ID NO.24)：EQKLISEEDL。
所述CaptureSelect C-tag具有如SEQ ID NO.25所示的氨基酸序列，所述SEQ ID NO.25所示的氨基酸序列如下：
CaptureSelect C-tag(SEQ ID NO.25)：EPEA。
所述FLAG-tag具有如SEQ ID NO.26所示的氨基酸序列，所述SEQ ID NO.26所示的氨基酸序列如下：
FLAG-tag(SEQ ID NO.26)：DYKDDDDK。
所述3×FLAG-tag具有如SEQ ID NO.27所示的氨基酸序列，所述SEQ ID NO.27所示的氨基酸序列如下：
3×FLAG-tag(SEQ ID NO.27)：DYKDHDGDYKDHDIDYKDDDDK。
所述Strep-tag具有如SEQ ID NO.28所示的氨基酸序列，所述SEQ ID NO.28所示的氨基酸序列如下：
Strep-tag(SEQ ID NO.28)：WSHPQFEK。
所述6×His-tag具有如SEQ ID NO.29所示的氨基酸序列，所述SEQ ID NO.29所示的氨基酸序列如下：
6×His-tag(SEQ ID NO.29)：HHHHHH。
所述V5-tag具有如SEQ ID NO.30所示的氨基酸序列，所述SEQ ID NO.30所示的氨基酸序列如下：
V5-tag(SEQ ID NO.30)：GKPIPNPLLGLDST。
所述S-tag具有如SEQ ID NO.31所示的氨基酸序列，所述SEQ ID NO.31所示的氨基酸序列如下：
S-tag(SEQ ID NO.31)：KETAAAKFERQHMDS。
所述HA-tag具有如SEQ ID NO.32所示的氨基酸序列，所述SEQ ID NO.32所示的氨基酸序列如下：
HA-tag(SEQ ID NO.32)：YPYDVPDYA。
所述VSV-G-tag具有如SEQ ID NO.33所示的氨基酸序列，所述SEQ ID NO.33所示的氨基酸序列如下：
VSV-G-tag(SEQ ID NO.33)：YTDIEMNRLGK。
所述GST-tag具有如SEQ ID NO.34所示的氨基酸序列，所述SEQ ID NO.34所示的氨基酸序列如下：
GST-tag(SEQ ID NO.34)：
Figure PCTCN2017113661-appb-000018
所述HaloTag具有如SEQ ID NO.35所示的氨基酸序列，所述SEQ ID NO.35所示的氨基酸序列如下：
HaloTag(SEQ ID NO.35)：
Figure PCTCN2017113661-appb-000019
所述XTEN-tag具有如SEQ ID NO.36所示的氨基酸序列，所述SEQ ID NO.36所示的氨基酸序列如下：
XTEN-tag(SEQ ID NO.36)：
Figure PCTCN2017113661-appb-000020
所述huEGFRt-tag具有一个截短的仅保留完整的西妥昔单抗(cetuximab(ErbituxTM))结合位点而不包含其胞外N-末端配体结合结构域和胞内的受体络氨酸激酶结构域的人类表皮生长因子受体多肽tEGFR，具 有如SEQ ID NO.37所示的氨基酸序列，所述SEQ ID NO.37所示的氨基酸序列如下：
huEGFRt-tag(SEQ ID NO.37)：
Figure PCTCN2017113661-appb-000021
所述能够标记疟原虫蛋白VAR2CSA的化合物指的是能够通过此化合物来识别疟原虫蛋白VAR2CSA，本领域技术人员可以根据疟原虫蛋白VAR2CSA进行选择，本发明的化合物可以为含有环辛炔基团的异硫氰酸荧光素(BCN-PEG4-FITC)和/或含有N-羟基琥珀酰亚胺的异硫氰酸荧光素(FITC-PEG4-NHS)。
所述BCN-PEG4-FITC化合物分子式如式I所示：
Figure PCTCN2017113661-appb-000022
所述FITC-PEG4-NHS化合物分子式如式II所示：
Figure PCTCN2017113661-appb-000023
根据本发明，所述嵌合抗原受体还包括铰链区、跨膜区和胞内信号区中的任意一种或至少两种的组合。
根据本发明，所述铰链区为常规铰链区，本领域技术人员可以根据需要进行选择，在此不作特殊限定，本发明采用人CD8α铰链区。
根据本发明，所述跨膜区为常规跨膜区，本领域技术人员可以根据需要进行选择，在此不作特殊限定，本发明采用人CD28跨膜区。
根据本发明，所述胞内信号区为常规胞内信号区，本领域技术人员可以根据需要进行选择，在此不作特殊限定，本发明采用为人CD27胞内信号区、人CD134胞内信号区、人CD28胞内信号区或人4-1BB (CD137)胞内信号区中的任意一种或至少两种的组合。
根据本发明，所述嵌合抗原受体的氨基末端含有一个CD8α信号肽；所述嵌合抗原受体的羧基末端还包含一个人CD3ζ胞内信号区。
第二方面，本发明提供编码如第一方面所述的嵌合抗原受体的核酸或与其具有至少60％同一性，优选至少80％同一性的核酸。
本发明中，所述核酸具有能够表达与VAR2CSA蛋白结合的氨基酸。
第三方面，本发明提供一种嵌合抗原受体表达细胞，所述表达细胞包含如第二方面所述的核酸。
优选地，所述细胞为免疫效应细胞，进一步优选为T细胞、B细胞、NK细胞、NKT细胞、树突状细胞或巨噬细胞中的任意一种或至少两种的组合。
第四方面，本发明提供一种重组载体，所述重组载体其包含有第二方面所述的核酸。
优选地，所述载体为重组克隆载体、重组真核表达质粒或重组慢病毒载体中的任意一种或至少两种的组合，优选为重组慢病毒载体。优选地，所述重组克隆载体选自但不限于pUC18、pUC19、pMD19-T、pGM-T载体、pUC57、pMAX或pDC315中的任意一种或至少两种的组合。
优选地，所述真核表达质粒选自但不限于pCDNA3系列载体、pCDNA4系列载体、pCDNA5系列载体、pCDNA6系列载体、pCI-neo系列载体、pEGFP系列载体、pSPT系列载体、pFLAG-CMV系列载体、pRL系列载体、pUC57载体、pMAX或pDC315中的任意一种或至少两种的组合。
优选地，所述重组慢病毒载体选自但不限于重组腺病毒载体、重组腺相关病毒载体、重组逆转录病毒载体、重组单纯疱疹病毒载体或重组痘苗病毒载体中的任意一种或至少两种的组合。
本发明中，通过将所述嵌合抗原受体的核酸构建体与所述载体进行重组，从而能够实现将所述重组后的载体转染到免疫细胞中获得表达该嵌合抗原受体的免疫细胞，实现嵌合抗原受体的功能。
第五方面，本发明提供一种重组病毒，其包含如第四方面所述的重组载体与包装辅助质粒共转染哺乳动物细胞得到的重组病毒；
第六方面，本发明提供一种嵌合抗原受体T细胞(CAR-T细胞)，其通过将如第五方面所述的重组病毒转染到T细胞中表达。
第七方面，本发明提供如第一方面所述的嵌合抗原受体、如第二方面所述的核酸、如第四方面所述的重组载体或如第五方面所述的重组病毒用于转染和扩增CAR-T细胞。
第八方面，本发明提供一种药物组合物，所述药物组合物包括第一方面所述的嵌合抗原受体、如第二方面所述的核酸、如第三方面所述的嵌合抗原受体表达细胞或如第四方面所述的表达载体，以及任选的药学上可接受的辅料。
第九方面，本发明提供如第一方面所述的嵌合抗原受体、如第二方面所述的核酸、如第三方面所述的嵌合抗原受体表达细胞或如第四方面所述的表达载体在制备治疗和/或预防自身免疫性疾病或肿瘤的药物中的用途。
优选地，所述肿瘤为实体瘤和/或血液瘤，所述肿瘤可以是能够被VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)特异性识别和结合的任意肿瘤组织和细胞，例如可以是人肺癌细胞系，包括NCI-H460(大细胞肺癌细胞系，ATCC#HTB177)，NCI-H520(鳞状细胞肺癌细胞系，ATCC#HTB182)以及A549(肺腺癌细胞系，ATCC#CCL185)都能够不同程度地被rVAR2蛋白特异性结合；rVAR2蛋白还能靶向结合人胎盘绒毛膜癌细胞系BeWo(ATCC#CCL98)；另外，B细胞淋巴瘤细胞系Raji(ATCC#CCL86)和急性骨髓性白血病细胞系KG-1a(ATCC#CCL246.1)也能够被rVAR2蛋白特异性结合；但是，rVAR2蛋白与健康人的PBMC和正常人的脐静脉内皮细胞(HUVEC，ATCC#PCS-100-010)结合为阴性。
第十方面，本发明提供一种治疗患有自身免疫性疾病和/或与肿瘤抗原的表达相关的疾病的受试者的方法，其包括对所述受试者施用有效量的包含第九方面所述的药物组合物的药物。
优选地，所述肿瘤为实体瘤和/或血液瘤，所述肿瘤可以是能够被VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)特异性识别和结合的任意肿瘤组织和细胞，例如可以是人肺癌细胞系，包括NCI-H460(大细胞肺癌细胞系， ATCC#HTB177)，NCI-H520(鳞状细胞肺癌细胞系，ATCC#HTB182)以及A549(肺腺癌细胞系，ATCC#CCL185)都能够不同程度地被rVAR2蛋白特异性结合；rVAR2蛋白还能靶向结合人胎盘绒毛膜癌细胞系BeWo(ATCC#CCL98)；另外，B细胞淋巴瘤细胞系Raji(ATCC#CCL86)和急性骨髓性白血病细胞系KG-1a(ATCC#CCL246.1)也能够被rVAR2蛋白特异性结合；但是，rVAR2蛋白与健康人的PBMC和正常人的脐静脉内皮细胞(HUVEC，ATCC#PCS-100-010)结合为阴性。
术语“变体”是指包含一个或几个至多个氨基酸的取代、缺失或添加的任何变体，条件是所述变体基本上保留原始序列所具有的相同功能。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的所述嵌合抗原受体可以识别VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)，所述VAR2CSA或者rVAR2蛋白通过结合肿瘤细胞表面的胎盘样硫酸软骨素pl-CSA从而具有靶向多种不同类型肿瘤细胞的能力，几乎95％(106/111)的来源于癌症患者的人类癌细胞系都能被靶向；
(2)本发明通过将嵌合抗原受体特异性结合VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)来间接的识别和杀伤表达含有胎盘样硫酸软骨素pl-CSA的表面抗原的肿瘤细胞，可以通过调控VAR2CSA蛋白的含量，从而进一步调控嵌合抗原受体的作用，当不存在VAR2CSA蛋白时，嵌合抗原受体也就不起作用，不会对人体健康细胞产生毒副作用；
(3)本发明嵌合抗原受体中的胞外识别域部分，即单链抗体(ScFv)部分能够被与其同源的单克隆抗体竞争性地结合其所靶向结合的位于VAR2CSA蛋白或者rVAR2蛋白上的结合域，可以通过调控VAR2CSA蛋白的单克隆抗体的含量，起到间接限制调节嵌合抗原受体细胞的功能的作用，有利于降低表达该嵌合抗原受体的T细胞等免疫细胞的毒副作用；
(4)本发明通过将嵌合抗原受体特异性结合VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)来间接的识别和杀伤表达含有胎盘样硫酸软骨素pl-CSA的表面抗原的肿瘤细胞，可以通过采用VAR2CSA蛋白或其相关结构域的重组蛋白rVAR2来偶联细胞毒素用来在免疫治疗后清除痊愈患者体内冗余的表达该嵌合抗原受体的细胞，降低脱靶的风险；
(5)本发明的所述嵌合抗原受体可以识别VAR2CSA蛋白或者VAR2CSA蛋白中能够与胎盘样硫酸软骨素(pl-CSA)结合的任意一个或至少两个结构域的重组蛋白(rVAR2)，所述VAR2CSA或者rVAR2蛋白特异性靶向结合的胎盘样硫酸软骨素pl-CSA，所述pl-CSA广泛且大量存在于肿瘤组织的细胞表面和细胞外基质中，使得表达该嵌合抗原受体的免疫细胞与VAR2CSA蛋白的协同作用下，具有潜在的靶向肿瘤微环境的作用，并且可能有助于表达该嵌合抗原受体的免疫细胞突破实体瘤的免疫抑制微环境，最终达到靶向杀伤实体瘤细胞的作用；
(6)本发明嵌合抗原受体可以作为治疗癌症的药物，其作用可以进行调控，为癌症治疗打开了一个新思路。
附图说明
图1(A)为原核表达纯化的带有Strep-tag蛋白标签的rVAR2重组蛋白的10％SDS-PAGE蛋白凝胶电泳分析结果，图1(B)为原核表达纯化的带有Strep-tag蛋白标签的rVAR2重组蛋白的10％SDS-PAGE蛋白凝胶电泳分析结果；
图2为酶联免疫吸附试验(ELISA)测定鼠源抗rVAR2蛋白抗体的功能，其中，NC为取未经免疫的小鼠的血清以1∶500倍稀释后作为ELISA测定的阴性对照，其余组别为实验组抗体的不同稀释倍数，所有对照组和实验组中的抗原(rVAR2重组蛋白)包被浓度为1μg/ml；
图3为流式检测rVAR2蛋白的肿瘤细胞靶向特异性；
图4为Western blot鉴定单克隆抗体5H4对rVAR2蛋白的结合作用，其中，A为原核表达纯化的rVAR2蛋白，B为真核表达纯化的rVAR2蛋白；
图5为12％SDS-PAGE蛋白凝胶电泳检测原核表达纯化的带有Strep-tag蛋白标签的ID1、DBL2X和ID2a多肽，其中，M，蛋白分子量标准；NC，阴性对照，空载体转染的大肠杆菌裂解液；ID2a，纯化的带有Strep-tag标签的ID2a蛋白，DBL2X，纯化的带有Strep-tag标签的DBL2X蛋白，ID1，纯化的带有Strep-tag标签的ID1蛋白；
图6为抗rVAR2单克隆抗体5H4的抗原表位筛选，其中，Mock为空白对照，检测体系除了没有添加单克隆抗体5H4以外，其余成分与实验组一致；HCS为hybridoma cell supernatant，小鼠B细胞杂交瘤表达的上清；
图7(A)为表达本发明中所述嵌合抗原受体的T细胞、VAR2CSA全长蛋白(或其pl-CSA结合结构域重组蛋白rVAR2)和表达pl-CSA抗原表位的肿瘤细胞三者之间的结合模式示意图；图7(B)为利用本发明嵌合抗原受体构建的几种不同类型的T细胞及其与VAR2CSA全长蛋白(或其pl-CSA结合结构域重组蛋白rVAR2)和表达pl-CSA抗原表位的肿瘤细胞三者之间的结合模式示意图；
图8基于5H4单链抗体的pLentiCART-anti-rVAR2的慢病毒表达载体质粒图谱；
图9(A)为10％SDS-PAGE蛋白凝胶电泳检测rVAR2重组蛋白在人血清中的稳定性；图9(B)为Western blot检测rVAR2重组蛋白在人血清中的稳定性；
图10为流式检测CART-anti-rVAR2(5H4 ScFv)细胞的慢病毒转染效率及其胞外抗原识别区(5H4 ScFv)的表达情况，其中，Mock为单独T细胞组，不加Alexa Fluor 647标记的羊抗鼠F(ab′)2IgG抗体处理；Negative Control为阴性对照组，T细胞加Alexa Fluor 647标记的羊抗鼠F(ab′)2IgG抗体处理；CART-anti-rVAR2(5H4 ScFv)为表达CART-anti-rVAR2(5H4 ScFv)的慢病毒转染T细胞后获得的CAR-T细胞表达组；
图11(A)为sCART-anti-rVAR2(5H4 ScFv)与Raji细胞共孵育过程中的IL-2的分泌水平检测；图11(B)为sCART-anti-rVAR2(5H4 ScFv)与Raji细胞共孵育过程中的IFN-γ的分泌水平检测；
图12为流式检测sCART-anti-rVAR2(5H4 ScFv)对肿瘤细胞Raji的体外杀伤能力，其中，Far Red+门中的信号表示Raji细胞的比例，而GFP+门中的信号表示CART-anti-rVAR2(5H4 ScFv)细胞的比例；
图13为体外实时动态监测sCART-anti-rVAR2(5H4 ScFv)对Raji细胞的杀伤作用，其中，白色箭头标记的细胞为Far Red标记的肿瘤细胞Raji，而未标记的细胞为共表达GFP报告基因的CART-anti-rVAR2(5H4 ScFv)细胞。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例1：VAR2CSA蛋白pl-CSA结合结构域的相关重组蛋白的表达及纯化
首先将编码VAR2CSA蛋白的pl-CSA结合结构域的相关重组蛋白(rVAR2)氨基酸序列的DNA分子克隆至一个原核或真核表达载体之中，再分别将构建好的原核表达载体转入大肠杆菌(例如BL21(DE3))中，将构建好的真核表达载体转入真核生物细胞(例如含有大T抗原的人胚肾细胞系HEK293T)中进行蛋白表达，收集蛋白表达细胞或培养基上清，再分别采用对应的蛋白标签亲和纯化介质进行蛋白纯化。其步骤如下：
Strep-tag亲和标签纯化体系：
首先离心收集表达带有Strep-tag标签的rVAR2重组蛋白的大肠杆菌，按每克细胞加入10毫升冰预冷的Buffer W(100mM Tris/HCl，pH 8.0，150mM NaCl，1mM EDTA)重悬大肠杆菌细胞，采用AH-1500超高压均质机(ATS Engineering Inc.)在100Mpa的压力下，低温破碎2-3次获得细胞裂解液。在14,000rpm，10min，4℃条件下离心收集细胞裂解液上清。用2倍Strep-Tactin树脂体积的Buffer W(100mM Tris/HCl，pH 8.0，150mM NaCl，1mM EDTA)洗涤并平衡树脂。将收集的细胞裂解液上清与平衡好的Strep-Tactin树脂孵育30min-1h后过柱，待穿透液全部流出后，分多次加入至少5倍Strep-Tactin树脂体积的冰预冷Buffer W(100mM Tris/HCl，pH 8.0，150mM NaCl，1mM EDTA)洗涤树脂，加入3倍Strep-Tactin树脂体积的冰预冷Buffer E(100mM Tris/HCl，pH 8.0，150mM NaCl，1mM EDTA，2.5mM desthiobiotin)分3次从Strep-Tactin树脂上洗脱并收集带有Strep-tag标签的rVAR2重组蛋白，再取一定量的带有Strep-tag蛋白标签的rVAR2重组蛋白纯化样品进行SDS-PAGE蛋白凝胶电泳分析，结果如图1(A)所示，带有Strep-tag蛋白标签的rVAR2重组蛋白的预测平均分子量约为74kDa。
所述带有Strep-tag蛋白标签的rVAR2重组蛋白的氨基酸序列如下(SEQ ID NO.38)：
Figure PCTCN2017113661-appb-000024
黑色加粗的氨基酸序列是在在基因克隆过程中引入的来自载体骨架DNA表达的氨基酸序列，而下划 线标记的氨基酸序列就是Strep-tag序列(SEQ ID NO.28)，通过它与Strep-Tactin树脂的亲和性来纯化带有Strep-tag蛋白标签的rVAR2重组蛋白。
3×FLAG-tag亲和标签纯化体系：
采用带有分泌型信号肽或非分泌型信号肽的真核(哺乳动物细胞)蛋白表达载体构建带有3×FLAG-tag蛋白标签的rVAR2重组蛋白的表达载体，将该表达载体通过电转或PEI转染的方式导入HEK293细胞或HEK293T细胞中进行带有3×FLAG-tag蛋白标签的rVAR2重组蛋白表达后分别收集细胞培养基上清(针对分泌型表达载体)或细胞(针对非分泌型载体)。在细胞培养基上清中加入5％的pH值调节缓冲液(1M Tris-HCl，3M NaCl，pH 7.4)后备用。在收集的细胞中按照106-107cells/ml的比例加入细胞裂解液Buffer L[50mM Tris HCl，pH 7.4，with 150mM NaCl，1mM EDTA，1％TRITON X-100，以及1％的蛋白酶抑制剂(Sigma，Cat.#P8340)]后室温孵育30min.12,000×g，4℃离心10min收集细胞裂解液上清，备用。
将准备好的含有3×FLAG-tag蛋白标签的rVAR2重组蛋白表达上清或者细胞裂解液上清与平衡好的ANTI-FLAG M2抗体偶联的亲和树脂或者ANTI-FLAG M2抗体偶联的磁珠(Sigma)冰上共孵育2h后过柱或用磁力架收集带有3×FLAG-tag蛋白标签的rVAR2重组蛋白结合的树脂或磁珠，用20倍树脂或磁珠体积的TBS缓冲液(50mM Tris HCl，150mM NaCl，pH 7.4)分三次洗涤收集到的树脂或磁珠，采用5倍树脂或磁珠体积的3×FLAG多肽洗脱液(用TBS缓冲液配制，3×FLAG多肽浓度为150ng/μl)洗脱并收集带有3×FLAG-tag蛋白标签的rVAR2重组蛋白，再取一定量的含有3×FLAG-tag蛋白标签的rVAR2重组蛋白纯化样品进行SDS-PAGE蛋白凝胶电泳分析，结果如图1(B)所示，含有3×FLAG-tag蛋白标签的rVAR2重组蛋白的预测平均分子量约为76kDa，而10％的SDS-PAGE凝胶电泳检测的分子量大小约为115kDa，预示着可能存在蛋白质的翻译后修饰。
所述带有3×FLAG-tag标签的rVAR2重组蛋白的氨基酸序列如下(SEQ ID NO.39)：
Figure PCTCN2017113661-appb-000025
黑色加粗的氨基酸序列是在在基因克隆过程中引入的来自载体骨架DNA表达的氨基酸序列，而下划线标记的就是3×FLAG-tag的氨基酸序列(SEQ ID NO.27)，通过它与ANTI-FLAG M2抗体偶联的树脂或磁珠的亲和性来纯化带有3×FLAG-tag蛋白标签的rVAR2重组蛋白。
实施例2：抗rVAR2蛋白鼠源多抗和单克隆抗体的获取
将原核表达纯化的rVAR2重组蛋白分别免疫3只4-6周龄的BALB/c小鼠，具体操作步骤如下：首先将纯化得到的rVAR2重组蛋白用磷酸盐缓冲液(PBS，pH7.4)缓冲液透析3次，超滤浓缩蛋白浓度至1μg/μl；取100μl rVAR2重组蛋白溶液(100μg)与100μl弗氏完全佐剂乳化后对小鼠进行背部多点注射进行初次免疫，后再分别用100μl rVAR2重组蛋白溶液(100μg)与100μl弗氏不完全佐剂乳化后对小鼠进行背部多点注射进行2-3次的加强免疫后，采用酶联免疫吸附试验(ELISA)检测免疫小鼠血清中多抗的功能与效价，结果如图2所示，以OD450值在1.0左右为标准，检测到的小鼠抗血清(多抗)效价最高约为1∶25000。
按照需要分别准备适量的来自小鼠腹腔的饲养细胞和小鼠骨髓瘤细胞SP2/0备用；取已经免疫的BALB/c小鼠，用异氟醚麻醉后摘除眼球采血，并分离血清作为抗体检测时的阳性对照血清。断颈致死已经免疫的BALB/c小鼠，手术取出其脾脏并置于已盛有10ml不完全培养基的平皿中，轻轻洗涤，并细心剥去周围结缔组织。将分离好的小鼠脾脏转移至另一个已盛有10mL不完全培养基平皿中的不锈钢筛网上，用注射器针芯研磨成细胞悬液使脾细胞进入平皿中的不完全培养基。用吸管吹打数次，制成单细胞悬液后计数。通常每只小鼠1×108-2.5×108个脾细胞。将1×108脾细胞与1×107骨髓瘤细胞SP2/0通过PEG处理融合制备杂交瘤，并铺板至含有饲养细胞的96-孔板中，采用HAT培养基[含有次黄嘌呤(hypoxantin)、氨基蝶呤(aminopterin)和胸腺嘧啶脱氧核苷(thymidin)三种物质的细胞培养基]培养并筛选杂交瘤细胞，镜检观察杂交瘤细胞生长情况，待其长至孔底面积1/10以上时吸取细胞培养上清供抗体检测，对阳性抗体检测孔的杂交瘤细胞采用有限稀释法做单克隆化，待其长至孔底面积1/10以上时吸取细胞培养上清再次进行抗体检测。选取分泌抗体与rVAR2重组蛋白的亲和性较高的单克隆化的杂交瘤细胞株，取部分细胞进行冻存；剩下的细胞，取一部分继续培养收集含有rVAR2重组蛋白单抗的上清。再取一部分进行RNA的提取并采用特定引物的RT-PCR法分析鼠源IgG抗体的重链和轻链的可变区编码序列。从中，选取了一株分泌单克隆抗体5H4的细胞株，其重链和轻链的可变区编码序列如表1所示，5H4的重链和轻链的可变区氨基酸序列如表2所示，5H4的重链和轻链的互补决定区(complementary determining region，CDR)序列如表3所示。
表1单克隆抗体5H4的重链可变区(VH)和轻链可变区(VL)的DNA编码序列
Figure PCTCN2017113661-appb-000026
Figure PCTCN2017113661-appb-000027
表2单克隆抗体5H4的重链可变区(VH)和轻链可变区(VL)的氨基酸序列
Figure PCTCN2017113661-appb-000028
表3单克隆抗体5H4的重链可变区(VH)和轻链可变区(VL)的互补决定区(complementary determining region，CDR)序列
Figure PCTCN2017113661-appb-000029
实施例3：流式检测rVAR2蛋白对肿瘤细胞的靶向性
分别取5×105个体外分离或培养的各个不同类型的细胞(包括正常细胞对照组和肿瘤细胞组)，利用纯化的原核或真核表达的rVAR2蛋白，以及通过免疫小鼠获得的抗rVAR2蛋白的鼠源多抗和购买的FITC染料标记的山羊抗鼠二抗(IgG H&L，Abcam，cat#ab6785)分别孵育处理45min，在孵育下一个试剂之前或试剂孵育完成之后用4℃预冷的含有0.02％NaN3、2％FBS的PBS溶液分别洗涤3次，每次间隔2min。最后采用流式细胞术检测rVAR2蛋白对多种不同类型的肿瘤细胞的靶向性，实验分组如下表4所示：
表4流式检测rVAR2蛋白对肿瘤细胞的靶向性实验分组
Figure PCTCN2017113661-appb-000030
结果如图3所示，从图3可以看出，rVAR2蛋白可特异性靶向多种不同类型的肿瘤细胞。例如人肺癌细胞系，包括NCI-H460(大细胞肺癌细胞系，ATCC#HTB177)，NCI-H520(鳞状细胞肺癌细胞系，ATCC#HTB182)以及A549(肺腺癌细胞系，ATCC#CCL185)都能够不同程度地被rVAR2蛋白特异性结合；rVAR2蛋白还能靶向结合人胎盘绒毛膜癌细胞系BeWo(ATCC#CCL98)；另外，B细胞淋巴瘤细胞系Raji(ATCC#CCL86)和急性骨髓性白血病细胞系KG-1a(ATCC#CCL246.1)也能够被rVAR2蛋白特异性结合；但是，rVAR2蛋白与健康人的PBMC和正常人的脐静脉内皮细胞(HUVEC，ATCC#PCS-100-010)结合为阴性。
实施例4：rVAR2重组蛋白的鼠源单克隆抗体5H4的功能验证
采用Western blot验证单克隆抗体5H4与rVAR2重组蛋白抗原的结合能力。一抗为纯化的单克隆抗体5H4，二抗为HRP标记的羊抗鼠抗体，结果如图4所示。
从图4可以看出，原核表达纯化的rVAR2蛋白，约74kDa，真核表达纯化的rVAR2蛋白，约115kDa，Westernblot鉴定结果表明单克隆抗体5H4能结合原核或真核表达纯化的rVAR2蛋白。
实施例5：抗rVAR2单克隆抗体5H4抗原表位的筛选
根据VAR2CSA与pl-CSA(胎盘样硫酸软骨素)结合的核心结构域rVAR2的结构与功能特性可将其细分为三个主要的结构域组分，包括ID1，DBL2X和ID2a(Clausen et al.，2012)。rVAR2蛋白及其结构域组分的氨基酸的序列如下：
rVAR2蛋白的氨基酸序列如下(SEQ ID NO.39)：
Figure PCTCN2017113661-appb-000031
Figure PCTCN2017113661-appb-000032
ID1多肽的氨基酸序列如下(SEQ ID NO.2)：
Figure PCTCN2017113661-appb-000033
DBL2X多肽的氨基酸序列如下(SEQ ID NO.3)：
Figure PCTCN2017113661-appb-000034
ID2a多肽的氨基酸序列如下(SEQ ID NO.4)：
Figure PCTCN2017113661-appb-000035
分别将ID1、DBL2X、ID2a多肽的DNA编码序列克隆至大肠杆菌表达载体中，经过蛋白表达纯化后获取了三者的蛋白，其SDS-PAGE蛋白凝胶电泳结果如图5所示，纯化的带有Strep-tag标签的ID2a蛋白，预测的平均分子量大小约为15kDa；纯化的带有Strep-tag标签的DBL2X蛋白，预测的平均分子量大小为40kDa；纯化的带有Strep-tag标签的ID1蛋白，预测的平均分子量大小为17kDa。
采用抗原特异性ELISA法初步确认抗rVAR2单克隆抗体5H4的抗原表位：将纯化的ID1、DBL2X、ID2a3种抗原肽分别用Na2CO3-NaHCO3缓冲液(pH 9.6)的稀释至终浓度为1μg/ml，分别按照100μl抗原/孔添加至96-孔酶标板中，于37℃包被2h；用PBS-T buffer[含有0.05％(v/v)Tween 20的PBS缓冲液，pH7.4]洗涤两次；拍干酶标板孔内液体，每孔加250μl 5％(v/v)脱脂牛奶/PBS-T buffer，37℃孵育1.5h；用PBS-T洗4次，拍干酶标板孔内液体，每孔加100μl 0.1％(v/v)脱脂牛奶/PBS-T buffer稀释的自杂交瘤上清中纯化的单克隆抗体5H4(一抗)，置于37℃，孵育60min；孵育完一抗后，用PBS-T洗4次，拍干酶标板孔内液体，每孔加100μl 0.1％(v/v)脱脂牛奶/PBS-T buffer稀释的按1∶8000稀释的HRP标记的羊抗鼠IgG(二抗)，37℃孵育45min；孵育完二抗后，用PBS-T buffer洗4次，拍干酶标板孔内液体，每孔加入100μl TMB显色底物，37℃，避光显色10min，每孔加50μl 1M硫酸终止反应，检测OD450。
ELISA结果如图6所示，只有ID2a抗原肽的包被孔的检测结果与rVAR2蛋白包被孔的检测结果一致， 表明抗rVAR2单克隆抗体5H4的抗原表位位于ID2a多肽表位之上。
实施例6：CAR-T细胞的构建及其功能机制
为了进一步提高这种基于VAR2CSA的广谱性CAR-T细胞的杀伤活性并增强其应用安全性。构建了一个以VAR2CSA或其pl-CSA的结合结构域重组蛋白(rVAR2)及其单链抗体(anti-VAR2CSA ScFv或anti-rVAR2 ScFv)制造的CAR-T(CART-anti-VAR2CSA或CART-anti-rVAR2)细胞为体系的可调控性CAR-T细胞：即利用全长VAR2CSA蛋白或rVAR2蛋白的肿瘤细胞靶向性来结合并标记肿瘤细胞，再用我们所开发的全长VAR2CSA蛋白或rVAR2蛋白特异性的鼠源单链抗体或人源化单链抗体来构建CART-anti-VAR2CSA或CART-anti-rVAR2，其作用机制为利用CART-anti-VAR2CSA或CART-anti-rVAR2间接地靶向杀伤全长VAR2CSA蛋白或rVAR2蛋白标记的肿瘤细胞，其示意图如图7所示。
将这种通过VAR2CSA全长蛋白或其pl-CSA结合结构域的重组蛋白与其单链抗体构建的CAR-T细胞一起，所构成的可调控性CAR-T细胞技术体系，命名为sCART-anti-VAR2CSA(包含VAR2CSA的全长蛋白的体系)或sCART-anti-rVAR2细胞(包含VAR2CSA蛋白的pl-CSA结合结构域重组蛋白的体系)。
以CART-anti-rVAR2(5H4 ScFv)为例，如图8所示，其负责识别rVAR2上的ID2a表位的单链抗体ScFv序列包括anti-rVAR2单克隆抗体5H4的VH链以及5H4的VL链以及两者之间的连接序列L(Linker)，linker序列包括但不限于GGGGSGGGGSGGGGS等氨基酸序列。
实施例7：rVAR2蛋白在人血清中的稳定性检测
为了确认rVAR2蛋白是否能够作为一个CART-anti-rVAR2细胞的介质来靶向杀伤肿瘤细胞，首先需要确定rVAR2蛋白能否在人的血液中稳定存在。
因此，从一位非小细胞肺癌患者(男性，年龄：64)捐献的2毫升血液中分离得到了约1ml血清，然后采用V底的96孔板，每孔加入25μl病人的血清与5μl经0.2μm蛋白低吸附滤膜过滤除菌的rVAR2重组蛋白混合至rVAR2重组蛋白的终浓度为0.2μg/μl，铺板后置于37℃，5％CO2培养箱中处理。
按照样品在培养箱中的放置天数(Day)分别取样，Day 0表示第0天，即取30μl刚刚混合好的样品，Day 1表示取30μl在37℃，5％CO2培养箱中处理一天(24h)的样品，以此类推，分别取Day 2、Day 3、Day 4、Day 5、Day 7、Day 9、Day 11、Day 14、Day 17和Day 22的样品，每取一份样品分别加入570μl SDS-PAGE蛋白上样缓冲液(2×)，煮沸3min后冷却后置于-20℃储存备用。阴性对照组(NC)，取肺癌患者的血清10μl，用SDS-PAGE蛋白上样缓冲液(2×)稀释20倍后煮沸3min，冷却后置于-20℃储存备用。阳性对照组(PC)，将纯化的rVAR2重组蛋白用SDS-PAGE蛋白上样缓冲液(2×)稀释至0.1μg/μl，煮沸3min后冷却后置于-20℃储存备用。
分两组，分别取准备好的样品5μl，上样至10％SDS-PAGE凝胶，电泳检测，取其中一组转PVDF膜，并采用抗Strep-tag单抗通过Western blot检测带有Strep-tag标签的rVAR2重组蛋白的稳定性。
结果如图9(A)-图9(B)所示，带有Strep-tag蛋白标签的rVAR2重组蛋白，平均分子量约74kDa，人血清白蛋白，平均分子量约66.5kDa，表明37℃，5％CO2培养箱处理条件下，rVAR2重组蛋白能够在肺癌患者的血清中稳定存在3周以上。
实施例8：CART-anti-rVAR2细胞的构建
首先构建CART-anti-rVAR2细胞，我们将抗rVAR2重组蛋白ID2a结构域的5H4 ScFv克隆到一个慢病毒表达载体中，该载体以EF1α启动子下游依次串联有CD8信号肽、5H4 ScFv、CD8a铰链区(CD8Hinge)、CD28跨膜序列(TM)、CD28共刺激因子、CD137(4-1BB)共刺激因子和CD3ζ结构域序列，示意图如图8所示。
采用PEI转染法将大量提取的CART-anti-rVAR2(5H4 ScFv)表达质粒与第三代慢病毒包装质粒pMDLg-pRRE∶pRSV-Rev∶pMD2.G按照3∶1∶1∶1的质量比混合后共转染HEK293T细胞，转染72h后收集细胞培养上清，将收集的培养上清用0.45μm的膜过滤后，经超滤浓缩至慢病毒滴度为1.6×108TU/ml-1×1010TU/ml，于-80℃冰箱中保存备用。接下来按照如下步骤生产CART-anti-rVAR2(5H4 ScFv)细胞：
(1)分离PBMC细胞
取志愿者捐献的新鲜全血50ml，800g离心10min；取白细胞层，用2％FBS稀释至8ml；吸取4ml LymphoPrep加入15ml离心管中，再将稀释血液小心沿管壁加至分层液上，保持两者界面清晰；800g离心20min(加速设置为6，降速设置为1)；用巴氏吸管轻轻吸出灰白色的单个核细胞，加入另一支已含有10ml RF-10(含有10％灭活FBS的RPMI 1640培养基)的离心管中，混匀；以500g离心5min，弃上清；加10mL RF-10重悬细胞，Trypan blue染色并计数，350g离心10min，弃上清。
(2)磁珠分选CD4+T和CD8+T细胞
采用
Figure PCTCN2017113661-appb-000036
CD4 Positive Isolation Kit(Invitrogen，Cat.#11331D)和
Figure PCTCN2017113661-appb-000037
CD8 Positive Isolation Kit(Invitrogen，Cat.#11333D)分别分离PBMC中的CD4+T细胞和CD8+T细胞。基本操作步骤如下：涡旋混匀磁珠，取25μl磁珠(Dynabeads)于试管中，加入1ml Buffer 1混匀，将试管放在磁力架上1min，收集上清。加25μl Buffer 1重悬磁珠备用；将PBMC细胞用Buffer 1重悬至密度为1×107个/ml；向1ml的PBMC细胞中加入25μl洗涤好的磁珠，2-8℃孵育20min，放在摇床上倾斜旋转；将试管放在磁力架上2min，收集上清；移开试管，加1ml Buffer 1，吹打混匀，放在磁力架上2min，收集上清，重复一次，将以上步骤收集的含有其他细胞的上清液转移到一个新的无菌试管中，用于后续从中分离CD8+T细胞；加100μl Buffer 2重悬磁珠结合的CD4+T细胞，加10μl DETACHaBEAD，室温下孵育45min使细胞从磁珠上释放；将试管放在磁力架上1min，含有细胞的上清转移到新的试管中，加500μl Buffer 2洗涤磁珠2-3次，收集含有部分CD4+T细胞的上清；加4ml Buffer 2，350g离心5min，去除包含DETACHaBEAD上清，用Buffer 2重悬收集到的CD4+T细胞；分选CD4+T细胞过程中收集的上清继续按照按试剂盒操作 收集CD8+T细胞。
(3)T细胞的培养
磁珠分选后的CD4+T和CD8+T细胞经350g离心10min；RF-10(含有10％灭活FBS的RPMI 1640培养基)重悬计数；按照1∶1的比例加CD4+T和CD8+T细胞培养板中培养，细胞密度为5×105个/ml；在T细胞专用培养基加入CD3/CD28抗体磁珠，加入磁珠的量按与细胞的比例1∶1加入；加rhIL-2，使终浓度为10ng/ml；每周计数2-3次，记录细胞的增殖情况。
(4)T细胞的慢病毒转染
磁珠分选后的CD4+T和CD8+T细胞经350g离心10min，加完全培养基重悬计数；按照1∶1的比例加CD4+T和CD8+T细胞于96孔板中培养，细胞密度为5×105个/ml，每孔细胞数为1×105个；洗涤磁珠，向培养皿中加入与细胞1∶1比例的磁珠；加rhIL-2，使终浓度为10μg/l；刺激24h后，感染细胞，加Polybrene，使终浓度为6μg/ml，混匀；16-24h后换液；3天后用流式检测T细胞感染效率，结果如图10所示，可见，流式检测的感染效率为45％左右，可以较高效制得CAR-T细胞。
实施例9：sCART-anti-rVAR2这种可调控性CAR-T与肿瘤细胞共孵育过程中的细胞因子分泌检测
为了检测sCART-anti-rVAR2这种可调控性CAR-T细胞是否对肿瘤细胞有潜在的杀伤功能，我们首先利用相关细胞因子检测试剂盒[R&D Systems，Human IFN-γELISA检测试剂盒(Cat.#dif50)和Human IL-2 ELISA检测试剂盒(Cat.#d2050)]对sCART-anti-rVAR2体系与肿瘤细胞共孵育过程中的细胞因子分泌水平按如下步骤进行了检测：
(1)细胞收集和预处理
分别收集效应细胞[CAR-T细胞：CART-anti-rVAR2(5H4 ScFv)]与靶细胞(癌细胞)，250×g离心4min，去上清，用无血清的1640培养基(10ml)洗涤细胞两次，再用1640培养基调整细胞密度至6×105个/ml；
(2)实验分组
实验组：效应细胞[CART-anti-rVAR2(5H4 ScFv)，CARTV2]与靶细胞(Raji，R)以1∶1的比例均匀铺于48孔板中，每种细胞设定为1.25×105个/孔/250μl，即总体积为500μl；其中效应细胞(或靶细胞)与rVAR2重组蛋白(rVAR2，V)提前共孵育组是先将rVAR2蛋白与效应细胞(或靶细胞)于37℃共孵育1h后再继续后续的操作。分别标注为：[CARTV2+V]+R，表示先将效应细胞与rVAR2重组蛋白共孵育，再加入靶细胞；或者[V+R]+CARTV2，表示先将rVAR2重组蛋白与靶细胞共孵育，再加入效应细胞。本实验中rVAR2重组蛋白的使用浓度为18nM。
对照组：普通T细胞(Normal T Cells，T)加靶细胞组，即T+R组，将每种细胞设定为1.25×105个/孔/250μl，不足部分用250μl RPMI-1640培养基补齐，总体积保持为500μl。
效应细胞加靶细胞组，即CARTV2+R组；
普通T细胞与rVAR2重组蛋白共孵育，再加入靶细胞组，即[T+V]+R组；
rVAR2重组蛋白与靶细胞共孵育，再加入普通T细胞组，即[V+R]+T组；
各组细胞分别于37℃培养箱共孵育24h后，收集上清，待测。
(3)细胞因子检测：
1)往ELISA板上每个孔加入100μl实验稀释液(Assay Diluent)；
2)将样品以及浓度梯度稀释的标准品加入已包被抗体的ELISA孔板中，每孔100μl，除去气泡室温孵育2h；
3)移去孔内液体，每孔加300μl漂洗缓冲液洗涤，洗涤三次，最后一次吸干漂洗缓冲液；
4)加200μl对应的检测抗体，室温孵育2h；
5)重复步骤3)；
6)加200μl底物液(A+B)，室温避光孵育30min；
7)加50μl终止液，可见溶液由蓝色变为黄色(如果颜色为绿色或者没有改变颜色，轻拍使其混匀)；
8)30min内用酶标仪于450nm处检测吸光值；
9)根据标准曲线计算样品浓度。
检测结果如图11(A)-图11(B)所示，sCART-anti-rVAR2体系与肿瘤细胞B细胞淋巴瘤细胞系Raji(ATCC#CCL86)或者NCI-H460(大细胞肺癌细胞系，ATCC#HTB177)共孵育过程中会分泌大量的IFN-γ和IL-2，提高抗肿瘤活性，这也预示着sCART-anti-rVAR2体系对不同类型的肿瘤细胞具有潜在的杀伤功能。
实施例10：Far Red法验证sCART-anti-rVAR2这种可调控性CAR-T细胞对肿瘤细胞的杀伤功能
首先采用Far Red法，即CellTraceTM Far Red Cell Proliferation Kit(Invitrogen，Cat.#C34564)试剂盒对靶细胞进行标记：(1)取适量肿瘤细胞，300×g离心3min后去掉上清；(2)将细胞用PBS重悬后，300×g离心3min，去掉上清，重复一次本步骤；(3)用PBS重悬至细胞密度为1×106个/ml；(4)每1ml体系细胞中加入1μl浓度为200μM的Far-Red，37℃水浴孵育20min；(6)孵育结束后，加入5倍体积的RF-10培养基(含有10％灭活FBS的RPMI 1640培养基)终止反应(5min)；(7)300g离心3min，去上清；(8)用T细胞扩增培养基重悬至5×105/ml。
再取CAR-T细胞经300×g离心3min，去上清；用PBS重悬后，300×g离心3min，去上清，重复一次；用T细胞扩增培养基(Gibco，Cat.#A10485)重悬至5×105个/ml；
为了验证效应细胞[CAR-T细胞：CART-anti-rVAR2(5H4 ScFv)]在rVAR2重组蛋白的介导下对靶细胞(癌细胞Raji)的杀伤能力，将实验分为以下几组，具体如表5所示，在各组中，rVAR2重组蛋白无论是先与哪种细胞共孵育，其蛋白的摩尔浓度都是相应的与其共孵育的细胞的500倍；而效应细胞与靶细胞的效靶比为2∶1。
表5Far Red法检测sCART-anti-rVAR2(5H4 ScFv)对肿瘤细胞的杀伤功能实验分组
Figure PCTCN2017113661-appb-000038
Figure PCTCN2017113661-appb-000039
37℃，5％CO2培养箱中孵育16小时后，300g离心3min收集细胞；将离心下来的细胞用含有2％灭活的FBS和0.02％NaN3的PBS buffer清洗两次后，再用该PBS buffer重悬细胞；上流式细胞仪分别检测Far-Red的信号(激发波长630nm，发射波长661nm)。
体外实验结果如图12所示，表明sCART-anti-rVAR2(5H4 ScFv)体系对Raji细胞具有杀伤作用，并且只要rVAR2重组蛋白与T细胞存在的条件下，对Raji细胞都有一定的杀伤作用，推测正常的T细胞对rVAR2重组蛋白标记的靶细胞也有一定的识别杀伤作用。
实施例11：体外实时动态监测sCART-anti-rVAR2这种可调控性CAR-T细胞对肿瘤细胞的杀伤作用
首先采用与实施例10中同样的Far-Red法对靶细胞Raji进行染色标记，再用相应的T细胞培养基重悬后将Raji细胞按5000个/100μl孔铺在96-孔培养板上，按照效靶比4∶1的比例加入制备的CART-anti-rVAR2(5H4 ScFv)细胞，由于慢病毒转染效率约为45％以上，实际效靶比约为2∶1-4∶1之间，并且按照加入效靶细胞间连接介质的rVAR2重组蛋白的先后次序分为两组：先将rVAR2重组蛋白与CART-anti-rVAR2细胞(rVAR2重组蛋白终浓度为18nM，CART-anti-rVAR2细胞为20000个/100μl)于37℃共孵育1h后，再加入到肿瘤细胞中，命名为[CART-anti-rVAR2(5H4 ScFv)+rVAR2]+Raji组；或者先将rVAR2重组蛋白(rVAR2重组蛋白终浓度为18nM)与Raji细胞于37℃共孵育1h后，再加入20000个/100μl的CART-anti-rVAR2细胞，命名为[Raji+rVAR2]+CART-anti-rVAR2(5H4 ScFv)组。另外，因为Raji是B细胞淋巴瘤细胞系，其细胞表面高表达CD19，因此，可以用靶向CD19的CAR-T细胞(CART-CD19+细胞，Porter et al.，N Engl J Med.2011，365(8)：725-33；Grupp et al.，N Engl J Med.2013；368(16)：1509-18.)作为阳性对照来衡量sCART-anti-rVAR2(5H4 ScFv)体系的功能，所以我们采用同样的细胞数目和效靶比设定了CART-CD19++Raji作为阳性对照组。
将所有实验组和对照组放置在37℃，5％CO2培养箱中培养，利用JuLI Stage全自动活细胞监控系统实时观测sCART-anti-rVAR2(5H4 ScFv)对肿瘤细胞的杀伤活性。
结果如图13所示，表明可调控性嵌合抗原受体T细胞体系sCART-anti-rVAR2(5H4 ScFv)对肿瘤细胞Raji的体外杀伤效果明显，但需要的时间相对于CART-CD19+更长，预示着其杀伤作用更温和，可能更有助于降低CAR-T细胞的毒副反应，有利于增强其安全性。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (14)

  1. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包含识别疟原虫蛋白VAR2CSA、疟原虫蛋白VAR2CSA上的蛋白标签或能够标记疟原虫蛋白VAR2CSA的化合物中的任意一种或至少两种的组合的结构域。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述识别疟原虫蛋白VAR2CSA的结构域包含具有抗VAR2CSA蛋白抗体的重链可变区和轻链可变区；
    优选地，所述VAR2CSA蛋白抗体的抗原为DBL1X、ID1、DBL2X、ID2a、ID2b、DBL3X、ID3、DBL4ε、ID4、DBL5ε、ID5或DBL6ε中的任意一种或至少两种的组合，优选为ID1、DBL2X和ID2a的组合；
    优选地，所述DBL1X包含具有如SEQ ID NO.1所示的氨基酸序列；
    优选地，所述ID1包含具有如SEQ ID NO.2所示的氨基酸序列；
    优选地，所述DBL2X包含具有如SEQ ID NO.3所示的氨基酸序列；
    优选地，所述ID2a包含具有如SEQ ID NO.4所示的氨基酸序列；
    优选地，所述ID2b包含具有如SEQ ID NO.5所示的氨基酸序列；
    优选地，所述DBL3X包含具有如SEQ ID NO.6所示的氨基酸序列；
    优选地，所述ID3包含具有如SEQ ID NO.7所示的氨基酸序列；
    优选地，所述DBL4ε包含具有如SEQ ID NO.8所示的氨基酸序列；
    优选地，所述ID4包含具有如SEQ ID NO.9所示的氨基酸序列；
    优选地，所述DBL5ε包含具有如SEQ ID NO.10所示的氨基酸序列；
    优选地，所述ID5包含具有如SEQ ID NO.11所示的氨基酸序列；
    优选地，所述DBL6ε包含具有如SEQ ID NO.12所示的氨基酸序列。
  3. 根据权利要求1或2所述的嵌合抗原受体，所述抗VAR2CSA蛋白抗体为通过15-20个氨基酸的短肽连接而成的单链抗体；
    优选地，所述疟原虫蛋白VAR2CSA的结合域包含具有抗VAR2CSA蛋白的单链抗体的重链可变区和轻链可变区的CDR序列；
    优选地，所述具有单链抗体的重链可变区的CDR具有以下序列：CDR1如SEQ ID NO.13所示的氨基酸序列；CDR2如SEQ ID NO.14所示的氨基酸序列；CDR3如SEQ ID NO.15所示的氨基酸序列；
    优选地，所述具有单链抗体的轻链可变区的CDR具有以下序列：CDR1如SEQ ID NO.16所示的氨基酸序列；CDR2如SEQ ID NO.17所示的氨基酸序列；CDR3如SEQ ID NO.18所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的嵌合抗原受体，其特征在于，所述VAR2CSA的结合域的单链抗体的重链可变区包含具有如SEQ ID NO.19所示的氨基酸序列或与其具有至少70％以上氨基酸序列同一性，优选为90％以上氨基酸同一性的变体；
    优选地，所述VAR2CSA的结合域的单链抗体的轻链可变区包含具有如SEQ ID NO.20所示的氨基酸序列或与其具有至少70％以上氨基酸序列同一性，优选为90％以上氨基酸同一性的变体；
    优选地，所述VAR2CSA的结合域的重链可变区包含具有如SEQ ID NO.21所示的核苷酸序列或与其具有至少60％以上核苷酸序列同一性，优选为80％以上核苷酸同一性的变体；
    优选地，所述VAR2CSA的结合域的轻链可变区包含具有如SEQ ID NO.22所示的核苷酸序列或与其具有至少60％以上核苷酸序列同一性，优选为80％以上核苷酸同一性的变体；
    优选地，所述疟原虫蛋白VAR2CSA上的蛋白标签为PNE-tag、人myc-tag CaptureSelect C-tag、FLAG-tag、3×FLAG-tag、Strep-tag、6×His-tag、V5 tag、S-tag、HA-tag、VSV-G-tag、GST-tag、HaloTag、XTEN-tag或huEGFRt-tag蛋白标签中的任意一个或至少两个的组合。
  5. 根据权利要求1-4中任一项所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括铰链区、跨膜区和胞内共刺激信号区中的任意一种或至少两种的组合；
    优选地，所述铰链区为人CD8α铰链区；
    优选地，所述跨膜区为人CD28跨膜区；
    优选地，所述胞内信号区为人CD27胞内信号区、人CD134胞内信号区、人CD28胞内信号区或人4-1BB胞内信号区中的任意一种或至少两种的组合；
    优选地，所述嵌合抗原受体的氨基末端含有一个CD8α信号肽；
    优选地，所述嵌合抗原受体的羧基末端还包含一个人CD3ζ胞内信号区。
  6. 编码如权利要求1-5中任一项所述的嵌合抗原受体的核酸或与其具有至少60％同一性，优选至少80％同一性的核酸。
  7. 一种嵌合抗原受体表达细胞，其特征在于，所述表达细胞包含如权利要求6所述的核酸；
    优选地，所述细胞为免疫效应细胞，进一步优选为T细胞、B细胞、NK细胞、NKT细胞、树突状细胞或巨噬细胞中的任意一种或至少两种的组合。
  8. 一种重组载体，其特征在于，所述重组载体其包含有权利要求6所述的核酸；
    优选地，所述载体为重组克隆载体、重组真核表达质粒或重组慢病毒载体中的任意一种或至少两种的组合，优选为重组慢病毒载体；
    优选地，所述重组克隆载体包括pUC18、pUC19、pMD19-T、pGM-T载体、pUC57、pMAX或pDC315中的任意一种或至少两种的组合；
    优选地，所述真核表达质粒包括pCDNA3系列载体、pCDNA4系列载体、pCDNA5系列载体、pCDNA6系列载体、pCI-neo系列载体、pEGFP系列载体、pSPT系列载体、pFLAG-CMV系列载体、pRL系列载体、pUC57载体、pMAX或pDC315中的任意一种或至少两种的组合；
    优选地，所述重组慢病毒载体包括重组腺病毒载体、重组腺相关病毒载体、重组逆转录病毒载体、重组单纯疱疹病毒载体或重组痘苗病毒载体中的任意一种或至少两种的组合。
  9. 一种重组病毒，其包含如权利要求8所述的重组载体与包装辅助质粒共转染哺乳动物细胞得到的重组病毒。
  10. 一种嵌合抗原受体T细胞，其通过将如权利要求8所述的重组病毒转染到T细胞中表达。
  11. 如权利1-5中任一项所述的嵌合抗原受体、如权利要求6所述的核酸、如权利要求8所述的重组载体或如权利要求9所述的重组病毒用于转染和扩增CAR-T细胞。
  12. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-5中任一项所述的嵌合抗原受体、如权利要求6所述的核酸、如权利要求7所述的嵌合抗原受体表达细胞或如权利要求8所述的表达载体，以及任选的药学上可接受的辅料。
  13. 如权利要求1-5中任一项所述的嵌合抗原受体、如权利要求6所述的核酸、如权利要求7所述的嵌合抗原受体表达细胞或如权利要求8所述的表达载体在制备治疗和/或预防自身免疫性疾病或肿瘤的药物中的用途；
    优选地，所述肿瘤为实体瘤和/或血液瘤。
  14. 一种治疗患有自身免疫性疾病和/或与肿瘤抗原的表达相关的疾病的受试者的方法，其包括对所述受试者施用有效量的包含权利要求12所述的药物组合物的药物；
    优选地，所述肿瘤为实体瘤和/或血液瘤。
PCT/CN2017/113661 2017-11-29 2017-11-29 一种嵌合抗原受体及其应用 Ceased WO2019104562A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US17/051,747 US12221475B2 (en) 2017-11-29 2017-11-29 Chimeric antigen receptor and application thereof
GB2015416.7A GB2585607B (en) 2017-11-29 2017-11-29 Chimeric antigen receptor and application thereof
EP17933216.8A EP3744738A4 (en) 2017-11-29 2017-11-29 CHIMERA ANTIGEN RECEPTOR AND USES THEREOF
AU2017441551A AU2017441551B2 (en) 2017-11-29 2017-11-29 Chimeric antigen receptor and application thereof
CA3098330A CA3098330C (en) 2017-11-29 2017-11-29 Chimeric antigen receptor and application thereof
CN201780001820.4A CN110325551B (zh) 2017-11-29 2017-11-29 一种嵌合抗原受体及其应用
PCT/CN2017/113661 WO2019104562A1 (zh) 2017-11-29 2017-11-29 一种嵌合抗原受体及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/113661 WO2019104562A1 (zh) 2017-11-29 2017-11-29 一种嵌合抗原受体及其应用

Publications (1)

Publication Number Publication Date
WO2019104562A1 true WO2019104562A1 (zh) 2019-06-06

Family

ID=66664686

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/113661 Ceased WO2019104562A1 (zh) 2017-11-29 2017-11-29 一种嵌合抗原受体及其应用

Country Status (7)

Country Link
US (1) US12221475B2 (zh)
EP (1) EP3744738A4 (zh)
CN (1) CN110325551B (zh)
AU (1) AU2017441551B2 (zh)
CA (1) CA3098330C (zh)
GB (1) GB2585607B (zh)
WO (1) WO2019104562A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114075298B (zh) * 2022-01-07 2022-04-29 广州中科蓝华生物科技有限公司 一种索烃化的var2csa重组蛋白及其制备方法和应用
CN116874578B (zh) * 2022-12-09 2024-07-19 中山大学肿瘤防治中心(中山大学附属肿瘤医院中山大学肿瘤研究所) 一种var2csa重组蛋白及其制备方法和应用
WO2025222126A1 (en) * 2024-04-19 2025-10-23 Vanderbilt University Var2csa antibodies and methods of use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012082841A2 (en) * 2010-12-14 2012-06-21 University Of Maryland, Baltimore Universal anti-tag chimeric antigen receptor-expressing t cells and methods of treating cancer
WO2013117705A1 (en) * 2012-02-09 2013-08-15 Var2 Pharmaceuticals Aps Targeting of chondroitin sulfate glycans
CN105753991A (zh) 2015-11-26 2016-07-13 广州中科蓝华生物科技有限公司 一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用
WO2016135291A1 (en) * 2015-02-26 2016-09-01 Var2 Pharmaceuticals Aps Immunotherapeutic targeting of placental-like chondroitin sulfate using chimeric antigen receptors (cars) and immunotherapeutic targeting of cancer using cars with split-protein binding systems
WO2017179015A1 (en) * 2016-04-15 2017-10-19 Glaxosmithkline Intellectual Property Development Limited Compositions for the treatment of cancer

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013051718A1 (ja) * 2011-10-07 2013-04-11 国立大学法人三重大学 キメラ抗原受容体
CN102775500A (zh) 2012-08-03 2012-11-14 郑骏年 嵌合抗原受体iRGD-scFv(G250)-CD8-CD28-CD137-CD3ζ及其用途
SG11201605260VA (en) * 2013-12-27 2016-07-28 Zymeworks Inc Var2csa-drug conjugates
CN106977607A (zh) * 2016-01-18 2017-07-25 广州中科蓝华生物科技有限公司 一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012082841A2 (en) * 2010-12-14 2012-06-21 University Of Maryland, Baltimore Universal anti-tag chimeric antigen receptor-expressing t cells and methods of treating cancer
WO2013117705A1 (en) * 2012-02-09 2013-08-15 Var2 Pharmaceuticals Aps Targeting of chondroitin sulfate glycans
WO2016135291A1 (en) * 2015-02-26 2016-09-01 Var2 Pharmaceuticals Aps Immunotherapeutic targeting of placental-like chondroitin sulfate using chimeric antigen receptors (cars) and immunotherapeutic targeting of cancer using cars with split-protein binding systems
CN105753991A (zh) 2015-11-26 2016-07-13 广州中科蓝华生物科技有限公司 一种抗胎盘样硫酸软骨素的嵌合抗原受体及其应用
WO2017179015A1 (en) * 2016-04-15 2017-10-19 Glaxosmithkline Intellectual Property Development Limited Compositions for the treatment of cancer

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
"GBD 2015 Mortality and Causes of Death Collaborators", LANCET, vol. 388, no. 10053, 2016, pages 1459 - 1544
AYRES PEREIRA ET AL., PLOS PATHOG, vol. 12, no. 8, 2016, pages e1005831
BOUSEMA ET AL., NAT REV MICROBIOL, vol. 12, no. 12, 2014, pages 833 - 146
BRENTJENS ET AL., BLOOD, vol. 118, no. 18, 2011, pages 1255 - 63
CAO, Y. ET AL.: "Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer", ANGEWANDTE CHEMIE -INTERNATIONAL EDITION, vol. 55, no. 26, 20 June 2016 (2016-06-20), XP055519426, ISSN: 1521-3773, DOI: doi:10.1002/anie.201601902 *
CHMIELEWSKI ET AL., IMMUNOL REV, vol. 257, no. 1, 2014, pages 83 - 90
DAVILA ET AL., PLOS ONE, vol. 8, no. 4, 2013, pages e61338
FESNAK ET AL., NAT REV CANCER, vol. 16, no. 9, 2016, pages 566 - 81
GALETTO ET AL., BLOOD, vol. 124, no. 21, 2014, pages 1277 - 1287
GARGETT AND BROWN, FRONT.PHARMACOL, vol. 5, no. 235, 2014, pages 1 - 7
GARGETTBROWN, FRONT.PHARMACOL., vol. 5, no. 235, 2014, pages 1 - 7
GILL ET AL., BLOOD REV, vol. 30, no. 3, 2016, pages 157 - 67
GRUPP ET AL., ASH ANNU MEET ABSTR, vol. 120, no. 21, 2012, pages 2604
GRUPP ET AL., N ENGL J MED, vol. 368, no. 16, 2013, pages 1509 - 18
HACEIN-BEY-ABINA ET AL., N ENGL J MED, vol. 348, no. 3, 2003, pages 255 - 6
HACEIN-BEY-ABINA ET AL., N ENGL J MED, vol. 363, no. 4, 2010, pages 355 - 64
JENSEN ET AL., BIOL BLOOD MARROW TRANSPLANT, vol. 16, no. 9, 2010, pages 1245 - 56
KIEBACK ET AL., PROC NATL ACAD SCI USA, vol. 105, no. 2, 2008, pages 623 - 628
KLEBANOFF ET AL., NAT MED, vol. 22, no. 1, 2016, pages 26 - 36
KOCHENDERFER ET AL., BLOOD, vol. 122, no. 25, 2013, pages 4129 - 39
LEDFORD, NATURE, vol. 538, no. 7624, 2016, pages 150 - 151
LIM, JUNE, CELL, vol. 168, no. 4, 2017, pages 724 - 740
MAUDE ET AL., N ENGL J MED, vol. 371, no. 16, 2014, pages 1507 - 17
MESIANO ET AL., EXPERT OPIN BIOL THER, vol. 12, no. 6, 2012, pages 673 - 84
MORSUT ET AL., CELL, vol. 164, no. 4, 2016, pages 780 - 91
NEWICK ET AL., ANNU REV MED, vol. 68, 2017, pages 139 - 152
PASTERNAKDZIKOWSKI, INT J BIOCHEM CELL BIOL, vol. 41, no. 7, 2009, pages 1463 - 6
PASZKIEWICZ ET AL., J CLIN INVEST, vol. 126, no. 11, 2016, pages 4262 - 4272
PAUKEN ET AL., SCIENCE, vol. 354, no. 6316, 2016, pages 1160 - 1165
PORTER ET AL., N ENGL J MED, vol. 365, no. 8, 2011, pages 725 - 33
RODGERS ET AL., PROC NATL ACAD SCI US A, vol. 113, no. 4, 2016, pages E459 - 68
ROSENBERG ET AL., NAT MED, vol. 10, no. 9, 2004, pages 909 - 15
ROSENBERGRESTIFO, SCIENCE, vol. 350, no. 6258, 2015, pages aab4077 - 8
SALANTI ET AL., CANCER CELL, vol. 28, no. 4, 2015, pages 500 - 14
SALANTI ET AL., J EXP MED, vol. 200, no. 9, 2004, pages 1197 - 203
SALANTI ET AL., MOLMICROBIO, vol. 49, no. 1, 2003, pages 179 - 91
See also references of EP3744738A4
SEILER ET AL., EUR UROL, vol. 72, no. 1, 2017, pages 142 - 150
TEY, CLIN TRANSL IMMUNOLOGY, vol. 3, no. 6, 2014, pages e17
VOGLER ET AL., MOL THER, vol. 18, no. 7, 2010, pages 1330 - 1338

Also Published As

Publication number Publication date
AU2017441551B2 (en) 2024-05-16
CA3098330A1 (en) 2019-06-06
EP3744738A1 (en) 2020-12-02
EP3744738A4 (en) 2021-12-15
AU2017441551A1 (en) 2021-05-13
CN110325551A (zh) 2019-10-11
CN110325551B (zh) 2020-11-20
US12221475B2 (en) 2025-02-11
CA3098330C (en) 2024-01-02
US20210221880A1 (en) 2021-07-22
GB202015416D0 (en) 2020-11-11
GB2585607A (en) 2021-01-13
GB2585607B (en) 2022-09-07

Similar Documents

Publication Publication Date Title
US11692034B2 (en) CD47-CAR-T cells
CN109415409B (zh) Flag标记的cd19-car-t细胞
CN108373504B (zh) Cd24特异性抗体和抗cd24-car-t细胞
JP7091447B2 (ja) 新型なｓｃＦｖアミノ酸配列、それを含むキメラ抗原受容体、およびその使用
CN113784986B (zh) 间皮素特异性嵌合抗原受体及表达它的t细胞
WO2017219934A1 (zh) 一种高效稳定表达抗体的杀伤性细胞及其用途
WO2017219936A1 (zh) 一种高效稳定表达激活型抗体的car-t细胞及其用途
WO2017219937A1 (zh) 一种高效稳定表达抑制性抗体的car-t细胞及其用途
US20240293457A1 (en) Claudin18.2 chimeric antigen receptor and use thereof
US20220323598A1 (en) Anti-ror1 monoclonal antibody, functional fragment thereof, gene, drug delivery composition, and pharmaceutical composition
US12448453B2 (en) Fully human anti-human CD22 chimeric antigen receptor and application thereof
CN110325551B (zh) 一种嵌合抗原受体及其应用
US20210380688A1 (en) Antibodies for treating malignant tumors and uses thereof
US20220389076A1 (en) Modified immune cell and use thereof
CA3245280A1 (en) CHIMERIC ANTIGEN RECEPTOR WITH NANOBODY ANTI-MESOTHELIN AND ITS USE
US20250288612A1 (en) Chimeric antigen receptor against programmed death ligand 1 (pd-l1) and application thereof
WO2025189420A1 (en) Chimeric antigen receptor against programmed deathligand 1 (pd-l1) and application thereof
US20250288674A1 (en) Chimeric antigen receptor against programmed death ligand 1 (pd-l1) and application thereof
Renner et al. Enhanced solid tumor cell targeting by a neoepitope-encoding oncolytic measles virus combined with CAR therapy
WO2025252993A1 (en) Chimeric antigen receptor targeting glioma-associated antigens
HK40079524B (zh) 全人源抗人cd22的嵌合抗原受体及其应用
HK40079524A (zh) 全人源抗人cd22的嵌合抗原受体及其应用
CN121591905A (zh) 一组靶向gpc3不同表位的单克隆抗体及其在肝癌免疫治疗中的应用
CN116143924A (zh) 靶向人Claudin18.2蛋白的人源化单克隆抗体及其应用
CN118234751A (zh) 免疫反应性分子及其用途

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: 17933216

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 202015416

Country of ref document: GB

Kind code of ref document: A

Free format text: PCT FILING DATE = 20171129

WWE Wipo information: entry into national phase

Ref document number: 2015416.7

Country of ref document: GB

ENP Entry into the national phase

Ref document number: 2017933216

Country of ref document: EP

Effective date: 20200629

ENP Entry into the national phase

Ref document number: 3098330

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2021515256

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2017441551

Country of ref document: AU

Date of ref document: 20171129

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: JP

WWG Wipo information: grant in national office

Ref document number: 2015416.7

Country of ref document: GB