WO2020083282A1 - Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗 - Google Patents

Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗 Download PDF

Info

Publication number
WO2020083282A1
WO2020083282A1 PCT/CN2019/112530 CN2019112530W WO2020083282A1 WO 2020083282 A1 WO2020083282 A1 WO 2020083282A1 CN 2019112530 W CN2019112530 W CN 2019112530W WO 2020083282 A1 WO2020083282 A1 WO 2020083282A1
Authority
WO
WIPO (PCT)
Prior art keywords
region
nucleic acid
lymphocytes
acid molecule
lentivirus
Prior art date
Application number
PCT/CN2019/112530
Other languages
English (en)
French (fr)
Inventor
黄雪芬
陈周世
陈思毅
Original Assignee
艾生命序公司
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 艾生命序公司 filed Critical 艾生命序公司
Publication of WO2020083282A1 publication Critical patent/WO2020083282A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • 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
    • 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
    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N5/10—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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • 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
    • C07K2319/00—Fusion polypeptide
    • 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
    • C07K2319/33—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
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the field of biomedicine.
  • the present invention relates to T lymphocytes, lentiviruses, transgenic lymphocytes, constructs, therapeutic compositions for treating cancer, and methods for improving lymphocyte activity and therapeutic safety.
  • MSLN Mesothelin
  • Mesothelin is a differentiation antigen, and its expression in normal human tissues is limited to mesothelial cells in the pleura, pericardium, and peritoneum lining.
  • mesothelin is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancer and about 50% of lung adenocarcinoma and other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer , Esophageal cancer, breast cancer.
  • the mesothelin gene encodes a precursor protein of 71KDa, which is then processed into a shedding fragment of 31KDa and a protein fragment of 40KDa.
  • the shedding fragment of 31KDa is called megakaryocyte promoting factor (MPF), and the protein fragment of 40KDa is Called mesothelin, mesothelin is fixed on the cell membrane by the anchoring effect of glycosyl-phosphatidylinositol (GPI).
  • MPF megakaryocyte promoting factor
  • GPI glycosyl-phosphatidylinositol
  • mesothelioma is divided into pleural mesothelioma and peritoneal mesothelioma.
  • Pleural mesothelioma is a primary tumor of the pleura, which has a limited type (mostly benign) and a diffuse type (all malignant). Among them, diffuse malignant mesothelioma is one of the worst tumors in the prognosis of the chest.
  • Peritoneal mesothelioma refers to tumors that originate in peritoneal mesothelial cells. The clinical manifestations are not characteristic. Common symptoms and signs include: abdominal pain, ascites, abdominal distension, and abdominal mass.
  • the treatment methods include palliative treatment, surgical treatment, chemotherapy and radiotherapy. It is generally believed that for stage I patients with relatively limited tumors, radical pleural pneumonectomy is advocated. For patients with stage II, III, and IV, radical surgery is no longer meaningful, only palliative surgery is performed. In fact, most patients are already in stage II or above when the disease is clearly diagnosed. The rapid growth of pleural effusion often leads to severe breathing difficulties. Palliative surgery can only temporarily improve the quality of life of these advanced patients, but it cannot be cured.
  • mesothelin is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancer and about 50% of lung adenocarcinoma and other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer, esophagus Cancer, breast cancer. Therefore, mesothelin represents a very attractive target in the field of tumor immunotherapy.
  • activated cytotoxic T lymphocytes can express a negatively regulated regulatory structure, that is, express immune checkpoint molecules on the surface or inside the cell, such as programmed cell death 1 Receptor (PD-1) can be expressed on activated CTLs, which interacts with programmed death ligand 1 (PD-L1) expressed on tumor cells and can inhibit anti-tumor T cell responses.
  • PD-1 programmed cell death 1 Receptor
  • many tumor cells, including tumor cells with high expression of mesothelin highly express PD-L1.
  • the combination of PD-L1 and PD-1 leads to the downregulation of the proliferative response of CTLs, the reduction of cytokine secretion and the inability of T cells or Apoptosis. In turn, the effect of tumor immunotherapy is greatly reduced.
  • the inventors proposed a transgenic lymphocyte that can express and secrete anti-PD-L1 antibodies, express a chimeric antigen receptor that recognizes MSLN, and non-functional EGFR.
  • PD- Anti-mesothelin CAR-T cells secreted by L1 antibody.
  • the inventors found in experiments that the anti-mesothelin CAR-T cells proposed by the present invention can suppress the immune escape of tumor cells, have a specific killing ability against tumors expressing high mesothelin, and have a normal MSLN expression level of mesothelium. The cells are not killed, and the anti-mesothelin CAR-T cells proposed in this application can express non-functional EGFR, which significantly increases the safety of the CAR-T cell therapy.
  • the present invention proposes a T lymphocyte.
  • the T lymphocytes co-express anti-PD-L1 fusion antibody, non-functional EGFR, and chimeric antigen receptor, wherein the chimeric antigen receptor includes: an extracellular region, the extracellular The region includes a heavy chain variable region and a light chain variable region of a single chain antibody, the single chain antibody specifically recognizes tumor antigen mesothelin; a transmembrane region, the transmembrane region is connected to the extracellular region, so The transmembrane region includes a transmembrane segment of CD8 and is embedded in the cell membrane of the T lymphocyte; an intracellular region, the intracellular region is connected to the transmembrane region, and the intracellular region includes 4-1BB Intracellular segment and CD3 ⁇ chain; the anti-PD-L1 fusion antibody includes: PD-L1 single chain antibody, IgG1 hinge region and IgG1 Fc region, the
  • the T lymphocytes according to the embodiments of the present invention can secrete anti-PD-L1 antibodies, and have the property of resisting tumor cell-mediated immunosuppression.
  • the proliferation ability in vitro, the proliferation and survival ability in tumor patients are significantly improved.
  • the cell's killing ability is significantly enhanced, especially for tumors with high expression of MSLN and PD-L1 molecules, and it has a significant targeted killing effect and high safety.
  • the present invention proposes a lentivirus.
  • the lentivirus carries the following nucleic acid molecules: (a) a nucleic acid molecule encoding an anti-PD-L1 fusion antibody, the anti-PD-L1 antibody includes: a PD-L1 single chain antibody, an IgG1 hinge region, and IgG1 Fc region, the PD-L1 single chain antibody is connected to the IgG1 Fc region through an IgG1 hinge region, the IgG Fc region has T250Q and M248L mutations; (b) a nucleic acid molecule encoding a chimeric antigen receptor, the The extracellular region of the chimeric antigen receptor recognizes the tumor antigen mesothelin; and (c) a nucleic acid molecule encoding non-functional EGFR.
  • the transgenic lymphocytes obtained by introducing the lentivirus according to the embodiment of the present invention into lymphocytes can express and secrete anti-PD-L1 antibodies, have the characteristics of resisting tumor cell-mediated immunosuppression, have the ability to proliferate in vitro, and are in tumor patients
  • the body's proliferation and survivability are significantly enhanced, and its ability to kill tumor cells is significantly enhanced, especially for tumors that highly express MSLN and PD-L1 molecules, and have a significant targeted killing effect and high safety.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 9.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 10.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 11.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 12.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 13.
  • the present invention proposes a lentivirus.
  • the lentivirus carries a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 14.
  • the transgenic lymphocytes obtained by introducing the lentivirus according to the embodiment of the present invention into lymphocytes can express and secrete anti-PD-L1 antibodies, have the characteristics of resisting tumor cell-mediated immunosuppression, have the ability to proliferate in vitro, and are in tumor patients
  • the proliferation and survivability of the body are significantly enhanced, and the killing capacity of the tumor cells is significantly enhanced, especially for tumors that highly express MSLN and PD-L1 molecules, and have a significant targeted killing effect, and higher safety.
  • the present invention provides a transgenic lymphocyte.
  • the transgenic lymphocytes co-express anti-PD-L1 fusion antibody, non-functional EGFR, and chimeric antigen receptor, the chimeric antigen receptor recognizes tumor antigen mesothelin, wherein Antigen receptors include: extracellular region; transmembrane region, the transmembrane region is connected to the extracellular region, and embedded in the cell membrane of the transgenic lymphocyte; intracellular region, the intracellular region and all The transmembrane region is connected, and the intracellular region includes an intracellular segment of an immunocostimulatory molecule; the anti-PD-L1 fusion antibody includes: a PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1 Fc region, the PD- The L1 single chain antibody is connected to the IgG1 Fc region through the IgG1 hinge region, which has T250Q and M248L mutations.
  • the transgenic lymphocytes' proliferation ability in vitro, proliferation and survivability in tumor patients and specific killing ability in tumor patients are greatly improved, especially for high expression of MSLN and PD-L1 molecules
  • the tumor has a significant directional killing effect and high safety.
  • the invention proposes a construct.
  • the construct includes: a first nucleic acid molecule that encodes an anti-PD-L1 fusion antibody, and the anti-PD-L1 fusion antibody includes: PD-L1 single chain antibody, IgG1 Hinge region and IgG1 Fc region, the PD-L1 single-chain antibody is connected to the IgG1 Fc region through the IgG1 hinge region, the IgG Fc region has T250Q and M248L mutations; a second nucleic acid molecule, the second nucleic acid molecule encodes A chimeric antigen receptor that recognizes the tumor antigen mesothelin; and a third nucleic acid molecule that encodes a non-functional EGFR, wherein the anti-PD-L1 fusion antibody, the The chimeric antigen receptor and the non-functional EGFR are as described above.
  • the construct according to the embodiment of the present invention After the construct according to the embodiment of the present invention is successfully introduced into the lymphocytes of the embodiment of the present invention, it can effectively express and secrete anti-PD-L1 antibody, express non-functional EGFR, and express a chimeric antigen receptor that specifically recognizes MSLN, thereby obtaining The targeted killing effect of lymphocytes on tumor cells, especially tumor cells with high expression of MSLN and PD-L1 molecules, is more significant and safe.
  • the present invention proposes a therapeutic composition for treating cancer.
  • the therapeutic composition includes: the above construct, lentivirus, T lymphocytes or transgenic lymphocytes.
  • the composition of any of the above therapeutic compositions can achieve the expression and secretion of anti-PD-L1 antibodies of transgenic lymphocytes or T lymphocytes and non-functional EGFR and chimeric antigen receptors that specifically recognize MSLN in transgenic lymphocytes or T lymphocytes Efficient expression in cells, the resulting transgenic lymphocytes or T lymphocytes have significant resistance to tumor cell-mediated immunosuppression, proliferation in vitro and in vivo of tumor patients and survival ability of tumor patients are greatly improved, transgenic lymphocytes or T lymphocytes
  • the targeted killing effect on tumor cells is stronger.
  • the targeted killing effect of the therapeutic composition for treating cancer according to the embodiments of the present invention on tumor cells is significantly enhanced, especially on tumor cells that highly express MSLN and PD-L1 molecules, and the safety is further improved.
  • the present invention provides a method for improving the activity of lymphocytes and the safety of treatment.
  • the lymphocytes carry a chimeric antigen receptor that recognizes the tumor antigen mesothelin
  • the method includes: causing the lymphocytes to express an anti-PD-L1 fusion antibody, and causing the lymphocytes to express non-functional EGFR.
  • the anti-PD-L1 fusion antibody, the lymphocyte, the chimeric antigen receptor, and the non-functional EGFR are as defined above, and the activity of the lymphocytes includes the ability of the lymphocytes to proliferate in vitro, in tumors At least one of the proliferation and viability of the patient and the targeted killing ability of the lymphocytes in the tumor patient.
  • lymphocytes express and secrete anti-PD-L1 antibodies, lymphocyte activation, proliferative response upregulation, increased cytokine secretion, enhanced anti-apoptotic ability, lymphocyte expansion in vitro, and proliferation in tumor patients And the survival ability of tumor patients is greatly improved.
  • Non-functional EGFR lacks the N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of the wild-type EGFR receptor and a complete sequence that binds to anti-EGFR antibodies.
  • Non-functional EGFR can serve as lymph Cell suicide marker.
  • the present invention is an example of the lymphocytes used in the treatment of tumor cells with high expression of MSLN, if the patient has a serious adverse reaction, the lymphocytes of the example of the present invention can be cleared by anti-EGFR antibodies, which can improve the example of the present invention The safety of lymphocytes in the treatment of tumor patients with high expression of MSLN and PD-L1 molecules.
  • the invention proposes a method of treating cancer.
  • the method includes administering to the patient the aforementioned construct, the aforementioned lentivirus, the aforementioned T lymphocyte or the aforementioned transgenic lymphocyte.
  • the method for treating cancer according to the embodiments of the present invention has a strong targeted killing effect on tumor cells, and particularly has good therapeutic effect and high safety on tumor patients that highly express MSLN and PD-L1 molecules.
  • FIG. 1 is a schematic structural view of a lentiviral vector co-expressing anti-PD-L1 scFv-Fc fusion antibody, anti-MSLN antigen-specific chimeric antigen receptor and non-functional EGFR according to an embodiment of the present invention
  • Fig. 2 is an embodiment of the present invention co-expressing anti-PD-L1 scFv-Fc fusion antibody, anti-MSLN antigen-specific chimeric antigen receptor and non-functional EGFR lymphocytes secrete anti-PD-L1 scFv-Fc fusion antibody in vitro And a graph showing the results of inhibiting the interaction between PD-1 and PD-L1;
  • FIG. 3 is a graph showing the ability of anti-PD-L1 scFv-Fc fusion antibody, anti-MSLN antigen-specific chimeric antigen receptor and non-functional EGFR lymphocytes to kill tumor cells in vitro according to an embodiment of the present invention
  • FIG. 4 is a graph showing the results of co-expression of anti-PD-L1 scFv-Fc fusion antibody, anti-MSLN antigen-specific chimeric antigen receptor, and non-functional EGFR lymphocytes to kill tumor cells in accordance with an embodiment of the present invention.
  • Fig. 5 shows the anti-PD-L1 secreted by co-expressing anti-PD-L1 scFv-Fc fusion antibody, anti-MSLN antigen-specific chimeric antigen receptor and non-functional EGFR lymphocytes injected into mouse serum according to an embodiment of the present invention ScFv-Fc fusion protein antibody inhibits the interaction between PD-1 and PD-L1.
  • the “anti-PD-L1 fusion antibody” in this application refers to a fusion protein of a PD-L1 single chain antibody, an IgG1 hinge region, and an IgG1 Fc region, where the IgG1 Fc region has T250Q and M248L Mutation, the fusion antibody has anti-PD-L1 activity and can specifically bind to PD-L1.
  • the present invention proposes a transgenic lymphocyte.
  • the transgenic lymphocytes co-express anti-PD-L1 fusion antibody, non-functional EGFR, and chimeric antigen receptor, the chimeric antigen receptor recognizes tumor antigen mesothelin, wherein Antigen receptors include: extracellular region; transmembrane region, which includes the transmembrane segment of CD8, the transmembrane region is connected to the extracellular region, and is embedded in the cell membrane of the transgenic lymphocytes ; Intracellular region, the intracellular region is connected to the transmembrane region, and the intracellular region includes an intracellular segment of immune costimulatory molecules; the anti-PD-L1 fusion antibody includes: PD-L1 single chain antibody, IgG1 hinge region and IgG1 Fc region, the PD-L1 single chain antibody is connected to the IgG1 Fc region through the IgG1 hinge region, and the IgG1 Fc region has T
  • the anti-PD-L1 fusion antibody is a secreted antibody.
  • the anti-PD-L1 fusion antibody is secreted out of the cell and specifically binds to PD-L1 on the cell surface, thereby effectively suppressing the immune escape mechanism mediated by PD-1: PD-L1.
  • Human antibodies include two immunoglobulin light chains and two immunoglobulin heavy chains.
  • the heavy and light chains are connected by a covalent or non-covalent bond, resulting in the formation of three independent protein regions-two Fab regions And an Fc region.
  • the Fab region and the Fc region are connected by an elastic link as a hinge region.
  • the Fab region in an antibody has the same structure.
  • the Fab region has a specific antigen-binding site, and the Fc region interacts with the ligand. This site can induce effector functions, including cellular Fc receptors and C1q complement Ingredients.
  • the physiological activity of a therapeutic antibody is mediated by two independent natural immunoglobulin mechanisms: the efficacy of the therapeutic antibody is caused by its specificity and bivalent binding to the target antigen (eg, blocking or neutralizing the target antigen or induction Apoptosis) can also be caused by effector functions activated by immune complexes formed by Fc and effector ligands (Fc receptors and Clq components).
  • target antigen eg, blocking or neutralizing the target antigen or induction Apoptosis
  • effector functions activated by immune complexes formed by Fc and effector ligands Fc receptors and Clq components
  • a single chain antibody is a genetically engineered antibody in which the VH and VL domains are connected to a flexible polypeptide linker. Compared with the Fab region of whole antibodies, single-chain antibodies show better tissue penetration pharmacokinetics and have complete antigen-binding specificity because the antigen-binding surface is not changed. However, the half-life of single-chain antibodies in the blood is short, because there is no Fc molecule fragment in the single-chain antibody, lacking no Fc fragment effect function.
  • the PD-L1 single chain antibody in the anti-PD-L1 fusion antibody according to the embodiment of the present invention is connected to the IgG1 Fc region through the IgG1 hinge region, therefore, the half-life of the PD-L1 fusion antibody in the blood is compared to that of The PD-L1 single chain antibody is significantly extended.
  • the Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).
  • the Fc fragment is used to link with the single-chain antibody to increase the half-life of the single-chain antibody in the blood, and has an Fc effect function.
  • Five types of immunoglobulins IgM, IgD and IgE, IgG, IgA, IgG subclasses
  • IgG subclasses IgG1, IgG2, IgG3, IgG4
  • Human serum has the highest IgG content.
  • the four subclasses IgG1, IgG2, IgG3 and IgG4 are highly conserved and differ in their constant regions, especially in the CH2 domain on the hinge. These regions are involved in binding IgG Fc receptor (Fc ⁇ R) and complement C1q. As a result, different subclasses have different effector functions, triggering FC ⁇ r-expressing cells, thereby causing phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. IgG1 and IgG3 can effectively trigger this classical pathway complement, but IgG2 and IgG4 are not so effective.
  • PD-L1 immune checkpoint molecules are often highly expressed in tumor cells.
  • a single-chain antibody expressing PD-L1 against tumor cells is fused with IgG1 Fc, the half-life of this single-chain antibody is extended, and the binding ability of this single-chain antibody to PD-L1 is enhanced, thereby blocking PD-1 Interact with PD-L1 to enhance CTL to kill tumor cells.
  • IgG1Fc can effectively trigger effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) to further kill tumor cells.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • FcRn nascent Fc receptor
  • T lymphocytes or transgenic lymphocytes in tumor patients in vivo and in vitro, and the ability to kill specific tumor cells in tumor patients are significantly enhanced, especially for the high expression of MSLN and PD-L1 molecules
  • the specific killing effect of the tumor cells is greatly improved, and the safety is also significantly improved.
  • the non-functional EGFR of the embodiments of the present invention lacks the N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of the wild-type EGFR receptor and the complete anti-EGFR EGFR antibody binding sequence, non-functional EGFR can be used as a suicide marker for lymphocytes.
  • Non-functional EGFR expressing lymphocytes can be eliminated in vivo by anti-EGFR antibodies. Therefore, the T lymphocytes of the embodiments of the present invention express non-functional EGFR.
  • the transgenic lymphocytes can be cleared by anti-EGFR antibodies, which can be further improved.
  • the PD-L1 single chain antibody has the amino acid sequence shown in SEQ ID NO: 1.
  • the IgG1 Fc region has the amino acid sequence shown in SEQ ID NO: 2.
  • the anti-PD-L1 fusion antibody has the amino acid sequence shown in SEQ ID NO: 3.
  • the intracellular segment of the immunocostimulatory molecule is independently selected from at least one of 4-1BB, OX-40, CD40L, CD27, CD30, CD28, CD3 and their derivatives.
  • the combination of the expression of the intracellular segment of the immunocostimulatory molecule and the expression of the anti-PD-L1 antibody in the embodiment of the present invention has the effect of positively regulating and enhancing the cellular immune response.
  • the expression of the intracellular segment of the immunocostimulatory molecule of the embodiment of the present invention The combination of the expression of non-functional EGFR and the expression of anti-PD-L1 antibody makes the targeted killing effect of the transgenic lymphocyte proliferation of the embodiments of the present invention on tumors more significant and safe.
  • the intracellular segment of the immune costimulatory molecule is the intracellular segment of 4-1BB or CD3;
  • the lymphocytes are CD3 + T lymphocytes, CD8 + T lymphocytes, natural killer cells or natural killer T cells.
  • CD3 + lymphocytes are total T cells
  • natural killer cells are a type of immune cells
  • non-specific recognition target cells natural killer T cells are T cell subsets with T cells and natural killer cell receptors.
  • the expression of anti-PD-L1 antibody and the expression of chimeric antigen receptor in the above-mentioned lymphocytes make the targeted immunity of the above-mentioned lymphocytes' cell immunity stronger, and the killing effect on tumor cells is more significant; And the expression of chimeric antigen receptors makes the cellular immune killing effect of the above-mentioned lymphocytes safer and more effective.
  • the invention proposes a construct.
  • the construct includes: a first nucleic acid molecule that encodes an anti-PD-L1 fusion antibody, and the anti-PD-L1 fusion antibody includes: PD-L1 single chain antibody, IgG1 Hinge region and IgG1 Fc region, the PD-L1 single-chain antibody is connected to the IgG1 Fc region through the IgG1 hinge region, the IgG1 Fc region has T250Q and M248L mutations; a second nucleic acid molecule, the second nucleic acid molecule encodes an embedded An antigen receptor, the chimeric antigen receptor recognizes the tumor antigen mesothelin; and a third nucleic acid molecule that encodes a non-functional EGFR.
  • the obtained transgenic lymphocytes can express and secrete anti-PD-L1 antibodies, and have a significant anti-tumor-mediated immunosuppression effect, and enhance anti-apoptosis ability and proliferation ability 2.
  • the ability of targeted killing is significantly improved, the safety of immune killing is significantly improved, the proliferation and survival ability in tumor patients and in vitro and the killing ability in tumor patients are greatly improved, especially for tumor cells that highly express MSLN and PD-L1 molecules.
  • the effect of sexual killing is particularly significant.
  • the anti-PD-L1 fusion antibody has the amino acid sequence shown in SEQ ID NO: 3.
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 4 or 5.
  • the chimeric antigen receptor with the amino acid sequence shown in SEQ ID NO: 5 is longer than the chimeric antigen receptor with the amino acid sequence shown in SEQ ID NO: 4 in the extracellular and intracellular segments of the CD8 molecular segment.
  • the amino acid sequence of the hinge region, transmembrane region and intracellular region of CD8 will affect the spatial configuration of CAR protein molecules, the formation of dimers and multimers, and then affect the binding with downstream signaling molecules, which will affect the degree of lymphocyte activation and cytokines Generate levels.
  • the chimeric antigen receptor having the amino acid sequence shown in SEQ ID NO: 5 includes: an extracellular region including a single chain antibody and a hinge region, and the single chain antibody includes a heavy chain variable region of the single chain antibody And the light chain variable region, the single chain antibody specifically recognizes the antigen human MSLN, the hinge region includes an extracellular segment containing human CD8 molecule and 3 additional amino acid residues AAA, the AAA is located in the human CD8 molecule cell N-terminal of the outer segment, the extracellular segment of the human CD8 molecule has 55 amino acid residues; a transmembrane region, the transmembrane region includes a human CD8 molecule transmembrane segment, the human CD8 molecule transmembrane segment and the cell
  • the hinge region of the outer region is connected and embedded in the cell membrane of the T lymphocyte; the intracellular region, which includes the intracellular segment of human CD8 molecule and the intracellular segment of 4-1BB molecule and the intracellular segment of CD3 ⁇ chain, The intracellular segment of the
  • SEQ ID NO: 5 the spatial configuration of the chimeric antigen receptor is different. Lymphocytes expressing the chimeric antigen receptor have more moderate cell proliferation, less cytokine secretion, and cytokine release Syndrome and neurotoxicity have fewer advantages.
  • the non-functional EGFR has the amino acid sequence shown in SEQ ID NO: 6.
  • the nucleic acid molecule encoding the anti-PD-L1 fusion antibody has the nucleotide sequence shown in SEQ ID NO: 7-9.
  • the nucleic acid molecule encoding the chimeric antigen receptor has the nucleotide sequence shown in SEQ ID NO: 10 or 11.
  • the nucleic acid molecule encoding non-functional EGFR has the nucleotide sequence shown in SEQ ID NO: 12.
  • the lentivirus carries a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NO: 13-18.
  • the structure of the nucleotide sequence shown in SEQ ID NO: 13 to 18 can be expressed as S-VL (LS1) LVH (LS1) -IgG1HC (T250Q / M248L) -2A-MSLN CAR- (ires) -tEGFR, where S: signal peptide leader, L means linker peptide Linker, LS1 means anti-PD-L1 Mab clone # LS1, VL means light chain variable region light, chain variable region, VH means heavy chain variable Region heavy, chain variable region, H represents IgG1 Fc hinge region hinge region, C represents IgG1 heavy chain stable region heavy chain constant region, T250Q / M248L: IgG Fc T250Q and M248L amino acid mutation, 2A: 2A connecting peptide.
  • S signal peptide leader
  • L linker peptide Linker
  • LS1 means anti-PD-L1 Mab clone # LS1
  • VL means light chain variable region
  • the MSLN CAR encoded in the nucleotide sequence shown in SEQ ID NO: 13, 15, 17 is shorter than the MSLN CAR encoded in the nucleotide sequence shown in SEQ ID NO: 14, 16, 18, SEQ ID NO: 13
  • the MSLN CAR encoded in the nucleotide sequence shown in 15, 17 is abbreviated as MSLN CAR (short) in this article
  • the MSLN CAR encoded in the nucleotide sequence shown in SEQ ID NO: 14, 16, 18 is abbreviated in this article It is MSLN CAR (86).
  • the inventor realized that the above-mentioned anti-PD-L1 antibody, chimeric antigen receptor, and non-functional EGFR are independently expressed in at least one of the following ways, where, it should be noted that here Expression refers to both protein expression and RNA transcription.
  • the internal ribosome entry site sequence of the embodiment of the present invention is set between the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule expressing non-functional EGFR, and the internal ribosome entry site
  • the dot has the nucleotide sequence shown in SEQ ID NO: 19.
  • the internal ribosome entry site is usually located in the 5 'untranslated region (UTR) of the RNA virus genome, so that the translation of a viral protein can be independent of the 5' cap structure, and the other protein usually initiates translation by the 5 'cap structure.
  • the expression of the two genes before and after IRES is usually proportional.
  • the introduction of the internal ribosome entry site sequence enables the independent expression of nucleic acid molecules encoding anti-PD-L1 antibodies, nucleic acid molecules encoding chimeric antigen receptors and nucleic acid molecules encoding non-functional EGFR.
  • the internal ribosome entry site sequence is used to effectively ensure the high expression of the chimeric antigen receptor and non-functional EGFR, so that the specific killing effect of lymphocytes on tumors with high expression of MSLN is more significant The safety of immune killing has been further improved.
  • Promoter a first promoter operably linked to a nucleic acid molecule encoding a chimeric antigen receptor; a second promoter operably linked to a nucleic acid molecule that silences an immune checkpoint; and The third promoter is operably linked to the nucleic acid molecule expressing non-functional EGFR.
  • the first promoter, the second promoter, and the third promoter used are independently selected from U6, CMV, H1, EF-1, LTR, RSV promoters, first and second
  • the introduction of the promoter and the third promoter enables the independent expression of nucleic acid molecules encoding anti-PD-L1 antibodies, nucleic acid molecules encoding chimeric antigen receptors and nucleic acid molecules expressing non-functional EGFR, thereby effectively inhibiting PD1 / PD -L1-mediated tumor immune escape or high expression of non-functional EGFR, and ensure the high expression of chimeric antigen receptor, so that the survival rate of lymphocytes in the tumor environment is greatly improved, and the targeting effect of lymphocytes is stronger, The specific killing effect on tumors is more significant, and the safety of immune killing is further improved.
  • Fourth nucleic acid molecule a fourth nucleic acid molecule is provided between the first nucleic acid molecule and the second nucleic acid molecule or the second nucleic acid molecule and the third nucleic acid molecule, and the fourth nucleic acid molecule encodes a connecting peptide, the The linker peptide can be cleaved in the lymphocyte.
  • the 2A linking peptide has the amino acid sequence shown in SEQ ID NO: 20-23.
  • E, G, R, G, S, L, L, T C, G, D, V, E, E, N, P, G, P (SEQ ID NO: 20).
  • the introduction of the fourth nucleic acid molecule and its corresponding expressed linker peptide makes non-functional EGFR, chimeric antigen receptor, and anti-PD-L1 antibody expressed in lymphocytes in a non-fused state.
  • the introduction of the connecting peptide in the embodiment of the present invention ensures the biological function of non-functional EGFR, chimeric antigen receptor and anti-PD-L1 antibody, which has a more specific tumor killing effect and safety higher.
  • the introduction of the internal ribosome entry site sequence, or the introduction of the first, second, third promoter or third nucleic acid molecule allows cells to express and secrete anti-PD-L1 antibodies, express non-functional EGFR and express chimeric antigens And the non-functional EGFR and chimeric antigen receptors are expressed on the lymphocyte membrane in a non-fused state, thereby effectively suppressing negative immune regulation and ensuring the biological function of the chimeric antigen receptors, effectively realizing the timely transgenic lymphocytes Clearance, so that the survival rate of lymphocytes in the tumor environment is greatly improved, the targeted killing effect of lymphocytes is more significant, and the safety of immune killing is further improved.
  • the vector of the construct of the embodiment of the present invention is a non-pathogenic viral vector.
  • the non-pathogenic viral vector greatly improves the replication and amplification efficiency of the construct in lymphocytes, and thus the proliferation and viability of lymphocytes in tumor patients of the embodiments of the present invention are greatly improved, and the targeting effect of lymphocytes is further enhanced , The killing effect on tumor cells is more significant, and the safety of immune killing is further improved.
  • the vector of the construct of the embodiment of the present invention is a viral vector
  • the viral vector is selected from at least one of a retroviral vector, a lentiviral vector, an adenovirus vector, or an adenovirus-associated viral vector.
  • the viral vectors of the embodiments of the present invention have a wide range of viral infections during the process of virus packaging and infection, which can infect both terminally differentiated cells and cells in the dividing stage, and can be integrated into the host Chromosomes can be dissociated from the host chromosomes to achieve a broad-spectrum and high-efficiency infection efficiency, so that cell surface or intracellular immune checkpoints are efficiently silenced and non-functional EGFR is efficiently expressed and chimeric antigen receptors are highly efficient in lymphocytes It is shown that the lymphocytes of the embodiments of the present invention have greatly improved the proliferation and survivability of tumor patients, the targeting effect of lymphocytes is further enhanced, the killing effect on tumor cells is more significant, and the immune killing safety of lymphocytes is further improved.
  • the inventor inserts the target nucleic acid into the viral genome at certain viral sequence positions, thereby generating replication-defective viruses.
  • the inventors further constructed packaging cell lines (containing gag, pol and env genes, but excluding LTR and packaging components).
  • the inventor introduced the recombinant plasmid containing the gene of interest, along with the lentiviral LTR and packaging sequence, into the packaging cell line.
  • the packaging sequence allows the recombinant plasmid RNA transcript to be packaged into viral particles and then secreted into the culture medium.
  • the inventors further collected the matrix containing the recombinant lentivirus, selectively concentrated it, and used it for gene transfer. Slow vectors can infect a variety of cell types, including dividing cells and non-dividing cells.
  • the lentivirus of the embodiment of the present invention is a compound lentivirus.
  • the common lentiviral genes gag, pol and env it also contains other genes with regulatory and structural functions.
  • Lentiviral vectors are well known to those skilled in the art. Lentiviruses include: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors are produced by multiple attenuation of HIV pathogenic genes, such as deleting all the genes env, vif, vpr, vpu, and nef, so that the lentiviral vector forms a biosafety vector.
  • the recombinant lentiviral vector can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression in vivo and in vitro.
  • two or more vectors with packaging functions can infect non-dividing cells.
  • the targeting of recombinant viruses is achieved through the binding of antibodies or specific ligands (targeting specific cell type receptors) to membrane proteins.
  • the targeting of the recombinant virus by inserting an effective sequence (including regulatory regions) into the viral vector, together with another gene encoding the ligand of the receptor on a specific target cell, the vector has a specific targeting.
  • Various useful lentiviral vectors, as well as vectors produced by various methods and operations, are used to alter cell expression.
  • the adeno-associated virus vectors (AAV) of the embodiments of the present invention may be constructed using DNA of one or more well-known serum-type adeno-associated virus vectors.
  • microgenes are also included in the embodiments of the present invention.
  • Minigene means a combination (selected nucleotide sequence and operable necessary related linking sequence) to guide transformation, transcription and / or expression of gene products in host cells in vivo or in vitro.
  • the application of "operably linked" sequences includes expression control sequences of consecutive target genes, and expression control sequences acting on trans or remote control of target genes.
  • the vectors of the embodiments of the present invention also include conventional control elements, in cell transfection with plasmid vectors or / and cell infection with viral vectors.
  • a large number of expression control sequences may be used.
  • the promoter is an RNA polymerase promoter selected from U6, H1, pol I, pol II and pol III.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is selected from promoters based on the selected vector.
  • the promoter when selecting a lentiviral vector, is U6, H1, CMV IE gene, EF-1 ⁇ , ubiquitin C or phosphoglycerol kinase (PGK) promoter.
  • Other conventional expression control sequences include selectable markers or reporter genes, including nucleotide sequences encoding geneticin, hygromycin, ampicillin or puromycin resistance.
  • Other components of the vector include the origin of replication.
  • the present invention provides a method for preparing the aforementioned T lymphocytes or transgenic lymphocytes.
  • the method includes: introducing the aforementioned construct or the aforementioned lentivirus into lymphocytes or T lymphocytes.
  • the introduction method may be selected from the group consisting of electroporation and virus infection of the host cell.
  • lymphocytes or T lymphocytes were successfully introduced into the above-mentioned lymphocytes or T-lymphocytes to achieve the expression of the chimeric antigen receptor against the antigen MSLN and the expression and secretion of anti-PD-L1 antibody and the expression of non-functional EGFR , So that the resulting lymphocytes or T lymphocytes have significant resistance to tumor-mediated immunosuppression, proliferation in tumor patients and in vitro and survival ability of tumor patients are greatly improved, lymphocytes or T lymphocytes against tumor cells, In particular, tumor cells with high expression of MSLN and PD-L1 have stronger targeted killing effect and high safety of immune killing.
  • the present invention proposes a therapeutic composition for treating cancer.
  • the therapeutic composition includes: the above construct, the above lentivirus, the above T lymphocyte or the above transgenic lymphocyte.
  • the composition of any one of the above therapeutic compositions can achieve high expression of the antigen MSLN chimeric antigen receptor in transgenic lymphocytes or T lymphocytes and expression and secretion of anti-PD-L1 antibodies in transgenic lymphocytes or T lymphocyte cells; And the expression of non-functional EGFR on the surface of transgenic lymphocytes or T lymphocytes, so that the resulting transgenic lymphocytes or T lymphocytes are amplified in vitro, proliferated in tumor patients, and the survival ability of tumor patients is greatly improved.
  • T lymphocytes have a stronger targeted killing effect on tumor cells highly expressing MSLN and PD-L1 molecules, and the safety of immune killing is higher.
  • the therapeutic composition of the embodiments of the present invention provided to patients is preferably applied to a biologically compatible solution or an acceptable pharmaceutical carrier.
  • the prepared various therapeutic compositions are suspended or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; isotonic saline solution or other more obvious formulations for those skilled in this field.
  • a pharmaceutically or physiologically acceptable carrier such as physiological saline; isotonic saline solution or other more obvious formulations for those skilled in this field.
  • the appropriate carrier depends to a large extent on the route of administration.
  • Other isotonic sterile injections with and without water and sterile suspensions with and without water are pharmaceutically acceptable carriers.
  • a sufficient number of viral vectors are transfected into targeted T cells and provide sufficient strength of the transgene to express and secrete anti-PD-L1 antibodies and express non-functional EGFR as well as express unique MSLN chimeric antigen receptors body.
  • the dosage of the therapeutic agent mainly depends on the treatment status, age, weight, and patient's health, which may cause patient variability.
  • a treatment method includes the use of one or more drug therapies.
  • the cancer includes mesothelioma.
  • the survival ability is greatly improved.
  • the targeted killing effect of lymphocytes or T lymphocytes on tumor cells with high expression of MSLN is stronger, especially on tumor cells with high expression of MSLN and PD-L1 molecules.
  • the immune killing effect of PD-L1 molecular tumor cells is safer and more effective.
  • the present invention provides a method for improving the activity of lymphocytes and the safety of treatment.
  • the lymphocytes of the embodiments of the present invention carry chimeric antigen receptors that recognize tumor antigens.
  • the method includes: causing the lymphocyte to express an anti-PD-L1 fusion antibody; and causing the lymphocyte to express a non-functional EGFR, the anti-PD-L1 fusion antibody, lymphocyte,
  • the chimeric antigen receptor, non-functional EGFR is as defined above.
  • the lymphocyte activity of the embodiments of the present invention includes at least one of lymphocyte proliferative ability in vitro, proliferation and survival ability in tumor patients, and lymphocyte killing ability in tumor patients.
  • the cell surface or intracellular immune checkpoints of the lymphocytes of the embodiments of the present invention are silenced, the lymphocytes are activated, the proliferative response is upregulated, the secretion of cytokines is increased, and the ability to resist apoptosis is enhanced.
  • the lymphocytes of the embodiments of the present invention are expanded and proliferated in vitro, and the targeted killing effect on tumor cells is significantly enhanced.
  • Non-functional EGFR lacks the N-terminal ligand binding region and intracellular receptor tyrosine kinase activity, but includes the transmembrane region of the wild-type EGFR receptor and a complete sequence that binds to anti-EGFR antibodies.
  • Non-functional EGFR can serve as lymph Cell suicide marker.
  • the present invention is an example of the lymphocytes used in the treatment of tumor cells that highly express MSLN and PD-L1 molecules. If the patient has serious adverse reactions, the lymphocytes of the examples of the present invention can be cleared by anti-EGFR antibodies, which can improve the cost The safety of the lymphocytes of the embodiments of the invention to treat tumor patients with high expression of MSLN and PD-L1 molecules.
  • a replication-defective lentiviral vector was generated, and the lentiviral vector was centrifuged and collected for transduction of human T lymphocytes.
  • lentiviral transgenic plasmid 15 ⁇ g of lentiviral transgenic plasmid, 5 ⁇ g of pVSV-G (VSV glycoprotein expression plasmid), 10 ⁇ g of pCMVR8.74 plasmid (Gag / Pol / Tat / Rev expression plasmid) and 174 ⁇ l of Express to each plate of cells -In (concentration is 1 microgram / microliter). The supernatant was collected at 24 hours and 48 hours, respectively, and centrifuged at 28,000 rpm (centrifuge rotor Beckman SW 32Ti, purchased from Beckman Coulter, Brea, CA) using an ultracentrifuge for 2 hours. Finally, the virus plasmid pellet was resuspended with 0.75 ml of RPMI-1640 medium.
  • Human primary T lymphocytes were isolated from healthy volunteer donors. Human T lymphocytes were cultured in RPMI-1640 medium and stimulated with beads coated with monoclonal antibodies against CD3 and CD28 (purchased from Invitrogen, Carlsbad, CA). 18-24 hours after the activation of human T lymphocytes, the T lymphocytes were transduced by spin-inoculation method. The transduction process is as follows: in 24-well plates, each well is covered with 0.5 ⁇ 10 6 T For lymphocytes, add 0.75 ml of the above resuspended virus supernatant and Polybrene (concentration 8 ⁇ g / ml) to the cells in each well.
  • the mixture of cells and virus plasmids was centrifuged in a tabletop centrifuge (purchased from Sorvall ST 40; Thermo Scientific) at a room temperature of 2500 rpm for 90 minutes.
  • Human recombinant interleukin-2 (IL-2; purchased from Novartis, Basel, Switzerland) is added to the T lymphocyte culture medium every 2 to 3 days.
  • the final concentration of IL-2 is 100-IU / ml in T lymph During cell culture, the cell density is maintained at 0.5 ⁇ 10 6 to 1 ⁇ 10 6 / ml.
  • T lymphocytes are dormant, such as slower cell growth and smaller cells, the cell growth rate and size are evaluated by Coulter Counter (purchased from Beckman Coulter), or transduced T lymphocytes At a planned time point, T lymphocytes can be used for functional analysis.
  • the flow cytometer used in the examples of the present application was BD FACS Canto II (purchased from BD Biosciences), and the flow cytometry data was analyzed using FlowJoversion 7.2.5 software (purchased from Tree Star, Ashland, OR).
  • anti-MSLN CAR T lymphocytes anti-MSLN CAR T lymphocytes
  • target test cells are labeled with 51Cr at 37 degrees Celsius for 1 hour. After labeling, the cells were washed with RPMI medium containing 10% fetal bovine serum (FCS). After washing, the cells were resuspended in the same medium, and the concentration of resuspended cells was 1 ⁇ 10 5 / ml.
  • FCS fetal bovine serum
  • T cells were added to the target test cell suspension at different target-efficiency cell ratios (T: E), and the cells were seeded in 96-wells with a volume of 200 microliters per well.
  • the cells were cultured in a 37 degree incubator for 4 hours. After 4 hours, 30 microliters of supernatant was taken from each well and placed in a 96-microwell plate of a counter for counting and analysis.
  • the analytical instrument is a top counting NXT micro scintillation counter (purchased from Packard Bioscience). The number of effector cells in all counting wells is calculated based on the total number of T cells.
  • the labeled target test cells are MSLN + PD-L1 + H226 cells or MSLN + PD-L1 + Panc1 target cells.
  • Example 2 Construction of a vector co-expressing anti-PD-L1 single chain antibody and IgG fusion protein containing amino acid mutations and anti-MSLN chimeric antigen receptor
  • the inventors cloned the sequence encoding the anti-human MSLN single chain antibody, the 4-1BB intracellular segment and the T cell receptor combination zeta-chain sequence into a lentiviral vector containing the EF-1 promoter ( lentiviral vector), during the cloning process, the selected restriction enzymes were XbaI and NotI, and NotI and XhoI. Through enzyme digestion, ligation, screening and amplification of the target plasmid, the expression of anti-MSLN was generated. Lentiviral plasmid (LV-MSLN CAR) with antigen receptor.
  • FIG. 1 is a schematic diagram of a lentiviral vector, including the sequence encoding the anti-PD-L1 single chain antibody (LS1 clone) and the IgG fusion protein containing amino acid mutations, the 2A peptide sequence, the sequence of the anti-MSLN chimeric antigen receptor, the IRES sequence and tEGFR sequence.
  • the lentiviral vector ⁇ LS1 / M-CAR contains the sequence encoding the anti-PD-L1 single chain antibody (LS1 clone), the 2A peptide sequence, the sequence against the MSLN chimeric antigen receptor, the IRES sequence and the tEGFR sequence.
  • the lentiviral vector ⁇ LS1-QL contains sequences encoding anti-PD-L1 single chain antibody (LS1 clone), IRES sequence and tEGFR sequence.
  • the lentiviral vector M-CAR contains the sequence encoding the MSLN chimeric antigen receptor, IRES sequence and tEGFR sequence.
  • Example 3 The vector ⁇ LS1-QL / M-CAR transduces T lymphocytes to secrete anti-PD-L1 single-chain antibodies and IgG fusion proteins containing amino acid mutations to inhibit PD-1: PD-L1
  • peripheral blood lymphocytes were taken from anonymous donors. Peripheral blood lymphocytes are separated by gradient centrifugation. The gradient centrifuge is Ficoll-Hypaque.
  • T lymphocyte activating factor magnetic beads CD3 / CD28 purchased from Invitrogen, Carlsbad, CA
  • the activated T lymphocytes are transduced with lentiviral vectors and cultured in vitro, as described in Example 1.
  • the transduced T cells the number of cells is 2 ⁇ 10 6 / well
  • culture supernatant was harvested for PD-1: PD-L1 action inhibition experiment.
  • 100 ng / well PD-L1 protein (Aerobiosystems, Boston, MA) was used to coat 96-well ELISA plates. 10ng biotin-labeled PD1 (Aerobiosystems, Boston, MA) was mixed with 100ul of various CAR-T supernatants or with 50ng of commercially purchased anti-hPD-L1 (Aerobiosystems, Boston, MA), supplemented to a volume of 200uL Add the mixture to the coated 96-well plate. The 96-well plate to which the mixed liquid was added was left at room temperature for 2 hours, and then washed thoroughly. Dilute streptavidin-HRP was added to the well plate and placed at room temperature for 1 hour while slowly shaking.
  • the experimental results are shown in Figure 2.
  • the results in Figure 2 show that the culture medium transduced with the lentiviral vector ⁇ LS1-QL / M-CAR or ⁇ LS1-QL T lymphocytes can significantly inhibit the effect of PD-1: PD-L1, while transducing the lentiviral vector M-CAR
  • the culture medium of T lymphocytes did not inhibit the effect of PD-1: PD-L1 (* P ⁇ 0.01; ⁇ LS1-QL / M-CAR vs. M-CAR).
  • T lymphocytes transduced with ⁇ LS1-QL / M-CAR can secrete anti-PD-L1 single-chain antibodies and IgG fusion proteins containing amino acid mutations to inhibit the effect of PD-1: PD-L1.
  • T lymphocytes co-expressing anti-PD-L1 single chain antibody and IgG fusion protein containing amino acid mutations and anti-MSLN chimeric antigen receptor have enhanced ability to dissolve tumor cells (in vitro experiment)
  • peripheral blood lymphocytes were taken from anonymous donors. Peripheral blood lymphocytes are separated by gradient centrifugation. The gradient centrifuge is Ficoll-Hypaque. T lymphocytes and T cell activating factor magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) were incubated at 5% CO 2 and 37 degrees Celsius for 72 hours. The medium was supplemented with 2mmol / L glutamine, 10% High temperature inactivated fetal bovine serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U / ml of penicillin / streptomycin RPMI medium 1640 (purchased from Invitrogen Gibco Cat. No. 12633-012).
  • FCS High temperature inactivated fetal bovine serum
  • T cells were planted on a cell culture dish lined with recombinant fibronectin fragments (FNch-296; Retronectin) and transduced with lentiviruses.
  • the transduced lentiviruses were ⁇ LS1-QL / M-CAR, ⁇ LS1 / M-CAR
  • the process of ⁇ LS1-QL, M-CAR or no-load (Control) transduction is as described in Example 1.
  • Transduced T cells were cultured in RPMI-1640 medium and induced expansion with recombinant human IL-2 factor (100 ng / ml; purchased from R & D Systems) for 7-10 days, and then subjected to functional test experiments.
  • the inventors measured the killing effect of T cells (effector cells) transduced with different lentiviruses on MSLN + PD-L1 + H226 cells or MSLN + PD-L1 + Panc1 target cells, using different ratios of target effect cells, and the measurement method used standard
  • the 4-hour 51 chromium release method and the 4-hour 51 chromium release method are as described in Example 1.
  • T lymphocytes transduced with the lentiviral vector ⁇ LS1-QL / M-CAR T lymphocytes are more effective in killing MSLN + PD-L1 + than T lymphocytes transduced with the lentiviral vector M-CAR H226 cells or MSLN + PD-L1 + Panc1 target cells.
  • Transduction of lentiviral vector ⁇ LS1-QL / M-CAR T lymphocytes can also kill MSLN + PD-L1 + H226 cells more effectively than T lymphocytes transduced with lentiviral vector ⁇ LS1 / M-CAR MSLN + PD-L1 + Panc1 target cells.
  • T lymphocytes transduced with no-load lentivirus have no significant killing effect on MSLN + PD-L1 + H226 cells or MSLN + PD-L1 + Panc1 target cells.
  • the statistical data represent the mean ⁇ SEM of three wells. * P ⁇ 0.01; ⁇ LS1-QL / M-CAR vs. ⁇ LS1 / M-CAR or M-CAR. This shows that T lymphocytes transduced with ⁇ LS1-QL / M-CAR can secrete anti-PD-L1 single-chain antibodies and IgG Fc fusion proteins containing amino acid mutations have a stronger ability to kill MSLN + PD-L1 + target cells in vitro.
  • T-lymphocytes co-expressing anti-PD-L1 single chain antibody and IgG fusion protein with amino acid mutation and anti-MSLN chimeric antigen receptor have enhanced anti-tumor ability (in vivo mouse tumor model experiment)
  • NSG mice Immune-deficient non-obese diabetes (NOD) -scid ⁇ mice (NSG) (SCID mouse background, and with IL-2 receptor gamma chain defects) were used to test the antitumor activity of various transduced T cells.
  • NSG mice 5 / group
  • MSLN + PD-L1 + H226 left panel
  • MSLN + PD-L1 + Panc1 tumor cells 6 to 9 days after the injection, when the tumor cell diameter reached 4-5 mm, the mice were intravenously injected with 1 ⁇ 10 7 different transduced T cells.
  • the volume of the tumor was measured with a caliper at the specified time.
  • Tumor volume Tu 2 volume (width 2 length).
  • transduction of the lentiviral vector ⁇ LS1-QL / M-CAR T lymphocytes can kill MSLN + PD-L1 + more effectively than T lymphocytes transduced with the lentiviral vector M-CAR. H226 cells or MSLN + PD-L1 + Panc1 tumor cells.
  • Transduction of lentiviral vector ⁇ LS1-QL / M-CAR T lymphocytes can also kill MSLN + PD-L1 + H226 cells more effectively than T lymphocytes transduced with lentiviral vector ⁇ LS1 / M-CAR MSLN + PD-L1 + Panc1 tumor cells.
  • T lymphocytes transduced with no-load lentivirus have no significant killing effect on MSLN + PD-L1 + H226 cells or MSLN + PD-L1 + Panc1 tumor cells.
  • P ⁇ 0.01; ⁇ LS1-QL / M-CAR vs. ⁇ LS1 / M-CAR or M-CAR.
  • T lymphocytes transduced with ⁇ LS1-QL / M-CAR can secrete anti-PD-L1 single-chain antibodies and IgG Fc fusion proteins containing amino acid mutations have a stronger ability to kill MSLN + PD-L1 + tumor cells in vivo.
  • Example 6 The carrier ⁇ LS1-QL / M-CAR transduced T lymphocytes treated the serum of mice to inhibit PD-1: PD-L1.
  • the NSG mice used in Example 5 were fed with transduced T cells for 48 hours. The serum was separated and mixed equally from 4 mice per group. 96-well ELISA plates were coated with 100 ng / well of commercially purchased PD-L1 protein (Aerobiosystems, Boston, MA). 10ng biotin-labeled PD1 (Aerobiosystems, Boston, MA) was mixed with 100ul of various CAR-T supernatants or with 50ng of commercially purchased anti-hPD-L1 (Aerobiosystems, Boston, MA), and the volume was added to 200uL after mixing And add it to the coated plum. The plate was incubated at room temperature for 2 hours and washed thoroughly.
  • the experimental results are shown in Figure 5.
  • the results in Figure 5 show that the serum of mice transduced with vector ⁇ LS1-QL / M-CAR transduced T lymphocytes can significantly inhibit the effect of PD-1: PD-L1, while the culture medium of the transfected lentiviral vector M-CAR T lymphocytes No inhibitory effect on PD-1: PD-L1 (* P ⁇ 0.01; ⁇ LS1-QL / M-CAR vs. M-CAR).
  • Carrier ⁇ LS1 / M-CAR transduced T lymphocytes treated the rat sera with only weak inhibition PD-1: PD-L1 effect (* P ⁇ 0.01; ⁇ LS1 / M-CAR).
  • T lymphocytes transduced with ⁇ LS1-QL / M-CAR can secrete anti-PD-L1 single-chain antibodies and IgG fusion proteins containing amino acid mutations, which can obviously inhibit PD-1: PD-L1 in vivo.

Abstract

本发明提出了PD-L1抗体分泌的抗间皮素CAR-T细胞肿瘤免疫治疗，该CAR-T细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，其中，所述抗PD-L1融合抗体由抗PD-L1单链抗体连接到含有氨基酸突变的IgG1Fc片段形成。

Description

PD-L1抗体分泌的抗间皮素CAR-T细胞肿瘤免疫治疗
优先权信息
本申请请求2018年10月24日向中国国家知识产权局提交的、专利申请号为201811246615.7的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物医药领域，具体地，本发明涉及T淋巴细胞、慢病毒、转基因淋巴细胞、构建体、用于治疗癌症的治疗组合物和提高淋巴细胞活性和治疗安全性的方法。
背景技术
间皮素(mesothelin，MSLN)是一种分化抗原，它在人类正常组织的表达仅限于的胸膜、心包膜和腹膜衬里的间皮细胞。然而，间皮素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。间皮素基因编码71KDa的前体蛋白，前体蛋白继而被加工成31KDa的脱落片段和40KDa的蛋白片段，31KDa的脱落片段被称为巨核细胞促进因子(MPF)，而40KDa的蛋白片段即被称为间皮素，间皮素通过糖基磷脂酰肌醇(glycosyl-phosphatidylinositol，GPI)的锚定作用固定在细胞膜上。
以间皮瘤为例，间皮瘤有胸膜间皮瘤和腹膜间皮瘤之分，胸膜间皮瘤是胸膜原发肿瘤，有局限型(多为良性)和弥漫型(都是恶性)之分，其中弥漫型恶性间皮瘤是胸部预后最坏的肿瘤之一。腹膜间皮瘤是指原发于腹膜间皮细胞的肿瘤。临床表现不具有特征性，常见的症状和体征有：腹痛、腹水、腹胀及腹部包块等。恶性胸膜间皮瘤的治疗，目前仍然没有有效的根治方法。治疗方法上，有姑息性治疗、外科治疗、化学治疗及放射治疗等，一般认为对于肿瘤相对局限的I期病人，主张做根治的胸膜肺切除术。对于Ⅱ、Ⅲ、Ⅳ期病人，根治性手术已经没有意义了，只有施行姑息性手术。事实上，多数病人到疾病明确诊断时，已处于II期以上。迅速增长的胸水常导致患者严重的呼吸困难，姑息性手术只能暂时提高这些晚期病人的生活质量，而无法根治。
由此可见，开发针对间皮素高表达肿瘤的治疗方法尤为迫切。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
间皮素却在多种人类癌症组织中高表达，包括几乎所有的间皮瘤和胰腺癌和约70％的卵巢癌和约50％的肺腺癌以及其他癌症，例如胆管癌，胃癌，肠癌，食管癌，乳腺癌。因此，间皮素代表着肿瘤免疫治疗领域中一个有着巨大吸引力的靶点。
但是在肿瘤的免疫治疗中，作为免疫负调节机制，激活的细胞毒性T淋巴细胞(CTLs)可以表达负调控的监管结构，即在细胞表面或细胞内表达免疫检查点分子，如程序性细胞死亡1受体(PD-1)可以表达在活化CTLs上，其与肿瘤细胞上表达的程序性死亡配体1(PD-L1)相互作用，可以抑制抗肿瘤T细胞反应。而恰恰许多肿瘤细胞，包括间皮素高表达肿瘤细胞均高表达PD-L1，PD-L1与PD-1的结合，导致CTLs增生性反应的下调，细胞因子的分泌减少和T细胞的无能或凋亡。进而导致肿瘤免疫治疗的效果大打折扣。
为了解决上述问题，发明人提出了一种可以表达和分泌抗PD-L1抗体、表达具有识别MSLN的嵌合抗原受体以及无功能EGFR的转基因淋巴细胞，在本申请中，称之为PD-L1抗体分泌的抗间皮素CAR-T细胞。发明人在实验中发现，本发明所提出的抗间皮素CAR-T细胞可以抑制肿瘤细胞的免疫逃逸，对高表达间皮素肿瘤的特异杀伤能力强，而对正常MSLN表达水平的间皮细胞没有杀伤，且本申请所提出的抗间皮素CAR-T细胞可表达无功能EGFR，显著增加了该CAR-T细胞治疗的安全性。
在本发明的第一方面，本发明提出了一种T淋巴细胞。根据本发明的实施例，所述T淋巴细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，其中，所述嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别肿瘤抗原间皮素；跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链；所述抗PD-L1融合抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，所述IgG1 Fc区具有T250Q和M248L突变。根据本发明的实施例的T淋巴细胞可以分泌抗PD-L1抗体，具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著提高，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN及PD-L1分子的肿瘤具有显著的定向杀伤作用，安全性高。
在本发明的第二方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带下列核酸分子：(a)编码抗PD-L1融合抗体的核酸分子，所述抗PD-L1抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰 链区与所述IgG1 Fc区相连，所述IgG Fc区具有T250Q和M248L突变；(b)编码嵌合抗原受体的核酸分子，所述嵌合抗原受体的胞外区识别肿瘤抗原间皮素；以及(c)编码无功能EGFR的核酸分子。将根据本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其可以表达和分泌抗PD-L1抗体，具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN及PD-L1分子的肿瘤具有显著的定向杀伤作用，安全性高。
在本发明的第三方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：9所示核苷酸序列的核酸分子。
在本发明的第四方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：10所示核苷酸序列的核酸分子。
在本发明的第五方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：11所示核苷酸序列的核酸分子。
在本发明的第六方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：12所示核苷酸序列的核酸分子。
在本发明的第七方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：13所示核苷酸序列的核酸分子。
在本发明的第八方面，本发明提出了一种慢病毒。根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：14所示核苷酸序列的核酸分子。
将根据本发明实施例的慢病毒导入淋巴细胞所得的转基因淋巴细胞，其可以表达和分泌抗PD-L1抗体，具有抵抗肿瘤细胞介导的免疫抑制的特性，在体外的增殖能力、在肿瘤病人体内的增殖和生存能力显著增强，对肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN及PD-L1分子的肿瘤具有显著的定向杀伤作用，且安全性更高。
在本发明的第九方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述转基因淋巴细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，其中，所述嵌合抗原受体包括：胞外区；跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；所述抗PD-L1融合抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，所述IgG1 Fc区具有T250Q和M248L突变。根据本发明实施例的转基因淋巴细胞的体外增殖能力、在肿瘤病人体内的增殖和 生存能力以及在肿瘤病人体内的对肿瘤细胞的特异性杀伤能力大大提高，尤其对高表达MSLN及PD-L1分子的肿瘤具有显著的定向杀伤作用，安全性高。
在本发明的第十方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体包括：第一核酸分子，所述第一核酸分子编码抗PD-L1融合抗体，所述抗PD-L1融合抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，所述IgG Fc区具有T250Q和M248L突变；第二核酸分子，所述第二核酸分子编码嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素；以及第三核酸分子，所述第三核酸分子编码无功能EGFR，其中，所述抗PD-L1融合抗体、所述嵌合抗原受体、所述无功能EGFR如前面所描述的。根据本发明的实施例构建体成功导入本发明实施例的淋巴细胞后，可有效表达和分泌抗PD-L1抗体，表达无功能EGFR以及表达特异性识别MSLN的嵌合抗原受体，从而所获得的淋巴细胞对肿瘤细胞，尤其是高表达MSLN及PD-L1分子的肿瘤细胞的定向杀伤作用更加显著，安全性高。
在本发明的第十一方面，本发明提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，所述治疗组合物包括：上述构建体、慢病毒、T淋巴细胞或者转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现转基因淋巴细胞或T淋巴细胞抗PD-L1抗体的表达和分泌和无功能EGFR和特异性识别MSLN的嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，所得转基因淋巴细胞或T淋巴细胞具有显著的抵抗肿瘤细胞介导的免疫抑制，在肿瘤病人体外和体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对肿瘤细胞的靶向杀伤作用更强。根据本发明实施例的治疗癌症的治疗组合物对肿瘤细胞的靶向杀伤作用显著增强，尤其是对高表达MSLN及PD-L1分子的肿瘤细胞的靶向杀伤作用显著增强、安全性进一步提高。
在本发明的第十二方面，本发明提出了一种提高淋巴细胞活性和治疗安全性的方法，所述淋巴细胞携带嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，根据本发明的实施例，所述方法包括：使所述淋巴细胞表达抗PD-L1融合抗体，以及使所述淋巴细胞表达无功能EGFR。所述抗PD-L1融合抗体、所述淋巴细胞、所述嵌合抗原受体、所述无功能EGFR如是如前所定义的，所述淋巴细胞活性包括所述淋巴细胞体外增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。根据本发明的实施例淋巴细胞表达和分泌抗PD-L1抗体，淋巴细胞活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强，淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高。上述抗PD-L1抗体的分泌 配合淋巴细胞嵌合抗原受体的抗原特异性功效，从而实现了有效抵抗肿瘤细胞介导的免疫抑制，对高表达MSLN的肿瘤细胞的靶向杀伤作用显著增强。无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR受体的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明是实施例的淋巴细胞在用于治疗治疗高表达MSLN的肿瘤细胞时，如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN及PD-L1分子的肿瘤病人的安全性。
在本发明的第十三方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，所述方法包括给患者给予前面所述的构建体、前面所述的慢病毒、前面所述的T淋巴细胞或者前面所述的转基因淋巴细胞。根据本发明实施例的治疗癌症的方法，对肿瘤细胞的靶向杀伤作用强，尤其对高表达MSLN及PD-L1分子的肿瘤病人的治疗效果好、安全性高。
附图说明
图1是根据本发明实施例的共表达抗PD-L1 scFv-Fc融合抗体，抗MSLN抗原特异性的嵌合抗原受体和无功能EGFR的慢病毒载体的结构示意图；
图2是根据本发明实施例的共表达抗PD-L1 scFv-Fc融合抗体，抗MSLN抗原特异性的嵌合抗原受体和无功能EGFR的淋巴细胞体外分泌抗PD-L1 scFv-Fc融合抗体及抑制PD-1与PD-L1相互作用的结果图；
图3是根据本发明实施例的共表达抗PD-L1 scFv-Fc融合抗体，抗MSLN抗原特异性的嵌合抗原受体和无功能EGFR的淋巴细胞体外杀伤肿瘤细胞能力的结果图；
图4是根据本发明实施例的共表达抗PD-L1 scFv-Fc融合抗体，抗MSLN抗原特异性的嵌合抗原受体和无功能EGFR的淋巴细胞体内杀伤肿瘤细胞能力的结果图；以及
图5是根据本发明实施例的共表达抗PD-L1 scFv-Fc融合抗体，抗MSLN抗原特异性的嵌合抗原受体和无功能EGFR淋巴细胞注射小鼠血清中，分泌的抗PD-L1 scFv-Fc融合蛋白抗体抑制PD-1与PD-L1相互作用的结果图。
发明详细描述
下面详细描述本发明的实施例，下面描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，如无特别说明，本申请所述的“抗PD-L1融合抗体”是指PD-L1单链 抗体、IgG1铰链区以及IgG1Fc区的融合蛋白，其中IgG1 Fc区具有T250Q和M248L突变，该融合抗体具有抗PD-L1活性，可以与PD-L1特异性结合。
转基因淋巴细胞
一方面，本发明提出了一种转基因淋巴细胞。根据本发明的实施例，所述转基因淋巴细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，其中，所述嵌合抗原受体包括：胞外区；跨膜区，所述跨膜区包括CD8的跨膜段，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；所述抗PD-L1融合抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，所述IgG1 Fc区具有T250Q和M248L突变。
根据本发明的实施例，所述抗PD-L1融合抗体是一种分泌型抗体。由此，抗PD-L1融合抗体分泌到细胞外，并且与细胞表面的PD-L1特异性结合，进而有效阻遏PD-1：PD-L1介导的免疫逃逸机制。
人类抗体包括两个免疫球蛋白轻链和两个免疫球蛋白重链，重链和轻链间以共价键或非共价键连接，进而导致形成三个独立的蛋白区-两个Fab区和一个Fc区。Fab区和Fc区通过作为铰链区的有弹性的连接部连接的。抗体中的Fab区是一样的结构，Fab区有一个特异性的抗原结合位点，Fc区与配体的相互作用位点，该位点能够诱导效应器功能，包括细胞Fc受体和C1q补体成份。治疗性抗体的生理学活性由两个独立的天然免疫球蛋白机制介导：治疗性抗体的效力由它的特异性和与目标抗原的二价结合引起(如，阻断或中和靶抗原或诱导细胞凋亡)，也可由Fc和效应器配体(Fc受体和C1q部件)形成的免疫复合体激活的效应器功能引起。
单链抗体(scFv)是一种基因工程抗体，其中VH和VL域与柔性多肽连接体相连。与整体抗体的Fab区相比，单链抗体表现出更好的组织渗透药动学，并且由于抗原结合表面未被改变而具有完全的抗原结合特异性。然而，单链抗体在血液中的半衰期短，因为单链抗体中没有Fc分子片段，缺少没有Fc片段效应功能。根据本发明实施例的抗PD-L1融合抗体中的PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，因此，所述PD-L1融合抗体在血液中的半衰期相较于单独的PD-L1单链抗体，显著延长。
抗体的Fc区介导其血清半衰期和效应功能，如补体依赖的细胞毒(CDC)，抗体依赖性细胞毒作用(ADCC)和抗体依赖性细胞吞噬(ADCP)。Fc片段用于与单链抗体连接，以增加单链抗体在血液中的半衰期，并具有Fc效应功能。五类免疫球蛋白(IgM、IgD和 IgE，IgG，IgA，IgG亚类)和四个IgG亚类(IgG1、IgG2、IgG3、IgG4)是存在于人类。人血清中IgG含量最高。四亚类IgG1、IgG2、IgG3和IgG4是高度保守的，在他们的恒定区不同，尤其是在铰链上CH2域。这些地区的参与结合IgG Fc受体(FcγR)和补体C1q。结果，不同的子类有不同的效应功能，在触发FCγr表达细胞，从而引起吞噬或抗体依赖性细胞介导的细胞毒作用，并激活补体。IgG1和IgG3能有效触发这一经典途径补体，但IgG2和IgG4并不那么有效。
PD-L1免疫检查点分子往往是在肿瘤细胞中高表达。根据本发明的实施例，针对肿瘤细胞表达PD-L1的单链抗体融合了IgG1 Fc，此单链抗体的半衰期延长，并且此单链抗体与PD-L1结合能力增强，从而阻断PD-1和PD-L1相互作用，增强CTL杀伤肿瘤细胞。IgG1Fc能够有效的触发效应功能，如补体依赖的细胞毒(CDC)，抗体依赖性细胞毒作用(ADCC)和抗体依赖性细胞吞噬(ADCP)，来进一步杀伤肿瘤细胞。
现在很多基因工程方法瞄准改建抗体的药动学/药效学性质。改进抗体的药代动力学性质，一个比较深入的研究领域是涉及研究Fc区与新生Fc受体(FcRn)的相互作用。FcRn在内涵体酸性(pH～6)环境下与免疫球蛋白(IgG)结合，当暴露在生理pH环境下，又将IgG释放如循环系统。在内涵体内不与IgG结合的FcRn，会在溶酶体中经历蛋白质降解。IgG进入循环系统和进入降解途径的比例，对决定IgG在循环系统中半衰期至关重要。已经有研究证据表明，通过使CH2和CH3FC区中的残基突变以提高IgG与FcRn结合特性，可显著提高抗体的半衰期。发明人发现，将IgG1,IgG4或IgG2抗体的Fc区428位的Met突变为Leu(M428L)，250位的Thr突变为Gln(T250Q)，可显著提高IgG在体内的半衰期。
本发明实施例的T淋巴细胞或转基因淋巴细胞在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的对特意性肿瘤细胞的杀伤能力显著增强，尤其对高表达MSLN及PD-L1分子的肿瘤细胞的特异性杀伤效果大大提高，并且安全性也显著提高。
根据本发明的实施例，本发明实施例的无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR受体的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。无功能EGFR表达淋巴细胞可被抗EGFR抗体在体内清除。从而，本发明实施例的T淋巴细胞表达无功能EGFR，在保证转基因淋巴细胞的靶向杀伤作用的前提下，如果病人出现严重不良反应，转基因淋巴细胞可被抗EGFR抗体清除，进而可进一步提高本发明实施例的转基因淋巴细胞或T淋巴细胞治疗高表达MSLN及PD-L1分子的肿瘤病人的安全性。
根据本发明的实施例，所述PD-L1单链抗体具有SEQ ID NO:1所示的氨基酸序列。
Figure PCTCN2019112530-appb-000001
Figure PCTCN2019112530-appb-000002
根据本发明的实施例，所述IgG1 Fc区具有SEQ ID NO:2所示的氨基酸序列。
Figure PCTCN2019112530-appb-000003
根据本发明的实施例，所述抗PD-L1融合抗体具有SEQ ID NO:3所示的氨基酸序列。
Figure PCTCN2019112530-appb-000004
根据本发明的实施例，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种。本发明实施例的免疫共刺激分子胞内段的表达以及抗PD-L1抗体表达的联合具有正向调控和增强细胞免疫应答的作用，本发明实施例的免疫共刺激分子胞内段的表达、无功能EGFR的表达以及抗PD-L1抗体表达的联合，使得本发明实施例的转基因淋巴细胞增殖对肿瘤的定向杀伤作用更加显著和安全。
根据本发明的实施例，所述免疫共刺激分子胞内段是4-1BB或CD3的胞内段；
根据本发明的实施例，所述淋巴细胞是CD3 +T淋巴细胞、CD8 +T淋巴细胞、自然杀伤细胞或自然杀伤T细胞。CD3 +淋巴细胞是总T细胞，自然杀伤细胞是免疫细胞的一种，非特异性识别靶细胞，自然杀伤T细胞是具有T细胞和自然杀伤细胞受体的T细胞亚群。上述淋巴细胞中表达抗PD-L1抗体和表达嵌合抗原受体，使得上述淋巴细胞的细胞免疫的靶 向杀伤性更强，对肿瘤细胞的杀伤作用效果更加显著；上述淋巴细胞表达无功能EGFR和表达嵌合抗原受体，使得上述淋巴细胞的细胞免疫杀伤作用更加安全有效。
构建体
在本发明的另一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体包括：第一核酸分子，所述第一核酸分子编码抗PD-L1融合抗体，所述抗PD-L1融合抗体包括：PD-L1单链抗体、IgG1铰链区以及IgG1 Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1 Fc区相连，所述IgG1Fc区具有T250Q和M248L突变；第二核酸分子，所述第二核酸分子编码嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素；以及第三核酸分子，所述第三核酸分子编码无功能EGFR。根据本发明实施例的构建体导入淋巴细胞后，获得的转基因淋巴细胞能够表达和分泌抗PD-L1抗体，具有了显著的抵抗肿瘤介导的免疫抑制的功效，抗调亡能力和增殖能力增强、定向杀伤能力显著提高，免疫杀伤安全性显著提高，在肿瘤病人体内和体外的增殖和生存能力以及在肿瘤病人体内的杀伤能力大大提高，尤其对高表达MSLN及PD-L1分子的肿瘤细胞特异性杀伤效果尤为显著。
根据本发明的实施例，所述抗PD-L1融合抗体具有SEQ ID NO:3所示的氨基酸序列。
根据本发明的实施例，所述嵌合抗原受体具有SEQ ID NO:4或5所示的氨基酸序列。
Figure PCTCN2019112530-appb-000005
Figure PCTCN2019112530-appb-000006
具有SEQ ID NO:5所示氨基酸序列的嵌合抗原受体相比具有SEQ ID NO:4所示氨基酸序列的嵌合抗原受体在CD8分子区段的胞外段和胞内段更长。CD8铰链区、跨膜区和胞内区氨基酸序列将影响CAR蛋白分子空间构型，双聚体和多聚体形成，进而影响与下游信号传导分子的结合，从而影响淋巴细胞激活程度和细胞因子产生水平。具有SEQ ID NO：5所示氨基酸序列的嵌合抗原受体包括：胞外区，所述胞外区包括单链抗体和铰链区，所述单链抗体包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别抗原人MSLN，所述铰链区包括含人CD8分子胞外段和3个附加氨基酸残基AAA，所述AAA位于所述人CD8分子胞外段的N端，所述人CD8分子胞外段具有55个氨基酸残基；跨膜区，所述跨膜区包括人CD8分子跨膜段，所述人CD8分子跨膜段与所述胞外区的铰链区相连，并且嵌入到所述T淋巴细胞的细胞膜中；胞内区，所述胞内区包括人CD8分子胞内段和4-1BB分子胞内段以及CD3ζ链胞内段，所述人CD8分子胞内段具有7个氨基酸残基，所述人CD8分子胞内段与所述人CD8分子跨膜段相连。相比于SEQ ID NO:4，SEQ ID NO:5嵌合抗原受体的空间构型不同，表达该嵌合抗原受体的淋巴细胞具有细胞增殖更加温和，细胞因子分泌更少，细胞因子释放综合征和神经毒性副作用更少的优势。
根据本发明的实施例，所述无功能EGFR具有SEQ ID NO:6所示的氨基酸序列。
Figure PCTCN2019112530-appb-000007
根据本发明的实施例，所述编码抗PD-L1融合抗体的核酸分子具有SEQ ID NO:7～9所示的核苷酸序列。
Figure PCTCN2019112530-appb-000008
Figure PCTCN2019112530-appb-000009
Figure PCTCN2019112530-appb-000010
Figure PCTCN2019112530-appb-000011
根据本发明的实施例，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:10或11所示的核苷酸序列。
Figure PCTCN2019112530-appb-000012
Figure PCTCN2019112530-appb-000013
Figure PCTCN2019112530-appb-000014
根据本发明的实施例，所述编码无功能EGFR的核酸分子具有SEQ ID NO:12所示的核苷酸序列。
Figure PCTCN2019112530-appb-000015
根据本发明的实施例，所述慢病毒携带具有SEQ ID NO：13～18任一所示核苷酸序列的核酸分子。
Figure PCTCN2019112530-appb-000016
Figure PCTCN2019112530-appb-000017
Figure PCTCN2019112530-appb-000018
Figure PCTCN2019112530-appb-000019
Figure PCTCN2019112530-appb-000020
Figure PCTCN2019112530-appb-000021
Figure PCTCN2019112530-appb-000022
Figure PCTCN2019112530-appb-000023
Figure PCTCN2019112530-appb-000024
Figure PCTCN2019112530-appb-000025
Figure PCTCN2019112530-appb-000026
Figure PCTCN2019112530-appb-000027
Figure PCTCN2019112530-appb-000028
Figure PCTCN2019112530-appb-000029
Figure PCTCN2019112530-appb-000030
Figure PCTCN2019112530-appb-000031
Figure PCTCN2019112530-appb-000032
根据本发明的实施例，上述SEQ ID NO:13～18所示核苷酸序列的结构可表示为S-VL(LS1)LVH(LS1)-IgG1 HC(T250Q/M248L)-2A-MSLN CAR-(ires)-tEGFR，其中，S:信号 肽signal leader,L表示连接肽Linker，LS1表示抗PD-L1 Mab clone#LS1，VL表示轻链可变区light chain variable region，VH表示重链可变区heavy chain variable region，H表示IgG1 Fc铰链区hinge region，C表示IgG1重链稳定区heavy chain constant region，T250Q/M248L:IgG Fc T250Q和M248L氨基酸突变，2A:2A连接肽。其中，SEQ ID NO:13、15、17所示核苷酸序列中编码的MSLN CAR短于SEQ ID NO:14、16、18所示核苷酸序列中编码的MSLN CAR，SEQ ID NO:13、15、17所示核苷酸序列中编码的MSLN CAR在本文中简写为MSLN CAR(short)，SEQ ID NO:14、16、18所示核苷酸序列中编码的MSLN CAR在本文中简写为MSLN CAR(86)。
根据本发明的实施例，发明人是通过如下方式的至少之一实现上述抗PD-L1抗体、嵌合抗原受体以及无功能EGFR分别独立地表达的，其中，需要说明的是，此处的表达既指蛋白的表达又指RNA转录。
内部核糖体进入位点序列(IRES)，本发明实施例的内部核糖体进入位点序列设置在编码嵌合抗原受体的核酸分子与表达无功能EGFR的核酸分子之间，内部核糖体进入位点具有SEQ ID NO：19所示的核苷酸序列。
Figure PCTCN2019112530-appb-000033
内部核糖体进入位点通常位于RNA病毒基因组的5’非翻译区(UTR)，这样一个病毒蛋白的翻译就可以不依赖于5‘帽子结构，另一个蛋白通常靠5’帽子结构起始翻译，IRES前后的两个基因的表达通常是成比例的。内部核糖体进入位点序列的引入使得编码抗PD-L1抗体的核酸分子、编码嵌合抗原受体的核酸分子与编码无功能EGFR的核酸分子分别独立的表达。根据本发明的实施例，本发明实施例采用内部核糖体进入位点序列有效保证了嵌合抗原受体和无功能EGFR的高效表达，使得淋巴细胞对高表达MSLN肿瘤的特异性杀伤 效果更加显著，免疫杀伤安全性进一步提高。
启动子：第一启动子，第一启动子与编码嵌合抗原受体的核酸分子可操作地连接；第二启动子，第二启动子与沉默免疫检查点的核酸分子可操作地连接；以及第三启动子，第三启动子与表达无功能EGFR的核酸分子可操作地连接。根据本发明的实施例，所采用的第一启动子、第二启动子和第三启动子分别独立地选自U6，CMV,H1，EF-1,LTR,RSV启动子，第一以及第二启动子和第三启动子的引入，使得编码抗PD-L1抗体的核酸分子、编码嵌合抗原受体的核酸分子和表达无功能EGFR的核酸分子分别独立的表达，从而有效抑制了PD1/PD-L1介导的肿瘤免疫逃逸或高效表达了无功能EGFR，并且保证了嵌合抗原受体的高效表达，使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向作用更强，对肿瘤的特异性杀伤作用更加显著，免疫杀伤的安全性进一步提高。
第四核酸分子:第四核酸分子设置在所述第一核酸分子与第二核酸分子或第二核酸分子与所述第三核酸分子之间，并且所述第四核酸分子编码连接肽，所述连接肽能够在所述淋巴细胞中被切割。2A连接肽具有SEQ ID NO：20～23所示的氨基酸序列。
E G R G S L L T C G D V E E N P G P(SEQ ID NO：20)。
A T N F S L L K Q A G D V E E N P G P(SEQ ID NO：21)。
Q C T N Y A L L K L A G D V E S N P G P(SEQ ID NO：22)。
V K Q T L N F D L L K L A G D V E S N P G P(SEQ ID NO：23)。
第四核酸分子及其相应表达的连接肽的引入使得无功能EGFR、嵌合抗原受体、抗PD-L1抗体成非融合状态表达在淋巴细胞中。根据本发明的实施例，本发明实施例连接肽的引入保证了无功能EGFR、嵌合抗原受体以及抗PD-L1抗体的生物学作用，其具特异性更强的肿瘤杀伤效果，安全性更高。
通过上述内部核糖体进入位点序列、或第一、第二、第三启动子或第三核酸分子的引入，使得细胞表达和分泌抗PD-L1抗体、表达无功能EGFR以及表达嵌合抗原受体，并且无功能EGFR和嵌合抗原受体呈非融合状态表达在淋巴细胞膜上，从而高效抑制了免疫负调控和保证了嵌合抗原受体的生物学作用，有效实现了转基因淋巴细胞的及时清除，从而使得淋巴细胞在肿瘤环境中的成活率大大提高，淋巴细胞的靶向杀伤作用更加显著，免疫杀伤的安全性进一步提高。
另外，根据本发明的实施例，本发明实施例的构建体的载体是非致病性病毒载体。非致病性病毒载体大大提高了构建体在淋巴细胞中的复制和扩增效率，进而本发明实施例的淋巴细胞在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著，免疫杀伤安全性进一步提高。
根据本发明的实施例，本发明实施例的构建体的载体是病毒载体，病毒载体选自反转录病毒载体、慢病毒载体、腺病毒载体或腺病毒关联病毒载体的至少之一。根据本发明的实施例，本发明实施例的病毒的载体在病毒包装和感染过程中，病毒感染范围广泛，既可感染终末分化细胞，又可感染处于分裂期的细胞，既可整合到宿主染色体，又可游离在宿主染色体之外，实现广谱而高效的感染效率，从而细胞表面或细胞内免疫检查点被高效沉默和无功能EGFR被高效表达以及嵌合抗原受体在淋巴细胞中高效表达，本发明实施例的淋巴细胞的在肿瘤病人体内的增殖和生存能力大大提高，淋巴细胞的靶向作用进一步增强，对肿瘤细胞的杀伤作用更加显著，淋巴细胞的免疫杀伤安全性进一步提高。
根据本发明的具体实施例，以构建一个慢病毒载体为例，发明人为了构建一个慢病毒载体，在某些病毒序列的位置，将目的核酸插入到病毒基因组中，从而产生复制缺陷的病毒。为了产生病毒体，发明人进而构建包装细胞系(包含gag，pol和env基因，但不包括LTR和包装成分)。发明人将含有目的基因的重组质粒，连同慢病毒LTR和包装序列，一起引入包装细胞系中。包装序列允许重组质粒RNA转录产物被包装到病毒颗粒中，然后被分泌到培养基中。进而发明人收集包含重组慢病毒的基质，有选择性地浓缩，并用于基因转移。慢载体可以感染多种细胞类型，包括可分裂细胞和不可分裂细胞。
另外，根据本发明的实施例，本发明实施例的慢病毒是复合慢病毒，除了常见的慢病毒基因gag，pol和env，还包含有调控和结构功能的其他基因。慢病毒载体是本领域技术人员所熟知的，慢病毒包括：人类免疫缺陷病毒HIV–1，HIV–2和猿猴免疫缺陷病毒SIV。慢病毒载体通过多重衰减艾滋病毒致病基因产生，例如全部删除基因env，vif，vpr，vpu和nef，使慢病毒载体形成生物安全型载体。重组慢病毒载体能够感染非分裂细胞，同时可用于体内和体外基因转移和核酸序列表达。例如：在合适的宿主细胞中，和带有包装功能(gag，pol，env，rev和tat)的两个或更多的载体一起，能够感染非分裂细胞。重组病毒的靶向性，是通过抗体或特定配体(靶向特定细胞类型受体)与膜蛋白的结合来实现的。同时，重组病毒的靶向性通过插入一个有效序列(包括调控区域)到病毒载体中，连同另一个编码了特定靶细胞上的受体的配体的基因，使载体具有了特定的靶向。各种有用的慢病毒载体，以及各种方法和操作等产生的载体，用于改变细胞的表达。
根据本发明的实施例，本发明实施例的腺关联病毒载体(AAV)可使用一种或多种为人熟知的血清类型腺关联病毒载体的DNA构建。另外，根据本发明的实施例，本发明实施例的也包含微基因。微基因意味着用组合(选定的核苷酸序列和可操作的必要的相关连接序列)来指导转化、转录和/或基因产物在体内或体外的宿主细胞中的表达。应用“可操作的连接”序列包含连续目的基因的表达控制序列，和作用于反式或远距离控制目的基因的表 达控制序列。
另外，本发明实施例的载体还包括常规控制元素，在和质粒载体一起的细胞转染或/和病毒载体一起的细胞感染中。大量的表达控制序列(包括天然的，可诱导和/或特定组织的启动子)可能被使用。根据本发明的实施例，启动子为选自U6,H1,pol I,pol II and pol III的RNA聚合酶启动子。根据本发明的实施例，启动子为组织特异型启动子。根据本发明的实施例，启动子为诱导型启动子。根据本发明的实施例，启动子为选自基于所选载体的启动子。根据本发明的实施例，当选择慢病毒载体时，启动子为U6,H1,CMV IE基因，EF-1α，泛素C或磷酸甘油激酶(PGK)启动子。其他常规表达控制序列包括可选标记或报告基因，包括编码遗传霉素，潮霉素，氨苄青霉素或嘌呤霉素耐药性等的核苷酸序列。载体的其他组件包括复制起点。
构建载体的技术为本领域技术人员所熟知的，这些技术包括常规克隆技术，
制备转基因淋巴细胞的方法
在本发明的另一方面，本发明提出了一种制备前面所述的T淋巴细胞或者转基因淋巴细胞的方法。根据本发明的实施例，该方法包括：将前面所述的构建体或者前面所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。引入方式可以选自电转或病毒感染宿主细胞的方式引入。本发明实施例的构建体或慢病毒成功引入上述淋巴细胞或者T淋巴细胞中，实现了针对抗原MSLN的嵌合抗原受体的表达和抗PD-L1抗体的表达和分泌以及无功能EGFR的表达，从而使得所得淋巴细胞或T淋巴细胞具有显著的抵抗肿瘤介导的免疫抑制的功效，在肿瘤病人体内和体外的增殖及肿瘤病人体内存活能力大大提高，淋巴细胞或T淋巴细胞对肿瘤细胞，尤其是高表达MSLN及PD-L1的肿瘤细胞的靶向杀伤作用更强，免疫杀伤的安全性高。
治疗癌症的治疗组合物
在本发明的另一方面，本发明的提出了一种用于治疗癌症的治疗组合物。根据本发明的实施例，该治疗组合物包括：上述构建体、上述慢病毒、上述T淋巴细胞或者上述转基因淋巴细胞。上述任意一种治疗组合物的组成均可实现针对抗原MSLN嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达和转基因淋巴细胞或T淋巴细胞细胞表达和分泌抗PD-L1抗体；以及无功能EGFR在转基因淋巴细胞或T淋巴细胞表面的表达，从而使得所得转基因淋巴细胞或T淋巴细胞在体外扩增、在肿瘤病人体内的增殖及肿瘤病人体内存活能力大大提高，转基因淋巴细胞或T淋巴细胞对高表达MSLN及PD-L1分子的肿瘤细胞的靶向杀伤作用更强，免疫杀伤的安全性更高。
根据本发明的实施例，提供给患者的本发明实施例的治疗组合物,较好的应用于生物兼 容溶液或可接受的药学运载载体。作为准备的各种治疗组合物被悬浮或溶解在医药上或生理上可接受的载体，如生理盐水；等渗的盐溶液或其他精于此道的人的比较明显的配方中。适当的载体在很大程度上取决于给药途径。其他有水和无水的等渗无菌注射液和有水和无水的无菌悬浮液，是医药上可接受的载体。
根据本发明的实施例，足够数量的病毒载体被转导入靶向T细胞中，并提供足够强度的转基因，表达和分泌抗PD-L1抗体和表达无功能EGFR以及表达特有的MSLN嵌合抗原受体。治疗试剂的剂量主要取决于治疗状况，年龄，体重，病人的健康程度，从而可能造成病人的变异性。
表达和分泌抗PD-L1抗体和表达无功能EGFR以及表达特有的针对抗原MSLN嵌合抗原受体的这些方法是联合治疗的一部分。这些病毒载体和用于过继免疫治疗的抗肿瘤T细胞，可以被单独或结合其他治疗癌症的方法一起执行。在合适的条件下，一个治疗方法的包括使用一个或多个药物疗法。
根据本发明的实施例，所述癌症包括间皮瘤。表达和分泌抗PD-L1抗体和无功能EGFR的表达，联合嵌合抗原受体在转基因淋巴细胞或T淋巴细胞中的高效表达，使得所得淋巴细胞或T淋巴细胞在间皮瘤的环境中的存活能力大大提高，淋巴细胞或T淋巴细胞对高表达MSLN的肿瘤细胞的靶向杀伤作用更强，尤其对高表达MSLN及PD-L1分子的肿瘤细胞的杀伤作用更加显著，对高表达MSLN及PD-L1分子的肿瘤细胞的免疫杀伤作用更加安全有效。
提高淋巴细胞活性和治疗安全性的方法
在本发明的另一方面，本发明提出了一种提高淋巴细胞活性和治疗安全性的方法，本发明实施例的淋巴细胞携带嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，根据本发明的实施例，该方法包括：使所述淋巴细胞表达抗PD-L1融合抗体；以及使所述淋巴细胞表达无功能EGFR，所述抗PD-L1融合抗体、淋巴细胞、嵌合抗原受体、无功能EGFR是如前所定义的。根据本发明的实施例，本发明实施例的淋巴细胞活性包括淋巴细胞体外增殖能力，在肿瘤病人体内的增殖和生存能力以及淋巴细胞在肿瘤病人体内的杀伤能力的至少一种。根据本发明的实施例，本发明实施例的淋巴细胞的细胞表面或细胞内免疫检查点被沉默，淋巴细胞被活化、增生性反应上调、细胞因子分泌增多、抗调亡能力增强。本发明实施例的淋巴细胞在体外扩增和增殖、对肿瘤细胞的靶向杀伤作用显著增强。
无功能EGFR缺少N-端配体结合区和细胞内受体酪氨酸激酶活性，但包括野生型EGFR受体的跨膜区和完整的与抗EGFR抗体结合的序列，无功能EGFR可作为淋巴细胞的自杀标记。本发明是实施例的淋巴细胞在用于治疗高表达MSLN及PD-L1分子的肿瘤细胞时， 如果病人出现严重不良反应，本发明实施例的淋巴细胞可被抗EGFR抗体清除，进而可提高本发明实施例的淋巴细胞治疗高表达MSLN及PD-L1分子的肿瘤病人的安全性。
下面将结合实施例对本发明的方案进行解释。
本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本发明实施例中所用到的细胞系和基本实验技术如下所述：
慢病毒的产生和人T淋巴细胞的转导
产生复制缺陷的慢病毒载体，并将慢病毒载体离心收集用于人T淋巴细胞的转导。下面简要介绍慢病毒载体的产生、收集的实验过程：将293T细胞铺在底面积为150-平方厘米的细胞培养皿中，并根据说明书，使用Express-In(购自Open Biosystems/Thermo Scientific，Waltham，MA)对293T细胞进行病毒转导。每盘细胞加入15微克的慢病毒转基因质粒、5微克的pVSV-G(VSV糖蛋白表达质粒)、10微克的pCMVR8.74质粒(Gag/Pol/Tat/Rev表达质粒)和174微升的Express-In(浓度为1微克/微升)。分别于24小时和48小时收集上清，并使用超速离心机在28，000rpm(离心机转子为Beckman SW 32Ti，购自Beckman Coulter，Brea，CA)的条件下离心2小时。最后用0.75ml的RPMI-1640培养基对病毒质粒沉淀进行重悬。
从健康志愿者供体上分离人原代T淋巴细胞。人T淋巴细胞培养在RPMI-1640培养基中并使用抗CD3和CD28的单克隆抗体包被的珠(购自Invitrogen，Carlsbad，CA)进行刺激激活。人T淋巴细胞激活后的18～24小时，采用自旋-接种的方法对T淋巴细胞进行转导，转导过程如下所述：在24-孔板中，每孔铺有0.5×10 6T淋巴细胞，向每孔细胞中加入0.75ml的上述重悬的病毒上清和Polybrene(浓度为8微克/ml)。细胞和病毒质粒的混合液在台式离心机(购自Sorvall ST 40；Thermo Scientific)中离心，离心条件是室温，2500rpm，时间为90分钟。人重组白细胞介素-2(IL-2；购自Novartis，Basel，Switzerland)每隔2～3天加入T淋巴细胞培养液中，IL-2的终浓度为100-IU/ml，在T淋巴细胞培养过程中，保持细胞的密度为0.5×10 6～1×10 6/ml。一旦被转导的T淋巴细胞出现休眠，例如细胞生长速度变慢和细胞变小，其中，细胞生长速度和大小是通过Coulter Counter(购自Beckman Coulter)评估的，或被转导的T淋巴细胞在某个计划的时间点上，T淋巴细胞即可用来做功能分析。
本申请的实施例中所用的流式细胞仪为BD FACSCanto II(购自BD Biosciences)，并且流式细胞分析数据使用FlowJo version 7.2.5软件(购自Tree Star，Ashland，OR)进行分析。
铬释放实验
实施例中应用4–小时51铬释放法分析评估抗MSLN嵌合抗原受体T细胞(抗MSLN CAR T淋巴细胞)的细胞毒活性。具体步骤如下：目标测试细胞用51Cr在37摄氏度下标记1小时。标记后，用含有10％胎牛血清(FCS)的RPMI培养基润洗细胞。润洗后，将细胞重悬在相同的培养基中，重悬细胞的浓度是1×10 5/ml。转导后T细胞以不同的靶效细胞比值(T：E)加入目标测试细胞悬浮液中，并将细胞种在96-孔中，每孔体积是200微升。将细胞在37度培养箱中培养4小时。4小时后，从每孔中取出30微升的上清放于计数器的96-微孔板进行计数分析。分析仪器是顶级计数NXT微闪烁计数器(购自Packard Bioscience)。所有计数孔中效应细胞的数目是基于T细胞总数来计算的。被标记的目标测试细胞是MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1靶细胞。
实施例2构建共表达抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白和抗MSLN嵌合抗原受体的载体
本实施例中，发明人将编码有抗人MSLN的单链抗体的序列、4-1BB胞内段和T细胞受体组合的ζ-链序列克隆到含有EF-1启动子的慢病毒载体(lentiviral vector)上，克隆过程中，选择的限制性酶切是XbaI和NotI双酶切，以及NotI和XhoI双酶切，通过酶切、连接、筛选和目的质粒的扩增，生成表达抗MSLN嵌合抗原受体的慢病毒质粒(LV-MSLN CAR)。抗PD-L1单链抗体(LS1clone)和含有氨基酸突变的IgG Fc融合蛋白的序列被克隆进LV-MSLN CAR载体质粒，构建成慢病毒载体αLS1-QL/M-CAR。图1是慢病毒载体的示意图，包含编码抗PD-L1单链抗体(LS1clone)和含有氨基酸突变的IgG Fc融合蛋白序列，2A肽序列，抗MSLN嵌合抗原受体的序列，IRES序列及tEGFR序列。慢病毒载体αLS1/M-CAR包含编码抗PD-L1单链抗体(LS1clone)序列，2A肽序列，抗MSLN嵌合抗原受体的序列，IRES序列及tEGFR序列。慢病毒载体αLS1-QL包含编码抗PD-L1单链抗体(LS1clone)序列，IRES序列及tEGFR序列。慢病毒载体M-CAR包含编码MSLN嵌合抗原受体的序列，IRES序列及tEGFR序列。
实施例3载体αLS1-QL/M-CAR转导T淋巴细胞分泌抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白来抑制PD-1:PD-L1作用
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。在T淋巴细胞激活因子磁珠CD3/CD28(购自Invitrogen，Carlsbad，CA)存在下，被激活的T淋巴细胞经过慢病毒载体转导、体外扩增 培养，方法如实施例1所述。在慢病毒载体转导后的3-7天，收获转导的T细胞(细胞个数是2×10 6/孔)培养上清，用于PD-1:PD-L1作用抑制实验。100ng/孔的PD-L1蛋白(Aerobiosystems,Boston,MA)被用于包被96-孔ELISA板。10ng生物素标记的PD1(Aerobiosystems,Boston,MA)与100ul各种CAR-T上清液混合或与50ng的商业购买的anti-hPD-L1(Aerobiosystems,Boston,MA)混合，补充体积至200uL，并将混合液加至包被好的96-孔板中。将加有混合液的96-孔板在室温下放置2小时，之后进行充分洗涤。将稀释的streptavidin-HRP加入孔板中，并在室温条件下放置1小时，同时进行缓慢震荡。1小时后，对孔板进行6-次洗涤，并将100μL TMB HRP底物加入，当蓝色在阳性对照孔中出现的时候，加入100uL 1N硫磺酸去终止反应。测量孔板在450nm处的OD值。抑制活性(The inhibition activity(％))＝(OD 450 of Mock-OD 450 of sample)/(OD 450 of Mock-OD 450 of background)x100％.
实验结果如图2所示。图2结果表明，转导了慢病毒载体αLS1-QL/M-CAR或αLS1-QL T淋巴细胞的培养液能显著抑制PD-1:PD-L1作用，而转导了慢病毒载体M-CAR T淋巴细胞的培养液没有抑制PD-1:PD-L1作用(*P<0.01；αLS1-QL/M-CAR vs.M-CAR)。这说明转导了αLS1-QL/M-CAR的T淋巴细胞能分泌抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白来抑制PD-1:PD-L1作用。
实施例4共表达抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白和抗MSLN嵌合抗原受体的T淋巴细胞有增强溶解肿瘤细胞能力(体外实验)
在本实施例中，外周血淋巴细胞取自不记名供血者。外周血淋巴细胞通过梯度离心进行分离，梯度离心机为Ficoll-Hypaque。T淋巴细胞与T细胞激活因子磁珠CD3/CD28(购自Invitrogen，Carlsbad，CA)在5％CO 2、37摄氏度下孵育培养72小时，培养基是加有2mmol/L谷氨酰胺，10％高温灭活的胎牛血清(FCS)(购自Sigma-Aldrich Co.)和100U/ml的青霉素/链霉素双抗的RPMI培养基1640(购自Invitrogen Gibco Cat.no.12633-012)。激活培养72小时后，用洗液润洗细胞，将磁珠洗去。将T细胞种在铺有重组纤连蛋白片段(FNch-296；Retronectin)细胞培养皿上，并用慢病毒转导，转导慢病毒分别为αLS1-QL/M-CAR,αLS1/M-CAR,αLS1-QL,M-CAR或空载(Control)转导过程如实施例1所述。转导后的T细胞培养在RPMI-1640培养基中并用重组人类IL-2因子(100ng/ml；购自R&D Systems)进行诱导扩增7-10天，然后进行功能测试实验。发明人测量转导了不同慢病毒的T细胞(效应细胞)对MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1靶细胞的杀伤作用，用不同靶效细胞比例，测量方法采用标准4–小时 51铬释放法，4–小时 51铬释放法如实施例1所述。
结果如图3所示。如图3所示，转导了慢病毒载体αLS1-QL/M-CAR T淋巴细胞比转导 了慢病毒载体M-CAR的T淋巴细胞，可更为有效的杀死MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1靶细胞。转导了慢病毒载体αLS1-QL/M-CAR T淋巴细胞比转导了慢病毒载体αLS1/M-CAR的T淋巴细胞，也可更为有效的杀死MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1靶细胞。空载慢病毒转导的T淋巴细胞(Control T淋巴细胞)对MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1靶细胞无明显杀伤作用。统计数据代表三个孔的平均值±SEM。*P<0.01；αLS1-QL/M-CAR vs.αLS1/M-CAR或M-CAR。这说明转导了αLS1-QL/M-CAR的T淋巴细胞能分泌抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白有更强体外杀伤MSLN +PD-L1 +靶细胞能力。
实施例5共表达抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白和抗MSLN嵌合抗原受体的T淋巴细胞有增强抗肿瘤能力(体内鼠肿瘤模型实验)
免疫缺陷型非肥胖糖尿病(NOD)-scidγ小鼠(NSG)(SCID小鼠背景，并且具有IL-2受体γ链缺陷)被用来测试各种转导T细胞的抗肿瘤活性。NSG小鼠(5只/每组)被注射5x10 5个MSLN +PD-L1 +H226(左图)或者MSLN +PD-L1 +Panc1肿瘤细胞。在注射后的6～9天，肿瘤细胞直径达到4-5mm时，小鼠静脉注射1x10 7不同的转导T细胞。在指定时间用卡尺测量肿瘤的体积。肿瘤体积(Tumor volume)＝W 2L(宽 2长).
结果如图4所示。如图4所示，转导了慢病毒载体αLS1-QL/M-CAR T淋巴细胞比转导了慢病毒载体M-CAR的T淋巴细胞，可更为有效的杀死MSLN +PD-L1 +H226细胞或MSLN +PD-L1 +Panc1肿瘤细胞。转导了慢病毒载体αLS1-QL/M-CAR T淋巴细胞比转导了慢病毒载体αLS1/M-CAR的T淋巴细胞，也可更为有效的杀死MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1肿瘤细胞。空载慢病毒转导的T淋巴细胞(Control T淋巴细胞)对MSLN +PD-L1 +H226 cells或MSLN +PD-L1 +Panc1肿瘤细胞无明显杀伤作用。*P<0.01；αLS1-QL/M-CAR vs.αLS1/M-CAR或M-CAR。这说明转导了αLS1-QL/M-CAR的T淋巴细胞能分泌抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白有更强体内杀伤MSLN +PD-L1 +肿瘤细胞能力。
实施例6载体αLS1-QL/M-CAR转导T淋巴细胞治疗鼠的血清有抑制PD-1:PD-L1作用。
实施例5中所用到的NSG小鼠在注射了转导T细胞后，饲养48小时。将血清分离，并从每组4只小鼠中均等地混合。100ng/孔的商业购买的PD-L1蛋白(Aerobiosystems,Boston,MA)包被96-孔ELISA板。10ng生物素标记的PD1(Aerobiosystems,Boston,MA)与100ul各种CAR-T上清混合或与50ng的商业购买的抗-hPD-L1(Aerobiosystems,Boston,MA)混合，混合后补充体积至200uL，并加入到包被好的板子李。板子在室温条 件下孵育2小时，并进行充分洗涤。之后将稀释的streptavidin-HRP加入到板子里，并在室温条件下孵育1小时，同时进行缓慢震荡。之后，将板子洗涤5次并加入100uL TMB HRP底物，当阳性对照孔转变为蓝色时加入100μL的1N硫酸终止反应。测量板子在450nm处的OD值。抑制率(The inhibition activity(％))＝(OD450 of Mock-OD450 of sample)/(OD450 of Mock-OD450 of background)x100％.
实验结果如图5所示。图5结果表明，载体αLS1-QL/M-CAR转导T淋巴细胞治疗鼠的血清能显著抑制PD-1:PD-L1作用，而转导了慢病毒载体M-CAR T淋巴细胞的培养液没有抑制PD-1:PD-L1作用(*P<0.01；αLS1-QL/M-CAR vs.M-CAR).载体αLS1/M-CAR转导T淋巴细胞治疗鼠的血清仅有弱的抑制PD-1:PD-L1作用(*P<0.01；αLS1/M-CAR)。这说明转导了αLS1-QL/M-CAR的T淋巴细胞能分泌抗PD-L1单链抗体和含有氨基酸突变的IgG Fc融合蛋白有明显体内抑制PD-1:PD-L1作用。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (24)

  1. 一种T淋巴细胞，其特征在于，所述T淋巴细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，
    其中，
    所述嵌合抗原受体包括：
    胞外区，所述胞外区包括单链抗体的重链可变区和轻链可变区，所述单链抗体特异性识别肿瘤抗原间皮素；
    跨膜区，所述跨膜区与所述胞外区相连，所述跨膜区包括CD8的跨膜段，并且嵌入到所述T淋巴细胞的细胞膜中；
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括4-1BB的胞内段以及CD3ζ链；
    所述抗PD-L1融合抗体包括：
    PD-L1单链抗体、IgG1铰链区以及IgG1Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1Fc区相连，所述IgG1Fc区具有T250Q和M248L氨基酸突变。
  2. 根据权利要求1所述的T淋巴细胞，其特征在于，所述抗PD-L1单链抗体具有SEQ ID NO:1所示的氨基酸序列。
  3. 根据权利要求1所述的T淋巴细胞，其特征在于，所述IgG1Fc区具有SEQ ID NO:2所示的氨基酸序列。
  4. 根据权利要求1所述的T淋巴细胞，其特征在于，所述抗PD-L1融合抗体具有SEQ ID NO:3所示的氨基酸序列。
  5. 一种慢病毒，其特征在于，所述慢病毒携带下列核酸分子：
    (a)编码抗PD-L1融合抗体的核酸分子，所述抗PD-L1融合抗体包括：
    PD-L1单链抗体、IgG1铰链区以及IgG1Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1Fc区相连，所述IgG1Fc区具有T250Q和M248L氨基酸突变；
    (b)编码嵌合抗原受体的核酸分子，所述嵌合抗原受体的胞外区识别肿瘤抗原间皮素；以及
    (c)编码无功能EGFR的核酸分子。
  6. 根据权利要求5所述的慢病毒，其特征在于，所述抗PD-L1融合抗体具有SEQ ID NO:3所示的氨基酸序列；
    任选地，所述嵌合抗原受体具有SEQ ID NO:4或5所示的氨基酸序列；
    任选地，所述无功能EGFR具有SEQ ID NO:6所示的氨基酸序列。
  7. 根据权利要求6所述的慢病毒，其特征在于，所述编码抗PD-L1融合抗体的核酸分子具有SEQ ID NO:7～9任一所示的核苷酸序列；
    任选地，所述编码嵌合抗原受体的核酸分子具有SEQ ID NO:10或11所示的核苷酸序列；
    任选地，所述编码无功能EGFR的核酸分子具有SEQ ID NO:12所示的核苷酸序列。
  8. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：13所示核苷酸序列的核酸分子。
  9. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：14所示核苷酸序列的核酸分子。
  10. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：15所示核苷酸序列的核酸分子。
  11. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：16所示核苷酸序列的核酸分子。
  12. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：17所示核苷酸序列的核酸分子。
  13. 一种慢病毒，其特征在于，所述慢病毒携带具有SEQ ID NO：18所示核苷酸序列的核酸分子。
  14. 一种转基因淋巴细胞，其特征在于，所述转基因淋巴细胞共表达抗PD-L1融合抗体、无功能EGFR以及嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，
    其中，
    所述嵌合抗原受体包括：
    胞外区；
    跨膜区，所述跨膜区与所述胞外区相连，并且嵌入到所述转基因淋巴细胞的细胞膜中；
    胞内区，所述胞内区与所述跨膜区相连，并且所述胞内区包括免疫共刺激分子胞内段；
    所述抗PD-L1融合抗体包括：
    PD-L1单链抗体、IgG1铰链区以及IgG1Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1Fc区相连，所述IgG1Fc区具有T250Q和M248L氨基酸突变。
  15. 根据权利要求14所述的转基因淋巴细胞，其特征在于，所述免疫共刺激分子胞内段独立地选自4-1BB、OX-40、CD40L、CD27、CD30、CD28、CD3以及他们的衍生物的至少一种；
    优选地，所述免疫共刺激分子胞内段是4-1BB、CD3的胞内段；
    任选地，所述淋巴细胞是CD3 +T淋巴细胞；
    任选地，所述淋巴细胞是CD8 +T淋巴细胞；
    任选地，所述淋巴细胞是自然杀伤细胞；
    任选地，所述淋巴细胞是自然杀伤T细胞。
  16. 一种构建体，其特征在于，所述构建体包括：
    第一核酸分子，所述第一核酸分子编码抗PD-L1融合抗体，所述抗PD-L1融合抗体包括：
    PD-L1单链抗体、IgG1铰链区以及IgG1Fc区，所述PD-L1单链抗体通过IgG1铰链区与所述IgG1Fc区相连，所述IgG1Fc区具有T250Q和M248L氨基酸突变；
    第二核酸分子，所述第二核酸分子编码嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素；以及
    第三核酸分子，所述第三核酸分子编码无功能EGFR，
    其中，所述抗PD-L1融合抗体、所述嵌合抗原受体、所述无功能EGFR是如权利要求1～7、14～15任一项中所定义的。
  17. 根据权利要求16所述的构建体，其特征在于，所述第一核酸分子、所述第二核酸分子和所述第三核酸分子被设置为在权利要求15～16任一项所述的淋巴细胞中表达所述抗PD-L1融合抗体、嵌合抗原受体和无功能EGFR，并且所述抗PD-L1融合抗体、嵌合抗原受体与所述无功能EGFR呈非融合形式。
  18. 根据权利要求16所述的构建体，其特征在于，进一步包括：
    第一启动子，所述第一启动子与所述第一核酸分子可操作地连接；
    第二启动子，所述第二启动子与所述第二核酸分子可操作地连接；以及
    第三启动子，所述第三启动子与所述第三核酸分子可操作地连接；
    任选地，所述第一启动子、所述第二启动子、所述第三启动子分别独立地选自U6，H1，CMV，EF-1，LTR或RSV启动子；
    任选地，进一步包括：
    内部核糖体进入位点序列，所述内部核糖体进入位点序列设置在所述第一核酸分子与所述第三核酸分子之间或所述第二核酸分子与所述第三核酸分子之间，所述内部核糖体进入位点具有SEQ ID NO：19所示的核苷酸序列；
    任选地，进一步包括:
    第四核酸分子，所述第四核酸分子设置在所述第一核酸分子与所述第二核酸分子之间 或所述第二核酸分子与所述第三核酸分子之间，并且所述第四核酸分子编码连接肽，所述连接肽能够在所述淋巴细胞中被切割；
    任选地，所述连接肽具有SEQ ID NO：20～23所示的氨基酸序列。
  19. 根据权利要求16所述的构建体，其特征在于，所述构建体的载体是非致病性病毒载体；
    任选地，所述病毒载体包括选自反转录病毒载体、慢病毒载体或腺病毒相关病毒载体的至少之一。
  20. 一种制备权利要求1～4任一项所述的T淋巴细胞或者权利要求14～15任一项所述的转基因淋巴细胞的方法，其特征在于，包括：
    将权利要求16～19任一项所述的构建体或者权利要求5～13任一项所述的慢病毒引入到淋巴细胞中或者T淋巴细胞。
  21. 一种用于治疗癌症的治疗组合物，其特征在于，包括：
    权利要求16～19任一项所述的构建体、权利要求5～13任一项所述的慢病毒、权利要求1～4任一项所述的T淋巴细胞或者权利要求14～15任一项所述的转基因淋巴细胞。
  22. 根据权利要求21所述的治疗组合物，其特征在于，所述癌症包括选自间皮瘤，胰腺癌，卵巢癌，胆管癌，肺癌，胃癌，肠癌，食管癌和乳腺癌的至少之一。
  23. 一种提高淋巴细胞活性和治疗安全性的方法，所述淋巴细胞携带嵌合抗原受体，所述嵌合抗原受体识别肿瘤抗原间皮素，其特征在于，所述方法包括：
    使所述淋巴细胞表达抗PD-L1融合抗体，以及
    使所述淋巴细胞表达无功能EGFR，
    所述抗PD-L1融合抗体、所述淋巴细胞、所述嵌合抗原受体、所述无功能EGFR如权利要求1～7、14～15任一项中所定义的，
    所述淋巴细胞活性包括所述淋巴细胞在体外的增殖能力、在肿瘤病人体内的增殖和生存能力以及所述淋巴细胞在肿瘤病人体内的定向杀伤能力的至少一种。
  24. 一种治疗癌症的方法，其特征在于，包括：
    给患者给予权利要求16～19任一项所述的构建体、权利要求5～13任一项所述的慢病毒、权利要求1～4任一项所述的T淋巴细胞或者权利要求14～15任一项所述的转基因淋巴细胞。
PCT/CN2019/112530 2018-10-24 2019-10-22 Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗 WO2020083282A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811246615.7 2018-10-24
CN201811246615.7A CN111088231A (zh) 2018-10-24 2018-10-24 Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗

Publications (1)

Publication Number Publication Date
WO2020083282A1 true WO2020083282A1 (zh) 2020-04-30

Family

ID=70331956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/112530 WO2020083282A1 (zh) 2018-10-24 2019-10-22 Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗

Country Status (2)

Country Link
CN (1) CN111088231A (zh)
WO (1) WO2020083282A1 (zh)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021009694A1 (en) * 2019-07-17 2021-01-21 National University Of Singapore Functional binders synthesized and secreted by immune cells
US11253547B2 (en) 2019-03-05 2022-02-22 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
US11365236B2 (en) 2017-03-27 2022-06-21 Nkarta, Inc. Truncated NKG2D chimeric receptors and uses thereof in natural killer cell immunotherapy
US11560548B2 (en) 2014-05-15 2023-01-24 National University Of Singapore Immune cells expressing membrane-bound interleukin 15 (mbIL15) and uses thereof
US11896616B2 (en) 2017-03-27 2024-02-13 National University Of Singapore Stimulatory cell lines for ex vivo expansion and activation of natural killer cells

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW202216990A (zh) * 2020-09-08 2022-05-01 中國大陸商亘喜生物科技（上海）有限公司 用於ｔ細胞工程化的組合物和方法
CN114107390B (zh) * 2021-11-05 2023-10-24 中国科学院精密测量科学与技术创新研究院 一种用于表达抗体IgG1的rAAV载体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106350533A (zh) * 2015-10-09 2017-01-25 上海宇研生物技术有限公司 Anti‑PD‑L1‑CAR‑T及其制备方法和应用
WO2018137295A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 共表达抗msln嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
WO2018137294A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途
WO2018137293A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 治疗间质素阳性肿瘤的治疗组合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2592216T3 (es) * 2008-09-26 2016-11-28 Dana-Farber Cancer Institute, Inc. Anticuerpos anti-PD-1, PD-L1 y PD-L2 humanos y sus usos
CN107827990B (zh) * 2017-10-30 2020-07-10 河北森朗生物科技有限公司 一种多肽、编码其的核酸、其修饰的t淋巴细胞及其应用
CN108642070B (zh) * 2018-04-11 2022-03-15 沈阳金石生物制药有限公司 特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106350533A (zh) * 2015-10-09 2017-01-25 上海宇研生物技术有限公司 Anti‑PD‑L1‑CAR‑T及其制备方法和应用
WO2018137295A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 共表达抗msln嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
WO2018137294A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途
WO2018137293A1 (zh) * 2017-01-25 2018-08-02 北京马力喏生物科技有限公司 治疗间质素阳性肿瘤的治疗组合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATTA-MANNAN A.: "Monoclonal antibody clearance. Impact of modulating the interaction of IgG with the neonatal Fc receptor", J BIOL CHEM., vol. 282, no. 3, 19 January 2007 (2007-01-19), pages 1709 - 1717, XP055043665 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11560548B2 (en) 2014-05-15 2023-01-24 National University Of Singapore Immune cells expressing membrane-bound interleukin 15 (mbIL15) and uses thereof
US11365236B2 (en) 2017-03-27 2022-06-21 Nkarta, Inc. Truncated NKG2D chimeric receptors and uses thereof in natural killer cell immunotherapy
US11896616B2 (en) 2017-03-27 2024-02-13 National University Of Singapore Stimulatory cell lines for ex vivo expansion and activation of natural killer cells
US11253547B2 (en) 2019-03-05 2022-02-22 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
WO2021009694A1 (en) * 2019-07-17 2021-01-21 National University Of Singapore Functional binders synthesized and secreted by immune cells
CN114286683A (zh) * 2019-07-17 2022-04-05 新加坡国立大学 由免疫细胞合成和分泌的功能性结合物

Also Published As

Publication number Publication date
CN111088231A (zh) 2020-05-01

Similar Documents

Publication Publication Date Title
WO2020083282A1 (zh) Pd-l1抗体分泌的抗间皮素car-t细胞肿瘤免疫治疗
CN110950953B (zh) 抗b7-h3的单克隆抗体及其在细胞治疗中的应用
EP3114147B1 (en) Chimeric antigen receptor
AU2007241023B2 (en) Methods and compositions for localized secretion of anti-CTLA-4 antibodies
JP7288503B2 (ja) 改変された抗ｃｄ１９ ｃａｒ－ｔ細胞
US9469684B2 (en) Therapeutic and diagnostic cloned MHC-unrestricted receptor specific for the MUC1 tumor associated antigen
CN108373504B (zh) Cd24特异性抗体和抗cd24-car-t细胞
Reul et al. Tumor-specific delivery of immune checkpoint inhibitors by engineered AAV vectors
JP2023134640A (ja) 固形癌のためのキメラ抗原受容体及びキメラ抗原受容体が発現されたｔ細胞
WO2022007804A1 (zh) T淋巴细胞及其应用
WO2017120996A1 (zh) 共表达抗EGFRvIII嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
WO2023046110A1 (zh) 联合表达CCR2b的工程化免疫细胞及其制备和应用
WO2017120998A1 (zh) 治疗脑胶质母细胞瘤的治疗组合物
WO2021043169A1 (zh) 特异性结合b细胞成熟抗原的抗体及其用途
WO2017028374A1 (en) Construct, genetically modified lymphocyte, preparation method and usage thereof
WO2018006881A1 (zh) 重组免疫检查点受体及其应用
JP2024504817A (ja) 二重特異性ｃｓ１－ｂｃｍａ ｃａｒ－ｔ細胞及びその適用
WO2021232200A1 (en) Il-12 armored immune cell therapy and uses thereof
WO2018137295A1 (zh) 共表达抗msln嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
WO2017133174A1 (zh) 治疗b细胞白血病及b细胞淋巴瘤的治疗组合物
WO2018137293A1 (zh) 治疗间质素阳性肿瘤的治疗组合物
WO2018137294A1 (zh) 共表达抗msln嵌合抗原受体和无功能egfr的转基因淋巴细胞及其用途
CN111944053B (zh) 抗bcma的car及其表达载体和应用
WO2017120997A1 (zh) 共表达抗EGFRvIII嵌合抗原受体和无功能EGFR的转基因淋巴细胞及其用途
US11680099B2 (en) Anti-PD-1/CD47 bispecific antibody and application thereof

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: 19876218

Country of ref document: EP

Kind code of ref document: A1

WA Withdrawal of international application
NENP Non-entry into the national phase

Ref country code: DE