WO2020019983A1 - Cellule génétiquement modifiée utilisée pour traiter une tumeur - Google Patents

Cellule génétiquement modifiée utilisée pour traiter une tumeur Download PDF

Info

Publication number
WO2020019983A1
WO2020019983A1 PCT/CN2019/095482 CN2019095482W WO2020019983A1 WO 2020019983 A1 WO2020019983 A1 WO 2020019983A1 CN 2019095482 W CN2019095482 W CN 2019095482W WO 2020019983 A1 WO2020019983 A1 WO 2020019983A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
car
genetically engineered
engineered cell
cell
Prior art date
Application number
PCT/CN2019/095482
Other languages
English (en)
Chinese (zh)
Inventor
张书元
徐卫
林鸿
Original Assignee
赛诺生（深圳）基因产业发展有限公司
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 赛诺生（深圳）基因产业发展有限公司 filed Critical 赛诺生（深圳）基因产业发展有限公司
Publication of WO2020019983A1 publication Critical patent/WO2020019983A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • 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
    • 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
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C07K2319/33—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 invention belongs to the technical field of biological genetic engineering, and particularly relates to a chimeric antigen receptor (CAR) capable of expressing a tumor antigen, and a genetically engineered cell for treating a tumor.
  • CAR chimeric antigen receptor
  • Biological treatment of tumor is the fourth largest treatment method after surgery, radiotherapy and chemotherapy. Because the development of traditional surgery, radiotherapy and chemotherapy has entered the plateau, people are increasingly looking at the biological treatment of tumors.
  • Gene and cell immunotherapy for cancer is a cutting-edge technology of tumor biotherapy, with advantages such as relatively small toxic and side effects, and significant effects. Industry insiders expect gene cell immunotherapy to have the potential to become the fifth-largest cancer treatment.
  • To achieve cellular immunotherapy for cancer we must first overcome the containment of malignant cells by the immune system. With the development and maturation of gene therapy technology and targeted precision monoclonal antibody biotechnology, gene therapy technology and targeted precision monoclonal antibody biotechnology provide an effective solution to the problem of immune system cells accommodating cancer cells, which has never been done before. Solution.
  • This method involves genetically modifying autologous T cells to reprogram T cells to recognize tumor-specific antigens, prompting T cells to activate and eliminate malignant cancer cells.
  • This is the principle of chimeric antigen receptor (CAR) modified T cell (CAR-T) immunotherapy.
  • Chimeric antigen receptor (CAR) chimeric proteins have two important functional regions.
  • the first is a monoclonal antibody fragment (scFv) tumor antigen target binding domain that is expressed on the surface of T cells, which allows CAR-T cells to specifically recognize cancer antigen targets present on the surface of cancer cells to achieve the purpose of precise treatment.
  • Monoclonal antibody fragments are composed of variable regions of the light and heavy chains of a monoclonal antibody linked by a flexible linker.
  • the second is a T-cell activating factor expressed in T cells. When the antibodies on the surface of CAR-T cells are connected to the cancer antigen target, T cells can be activated in a way that is not related to the major histocompatibility complex (MHC). Anti-cancer effect.
  • MHC major histocompatibility complex
  • the original CAR was designed to directly link the monoclonal antibody fragment (scFv) tumor antigen target binding domain to the intracellular signal domain of CD3zeta via a hinge and a T cell receptor transmembrane region.
  • scFv monoclonal antibody fragment
  • the first-generation CAR-T could not fully activate T cells in the immunosuppressed tumor microenvironment and lacked T Cell expansion.
  • the researchers found that when a costimulatory domain signal molecule was added before the CD3zeta of the CAR's intracellular domain, the activation, expansion and persistence of CAR-T cells could be significantly improved, greatly improving the resistance Tumor effect.
  • CD19 glycoprotein expressed on the surface of B-cell malignant tumor cells is a relatively ideal tumor antigen.
  • CD19 is a B-cell surface protein that has been expressed throughout the development of B cells.
  • CD19 is expressed on the surface of almost all B-cell malignancies, including chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia ALL, and many non-Hodgkin's lymphomas (NHL).
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia ALL
  • NHL non-Hodgkin's lymphomas
  • CD19-positive tumors have become the object of clinical research for CAR-T cell immunotherapy.
  • Novymtis Kymriah TM tisagenlecleucel
  • Kite Pharma's Yescarta TM axicabtagene ciloleucel
  • BCMA B-cell maturation antigen
  • CD33 and CD123 glycoproteins have significantly higher expression on the surface of myeloid leukemia cancer cells, and can also be used as tumor-specific antigens for the treatment of CART cells. Recently it has been used in clinical experiments to treat relapsed or refractory acute myeloid leukemia.
  • CAR-T cells Given that T cells can actively travel to almost every part of the body and have the ability to overcome tumor escape, CAR-T cells have the unique potential of eliminating solid tumors in addition to treating blood cancers. Because most potential solid tumor antigen targets are non-specific and also expressed to varying degrees in healthy tissues, selecting appropriate solid tumor targets is relatively difficult and challenging. To date, solid tumor targets for CAR-T cell therapy include GD2, IL13Ra2, mesothelin, EGFRvIII, HER2, etc. Some have entered phase I clinical trials in humans for the treatment of glioblastoma multiforme (GBM).
  • GBM glioblastoma multiforme
  • Cytokine release syndrome is the most common side effect experienced by tumor patients receiving autologous CAR-T cell immunotherapy. Cytokine release syndrome (CRS) is an obvious systemic inflammatory response caused by the abrupt release of a large number of cytokines caused by the activation and exponential expansion of CAR-T cells in vivo. This usually occurs within a few days of the initial CAR-T cell infusion.
  • CRS Cerebral disease senor fever and flu-like symptoms
  • severe CRS symptoms include vasodilation, shock-induced capillary leakage, and dyspnea, leading to the need for intensive care unit care. In rare cases, it can cause brain edema and even death in patients.
  • varying degrees of neurotoxicity have been reported in patients treated with CAR-T cells, including delirium, encephalopathy, and seizures.
  • Intensive clinical monitoring indicates that the severity of CRS in patients is related to many factors.
  • IL6 is one of the most specific factors among the many cytokines released, which may play a central role in the pathophysiology induced by CRS.
  • Natural killer (NK) cells are a small portion (about 10%) of human peripheral lymphocytes with specific innate immune functions. They can spontaneously mediate the elimination of "natural" cytotoxicity without requiring prior inspiration. Certain tumors and viruses infect cells. It was then named natural killer (NK) cells. It is an important part of the human immune system. Since almost all healthy cells express MHC class I on the cell surface, NK cells, under the control of their own Ig-like receptor (KIR) inhibitory receptors, have no damaging effect on healthy autocells that normally express class I MHC. Down-regulating the expression of class I MHC is a fairly common mechanism for tumor and virus-infected cells to escape TCR recognition and killing of lytic T cells.
  • KIR Ig-like receptor
  • NK cells kill target cells of NK cells. They are those with down-regulated or no class I MHC expression, which is the so-called "lost self" principle.
  • the immune mechanism of NK cells overcomes the lack of this potential T cell immune mechanism and plays an important complementary role.
  • the mechanism of killing target cells by NK cells is the release of perforin mediated by targeted exocytosis of cytolytic particles and its penetration of target cell membranes into the cytoplasm to induce apoptosis.
  • NK cells do not have a single activated receptor, but multiple co-activated receptors, including natural cytotoxic receptors (NCR), NKp30, NKp44, and NKp46, NKG2D, CD16, 2B4, etc.
  • NCR natural cytotoxic receptors
  • NKp30, NKp44, and NKp46 NKG2D, CD16, 2B4, etc.
  • These activating receptors initiate the release of cytolytic granules containing perforin and granzyme by linking signal transfer proteins such as DAP10, DAP12, and CD3 ⁇ , and mediate the release of cytokines and chemokines such as IFN- ⁇ and TNF- ⁇ Wait.
  • the activation activity of NK cells is strictly inhibited.
  • NK cells have many inhibitory receptors, including killer cell Ig-like receptors (KIR), CD94-NKG2A, and LILR. Ultimately, the degree of activation of NK cells is weighed against the balanced integration of activated and inhibited receptors. This is a fundamental difference from killer T cell activation.
  • KIR killer cell Ig-like receptors
  • CD94-NKG2A CD94-NKG2A
  • LILR LILR
  • the CAR-T cell products currently on the market and under clinical development are basically autologous cell products.
  • the production process of auto-CAR-T cell products is very complicated, and the production cycle takes 2-3 weeks.
  • auto-CAR-T cell products are patient-specific, they are not suitable for large-scale GMP production, and the products are expensive.
  • waiting for their CAR-T cells for 2-3 weeks will be a big medical decision, because the patient's condition may worsen during this period.
  • patients also face the possibility that their CAR-T cells will fail during the production process and face the loss of valuable time for tumor treatment. Therefore, it is necessary to develop "ready-made" non-autologous (allogenic) CAR cell products suitable for large-scale industrialization of GMP and at an affordable price.
  • the object of the present invention is to provide an allogeneic engineered cell for treating tumors suitable for large-scale industrialization of GMP.
  • the genetically engineered cells for treating tumors provided by the present invention are chimeric antigen receptors CAR capable of simultaneously expressing tumor antigens and immune cells that safely kill switch factors.
  • the immune cells are allogeneic human immune cells.
  • the immune cells are allogeneic human natural killer cells NK. Therefore, the genetically engineered cell of the present application is preferably a CAR-NK cell.
  • the genetically engineered cells for treating tumors of the present invention are added with a safety kill switch factor without an immune response, and the safety kill switch factor used is a truncated human epidermal growth factor receptor polypeptide without immunogenicity ( EGFRt).
  • EGFRt human epidermal growth factor receptor polypeptide without immunogenicity
  • CAR-NK cells can still be killed by the antibody-dependent cytotoxicity (ADCC) pathway.
  • ADCC antibody-dependent cytotoxicity
  • nucleotide sequence of the truncated human epidermal growth factor receptor polypeptide according to the present invention is shown in SEQ ID NO.9.
  • the chimeric antigen receptor CAR of the tumor antigen is combined with the safety killing switch factor through the C-terminal T2A cleavable chain, and the nucleotide sequence of the C-terminal T2A cleavable chain is shown in SEQ ID NO.8.
  • the molecular structure of the chimeric antigen receptor CAR of the tumor antigen in the genetically engineered cells of the present invention includes: a) a monoclonal antibody fragment scFv on the cell surface, a tumor antigen target binding domain, b) a hinge and a transmembrane domain, and (c) Endoplasmic signal transduction domain composed of T cell receptor CD3 ⁇ domain and co-stimulatory signals.
  • CAR-modified NK cells can be obtained by combining the monoclonal antibody fragment (scFv) domain on the cell surface with the intracellular CD3zeta activation domain (necessary cells for NK cell activation) Internal signal molecules) to increase the activation signal intensity.
  • scFv monoclonal antibody fragment
  • CD3zeta activation domain nuclear cells for NK cell activation
  • Internal signal molecules Internal signal molecules
  • the monoclonal antibodies are CD19, CD20, BCMA, CD22, CD33, CD47, CD123, CD133, CD138, ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), GD2, Mesothelin, Muc1 and Muc 16, and CEA (carcinoembryonic antigen).
  • ROR1 Receptor Tyrosine Kinase Like Orphan Receptor 1
  • GD2 Mesothelin
  • Muc1 and Muc 16 adenothelin
  • CEA carcinoembryonic antigen
  • the nucleotide sequence of the monoclonal antibody fragment scFv is as shown in SEQ ID No. 4, 16 or 19.
  • the hinge is derived from the DC8 alpha chain, and its nucleotide sequence is shown in SEQ ID NO.5.
  • the transcellular membrane domain and co-stimulatory signal are from CD28, and its nucleotide sequence is shown in SEQ ID NO. 6; The acid sequence is shown in SEQ ID NO.7.
  • the genetically engineered cell contains a gene capable of simultaneously expressing a CD19 chimeric antigen receptor, a CD33 chimeric antigen receptor, or a BCMA chimeric antigen receptor (the nucleotide sequences are shown in SEQ ID Nos. 11, 17, and 20, respectively). ) And the truncated EGFR polypeptide gene building block (the nucleotide sequence is shown in SEQ ID No. 9), the gene building block nucleotide sequence is shown in SEQ ID No. 1, 15, 18.
  • the present invention also provides a method for preparing the above-mentioned genetically engineered cell.
  • the recombinant vector containing the T2A fusion of the CAR encoding gene and the EGFRt encoding gene is transferred into NK cells, or the T2A fusion of the CAR encoding gene and the EGFRt encoding gene is introduced.
  • NK cells NK cells are capable of expressing both CAR and EGFRt; the EGFRt is a truncated human epidermal growth factor receptor polypeptide.
  • the recombinant vector is a recombinant lentiviral vector, a recombinant retroviral vector or an electroporated DNA plasmid vector.
  • the nucleotide sequence of the CAR-encoding gene is shown in SEQ ID No. 11, 17, or 20, and the nucleotide sequence of the EGFRt-encoding gene is shown in SEQ ID No. 9.
  • the obtained genetically engineered cell may be cultured on a large scale under suspension culture technology under GMP conditions.
  • the nucleotide sequence of the T2A fusion of the CAR-encoding gene and the EGFRt-encoding gene is as shown in SEQ ID No. 1, 15 or 18.
  • the invention provides the application of the genetically engineered cells or the genetically engineered cells prepared by the above-mentioned preparation method to the preparation of a tumor treatment medicine.
  • the dosage form of the tumor treatment drug is an injection, and the injection can be used for intravascular injection, intratumor injection, subcutaneous injection, organ injection, intrapleural injection or intraperitoneal injection.
  • the genetically engineered cells of the present invention can reasonably expect that the genetically engineered cells of the present invention can be combined with pharmaceutically acceptable carriers and excipients to prepare drugs, so the drugs containing the genetically engineered cells of the present invention are included. It belongs to the protection scope of the present invention.
  • the medicine is a medicine for treating tumors.
  • the drug is an injection
  • the injection can be used for intravascular injection, intratumoral injection, subcutaneous injection, organ injection, intrapleural injection or intraperitoneal injection.
  • NK cells are much less likely and severe to trigger GvHD within the receptor than T cells. This is related to the unique cell biological and immunological characteristics of NK cells. NK cell expansion is tightly controlled by multiple inhibitory receptors such as the killer immunoglobulin-like receptor (KIR), CD94 / natural killer cell group 2A (NKG2A), and other inhibitory receptors. NK cells have a shorter life in the body. It usually does not attack non-hematopoietic tissues such as the liver, kidneys, muscles and lungs. This is the immunological basis for the development of allogeneic NK-CAR cell products of the present invention.
  • the genetically engineered cells provided by the present invention have good stability and anti-tumor effects, and the cells carry a safe killing switch factor.
  • the genetically engineered cell of the present invention is an allogeneic cell.
  • the allogeneic cell is not restricted by the production of autologous cell products, and is suitable for large-scale industrial production of GMP. It can be prepared into clinical-grade cell therapy products for treating and preventing human cells. This kind of malignant tumor is widely used in clinical cancer treatment.
  • Figure 1 is a schematic diagram of the components of the CD19-CAR-EGFRt chimeric antigen receptor and the killer switch gene.
  • Figure 2 is a schematic diagram of the structure of CD19-CAR-EGFRt lentiviral vector plasmid 98708.
  • Fig. 3 is a result of detecting the gene transduction rate of NK-SBN cells by flow cytometry.
  • Figure 4 shows the expression of CD19-CAR protein molecule in SBN-C19-CAR-NK cells by immunoblot.
  • FIG. 5 shows the expression of EGFRt protein molecule in SBN-CD19-CAR-NK cells by immunoblot.
  • Figure 6 shows the results of RTCA detection of SBN-CD19-CAR-NK cells against HeLa-CD19 cancer cells.
  • Figure 7 shows the results of co-culture of SBN-CD19-CAR-NK cells and human normal cells MRC-5 detected by RTCA.
  • FIG. 8 is a kinetic curve of SBN-CD19-CAR-NK cells killing HeLa-CD19 cancer cells.
  • FIG. 9 is a kinetic curve of co-culture of SBN-CD19-CAR-NK cells and human normal cells MRC-5.
  • Figure 10 shows the results of SBN-CD19-CAR-NK cells sensitivity to cetuximab.
  • Figure 11 shows the results of RTCA detection of SBN-CD33-CAR-NK cells against cancer cell MOLM-13.
  • Figure 12 shows the results of RTCA detection of co-culture of SBN-CD33-CAR-NK cells and human normal cells MRC-5.
  • FIG. 13 is a kinetic curve of killing cancer cell MOLM-13 by SBN-CD33-CAR-NK cells.
  • FIG. 14 is a kinetic curve of co-culture of SBN-CD33-CAR-NK cells and human normal cells MRC-5.
  • Figure 15 shows the results of RTCA detection of SBN-BCMA-CAR-NK cells against cancer cell H929.
  • Figure 16 shows the results of RTCA detection of co-culture of SBN-BCMA-CAR-NK cells and human normal cells MRC-5.
  • FIG. 17 is a kinetic curve of SBN-BCMA-CAR-NK cells killing cancer cell H929.
  • FIG. 18 is a kinetic curve of co-culture of SBN-BCMA-CAR-NK cells and human normal cells MRC-5.
  • the designed CD19-CAR is based on the structure of the second-generation CAR, using scFv and CD28 of anti-CD19 tumor antigens derived from FMC63 mouse hybridomas as co-stimulatory molecules linked to the intracellular signal domain of CD3zeta. Choosing CD28 as a co-stimulatory molecule can quickly activate CAR-NK cells in vivo and eliminate tumor cells in a short time.
  • a hinge domain from the CD8 alpha chain is added between the scFv and CD28 co-stimulatory molecules of the anti-CD19 tumor antigen, and its nucleotide sequence is shown in SEQ ID NO.5. .
  • a gene expressing a T2A cleavable polypeptide was added to the 3 'end of the anti-CD19-CAR gene building block.
  • EGFRt human epidermal growth factor receptor polypeptide
  • SEQ ID NO. 9 The expressed truncated human epidermal growth factor receptor polypeptide removes the ability to bind to human epidermal growth factor ligand, and also loses the function of activating signal activity in the cytoplasm, but retains the cells associated with anti-EGFR monoclonal antibody drugs. Outside area. T2A and truncated human epidermal growth factor receptor gene fragments are cloned into the anti-CD19-CAR gene building block in accordance with the sequence of the gene transcription framework. Under the same driving factor, equivalent anti-CD19-CAR and truncated humans are achieved.
  • cetuximab an existing anti-EGFR monoclonal antibody drug marketed after fluorophore labeling, can be used to detect truncated human epidermal growth factor receptor polypeptide and anti-CD19- CAR protein expression on the cell surface.
  • Figure 1 shows a schematic diagram of the CD19-CAR-EGFRt chimeric antigen receptor and killer switch gene building blocks.
  • the length of the gene building block is 4193bp.
  • the CD19-CAR-EGFRt chimeric antigen receptor and killer switch gene fragments were synthesized by ordering genes from the US IDT company. Gene sequencing verified the correctness of the gene sequence, and the gene sequence is shown in SEQ ID NO.1.
  • Human elongation factor-1 ⁇ promoter was used to drive the expression of anti-CD19-CAR and EGFRt genes.
  • Nucleotide 1-1178 is the human elongation factor-1 ⁇ promoter factor gene sequence, as shown in SEQ ID NO.2.
  • Nucleotide 1197-1262 is a human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, as shown in SEQ ID NO.3.
  • the GMCSFR ⁇ signal sequence was added before the anti-CD19-CAR sequence to guide the expression of the anti-CD19-CAR protein on the cell surface.
  • Nucleotides 1263-1997 tumor antigen is an anti-CD19 mouse monoclonal FMC63V H and V L, the gene fragment comprises a linker sequence and V L, V H gene (nucleotides 1584-1637), such as SEQ ID NO.4 Show.
  • Nucleotide 2007-2141 is the pivot domain sequence of the CD8alpha chain, as shown in SEQ ID NO.5.
  • Nucleotides 2142-2462 are the sequences of the CD28 transmembrane region and the cytoplasmic co-stimulatory domain, as shown in SEQ ID NO.6.
  • Nucleotides 2463-2798 are the cytoplasmic signal domain sequence of CD3zeta, as shown in SEQ ID NO.7.
  • Nucleotides 2814-2885 are the sequences of T2A cleavable polypeptides, and their nucleotide sequences are shown in SEQ ID NO.8.
  • T2A cleavable peptides play the role of equivalent expression of anti-CD19-CAR protein and EGFRt polypeptide.
  • Nucleotides 2886-2951 are human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, as shown in SEQ ID NO.3.
  • the GMCFSR ⁇ signal sequence was added before the anti-EGFRt sequence to guide the expression of the anti-EGFRt polypeptide on the cell surface.
  • Nucleotides 2952-3962 are the sequence of the EGFRt polypeptide, as shown in SEQ ID NO.9.
  • Nucleotide 3963-4193 is the sequence of bovine growth hormone BGH-polyA, as shown in SEQ ID NO.10.
  • FIG. 1 shows a schematic diagram of the structure of the 98708 vector plasmid.
  • 98708 vector plasmid DNA was amplified by the usual molecular biology experimental methods.
  • 98708 vector plasmid DNA and four plasmid DNAs encoding the lentiviral gag / pol, rev, and VSV-G virus membrane shells were used to co-precipitate and infect HEK293T cells cultured in a petri dish to produce CD19-CAR-EGFRt recombinant lentivirus , Named LV-CD19-CAR-EGFRt.
  • a culture medium containing LV-CD19-CAR-EGFRt was collected. Centrifuge at 500g for 15 minutes to remove cell debris, and save the supernatant containing LV-CD19-CAR-EGFRt at -80 ° C until use.
  • NK-SBN is a genetically engineered natural killer cell line developed by Shenzhen Cybino Gene Technology Co., Ltd. It has been disclosed in Chinese patent (application number 201810392435.3) that it can grow without the addition of IL-2. Remove and thaw the cells from the liquid nitrogen tank. The NK-SBN cells were expanded with RPMI + 10% FBS medium at 37 ° C in a 5% CO 2 cell incubator. The LV-CD19-CAR-EGFRt virus prepared in Example 2 was used to transduce NK-SBN cells in the logarithmic growth period under different MOIs.
  • Anti-EGFR monoclonal antibody (Human EGFR (Cetuximab) Alexa) labeled with the same fluorophore 488-conjugated Antibody, R & D Systems, Cat # FAB9577G-100) stained cells, and screened and purified the gene-transduced NK-SBN cells by flow cytometry.
  • the NK-SBN cells transduced with the expanded and selected LV-CD19-CAR-EGFRt recombinant lentivirus were cultured in RPMI + 10% FBS medium at 37 ° C in a 5% CO 2 cell incubator. The cells were named SBN-CD19-CAR-NK.
  • Figure 4 shows the expression of the CD19-CAR protein molecule in SBN-CD19-CAR-NK cells.
  • lane A is human T cells (showing endogenous CD3zeta protein) (positive control);
  • lane B is SBN-CD19-CAR-NK cells (showing CD19-CAR protein);
  • lane C is NK-SBN ( NK cells did not express CD3zeta protein or CD19-CAR protein) (negative control).
  • the results confirmed the correct molecular weight of the expressed CD19-CAR protein, about 73 kDa.
  • human T cells showed an endogenous CD3zeta protein of the correct molecular weight, about 16 kDa.
  • a similar Western blot was used to analyze the expression of EGFRt protein on the surface of SBN-CD19-CAR-NK natural killer cells. Take 1 ⁇ 10 7 SBN-CD19-CAR-NK, NK-SBN (no transduction, negative control) cells, and human epidermal-like cancer cell A431 cells As a positive control. Prepare cells according to the manufacturer's instructions (Roche Applied Science) using a RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% Triton x 100, 1 mM PMSF) containing a protease inhibitor. Lysates. Protein concentration was measured using a BCA kit.
  • Figure 5 shows the expression of EGFRt protein molecules in SBN-CD19-CAR-NK cells.
  • lane 1 is SBN-CD19-CAR-NK cells (with EGFRt protein shown) (lane plus 10ug of cell protein);
  • lane 2 is SBN-CD19-CAR-NK cells (with EGFRt protein shown) (lane plus 2ug Cell protein);
  • lane 3 is NK-SBN cells (NK cells do not express EGFRt protein) (negative control) (lane added 2ug cell protein);
  • lane 4 is human epidermal-like cancer cell A431 cells (expressing EGFR protein) (positive control) ) (Lanes added 2ug of cell protein);
  • lane 5 is the standard molecular weight.
  • human epidermal-like cancer cell A431 cells showed an endogenous EGFR protein of the correct molecular weight, about 170 kDa.
  • HeLa-CD19 (Carnova, USA) stably expressing CD19 tumor antigen were used as model cancer cells (positive), and human normal fibroblast MRC-5 was used as a negative control.
  • the anti-cancer effect of SBN-CD19-CAR-NK cells was detected using real-time cytotoxicity analysis (RTCA) method.
  • RTCA detection system continuously measures the impedance caused by the cells growing between the microelectronic biosensing electrodes through a microelectronic biosensor. Impedance increases as the cell grows. Conversely, cell death results in a decrease in impedance.
  • HeLa-CD19 and MRC-5 cells were first seeded into a 92-well culture plate with a microelectronic biosensor for the RTCA detection system. After culturing for about 26 hours, SBN-CD19-CAR-NK cells obtained under the conditions of MOI 20 and 40 were added to the wells of the culture plate. The ratio of SBN-CD19-CAR-NK cells to HeLa-CD19 cancer cells is 10: 1. Similarly, the ratio of SBN-CD19-CAR-NK cells to MRC-5 cells is also 10: 1. Continue incubation for 80 hours to detect changes in cell growth.
  • Figures 6 and 7 show the results of RTCA detection of SBN-CD19-CAR-NK cells on cancer cells and human normal cells, respectively. Fig.
  • Fig. 9 respectively show the cell killing kinetic curves converted according to the results of RTCA detection.
  • SBN-CD19-CAR-NK cells have a rapid and powerful killing effect on cancer cells. After co-culturing with HeLa-CD19 cancer cells for 5 hours, 95% of the cancer cells have died. By 24 hours, the cancer cell death rate has reached 99%.
  • the killing effect of SBN-CD19-CAR-NK cells on cancer cells is stronger than that reported in the literature (Berahovich, R. et al. FLAG-tagged CD19-specific CAR-T cells, Eliminate CD19-bearing, solid tumors, cells, and cells in front of vivos In BioBio, Landmark, 22, 1644-1654, June 1, 2017), the killing effect of CD19-CART cells on cancer cells.
  • the cancer cell death rate reached only 50% -70%.
  • SBN-CD19-CAR-NK cells The outstanding anti-cancer effect of SBN-CD19-CAR-NK cells is related to the anti-cancer efficacy of their parental NK-SBN cells.
  • Gene transduction CD19-CAR further enhanced the anti-cancer effect of NK cells.
  • SBN-CD19-CAR-NK cells did not show significant toxicity to human normal cells while showing a high killing rate for cancer cells. After 24 hours of co-cultivation, the vast majority of cells remained viable.
  • the experimental results confirm that the anti-cancer effect of SBN-CD19-CAR-NK cells is safe and specific.
  • the EGFRt sequence in the genetic components of SBN-CD19-CAR-NK cells is not only convenient for flow cytometry to detect the expression of anti-CD19 chimeric antigen receptor (CAR) on the cell surface, it also serves as a safety kill switch The role of factors. When combined with an anti-EGFR monoclonal antibody drug (cetuximab), it can kill SBN-CD19-CAR-NK cells through the antibody-dependent cytotoxicity (ADCC) pathway. Because human epidermal growth factor receptor (EGFR) is not expressed on hematopoietic and lymphatic cells, using EGFRt as a safe kill switch factor is specific and has no harmful side effects on normal blood and lymphatic cells.
  • ADCC antibody-dependent cytotoxicity
  • SBN-CD19-CAR-NK cells (selected from MOI 20 cells) cultured in RPMI medium were divided into three groups. The first group was not added with monoclonal antibody (control group), the second group was added with 1, 5, and At 10 ⁇ g / mL cetuximab, the third group was added with 1, 5 and 10 ⁇ g / mL rituximab against CD20 (control group). Incubate for 90 minutes. Centrifuge the cells and wash the cells with PBS. DELFIA cytotoxicity detection kit (AD0116, PerkinElmer) was used to detect the killing effect of cetuximab on SBN-CD19-CAR-NK cells.
  • the kit firstly label the three groups of cells with DELFIA BATDA reagent and incubate at 37 ° C for 30 minutes.
  • the labeled cells were washed with PBS and diluted to 1 ⁇ 10 5 / mL with RPMI medium.
  • 5 ⁇ 10 5 newly screened human normal PBMCs peripheral blood mononuclear cells
  • the 96-well plate was placed in a 37 ° C / 5% CO2 cell incubator for 3 hours.
  • Use a multichannel pipette to mix the liquid from each well 5-10 times. Centrifuge the 96-well plate at 500 g for 5 minutes.
  • Killing rate (%) of cetuximab on SBN-CD19-CAR-NK cells fluorescence intensity of treated SBN-CD19-CAR-NK cells / fluorescence intensity of positive control cells.
  • Figure 10 shows the results of sensitivity of SBN-CD19-CAR-NK cells to cetuximab.
  • cetuximab concentration With the increase of cetuximab concentration, the mortality of SBN-CD19-CAR-NK cells has increased significantly.
  • the death of SBN-CD19-CAR-NK cells is specific to cetuximab.
  • Rituximab has no significant effect on SBN-CD19-CAR-NK cells.
  • the experimental results confirmed that the EGFRt gene and protein activity in SBN-CD19-CAR-NK cells can play a role in safely killing the switch factor.
  • genomic DNA was extracted from SBN-CD19-CAR-NK cells of different passage numbers by conventional laboratory methods. Quantitative PCR method was used to detect and determine the copy number of integrated CD19-CAR gene. RNaseP gene was used as the standard for quantitative PCR gene copy number calculation.
  • the left primer sequence is shown in SEQ ID NO. 12
  • the right primer sequence is shown in SEQ ID NO. 13
  • the probe sequence is shown in SEQ ID NO. 14.
  • PCR cycling method denaturation / activation: 95 ° C, 5 minutes, once; denaturation: 95 ° C, 15 seconds; annealing / extension: 60 ° C, 1 minute; cycling 40 times.
  • SBN-CD19-CAR-NK cells contain about 1 copy of the integrated CAR gene.
  • the CAR gene integrated in the cell maintained good stability during continuous long-term cell culture.
  • the scFv gene sequence of the anti-CD19 tumor antigen was replaced with the scFv gene of the anti-CD33 tumor antigen, and CD33 was constructed.
  • -CAR-EGFRt chimeric antigen receptor and killer switch genome In a similar way, NK-SBN cells can be transduced with this genome to obtain SBN-CD33-CAR-NK cells for the treatment of CD33-positive acute myeloid leukemia.
  • CD33-CAR-EGFRt chimeric antigen receptor and a fragment that safely kills the switch factor were synthesized by ordering genes from the US IDT company.
  • nucleotides 1263-2054 are the scFv sequences of anti-CD33 tumor antigens, including the linker sequences of VH and VL genes (nucleotides 1662-1706). The sequence of genes is shown in SEQ ID NO.16.
  • Nucleotide 1-1178 is the human elongation factor-1 ⁇ promoter gene sequence, SEQ ID NO.2.
  • Nucleotide 1197-1262 is a human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, SEQ ID NO.3.
  • the GMCSFR ⁇ signal sequence was added before the anti-CD33-CAR sequence to guide the expression of the anti-CD33-CAR protein on the cell surface.
  • Nucleotides 2064-2198 are the pivot domain sequences of the CD8alpha chain, SEQ ID NO.5.
  • Nucleotides 2199-2519 are sequences of the CD28 transmembrane region and cytoplasmic co-stimulatory domain, SEQ ID NO.6.
  • Nucleotides 2520-2855 are the cytoplasmic signal domain sequence of CD3zeta, SEQ ID NO.7.
  • Nucleotide 2871-2942 is the sequence of T2A cleavable polypeptide, SEQ ID NO.8.
  • T2A cleavable peptides play the role of equivalent expression of anti-CD33-CAR protein and EGFRt polypeptide.
  • Nucleotides 2886-2951 are human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, please refer to the gene SEQ ID NO.3.
  • the GMCSFR ⁇ signal sequence was added before the anti-EGFRt sequence in order to direct the expression of the anti-EGFRt polypeptide on the cell surface.
  • Nucleotide 3009-4019 is the sequence of the EGFRt polypeptide, SEQ ID NO.9.
  • Nucleotides 4020-4250 are the sequence of bovine growth hormone BGH-polyA, SEQ ID NO.10.
  • CD33-CAR-EGFRt recombinant lentivirus was produced by the method described above and used to transduce NK-SBN cells to produce SBN-CD33-CAR-NK cells.
  • CD33-positive MOLM-13 acute myeloid leukemia cells (DSMZ, Germany) were used as model cancer cells (positive), and human normal fibroblast MRC-5 was used as a negative control.
  • the anti-cancer effect of SBN-CD33-CAR-NK cells was detected using real-time cytotoxicity analysis (RTCA) method.
  • the RTCA detection system continuously measures the impedance caused by the cells growing between the microelectronic biosensing electrodes through a microelectronic biosensor. Impedance increases as the cell grows. Conversely, cell death results in a decrease in impedance.
  • MOLM-13 and MRC-5 cells were seeded into a 92-well culture plate with a microelectronic biosensor for the RTCA detection system.
  • NK-SBN cells and SBN-CD33-CAR-NK cells that do not express CD33-CAR at different ratios to MOLM-13 cancer cell culture plates. Includes 1: 1 and 5: 1 (ratio of effector cells to cancer cells).
  • NK-SBN cells and SBN-CD33-CAR-NK cells that do not express CD33-CAR at different ratios to MRC-5 cells are added to the wells of MRC-5 cell culture plates.
  • Figures 11 and 12 show the results of RTCA detection of the effects of SBN-CD33-CAR-NK cells on cancer cells and human normal cells, respectively.
  • Figures 13 and 14 show the cell killing kinetic curves converted according to the results of RTCA detection, respectively. The results show that SBN-CD33-CAR-NK cells have a rapid and powerful killing effect on cancer cells. After co-culturing SBN-CD33-CAR-NK cells and MOLM-13 cancer cells for 5 hours, more than 90% of the cancer cells have died.
  • SBN-CD33-CAR-NK cells The outstanding anti-cancer effect of SBN-CD33-CAR-NK cells is related to the anti-cancer efficacy of their parental NK-SBN cells.
  • Gene transduction CD33-CAR further enhanced the anti-cancer effect of NK cells.
  • SBN-CD33-CAR-NK cells showed a high killing rate for cancer cells, but did not show significant toxic effects on human normal cells. After 24 hours of co-cultivation, the vast majority of cells remained viable. The experimental results confirmed that the anti-cancer effect of SBN-CD33-CAR-NK cells is highly safe and specific.
  • the scFv gene sequence of the anti-CD19 tumor antigen was replaced with an anti-BCMA (B-cell mature antigen) tumor antigen.
  • scFv gene constructed the genome of BCMA-CAR-EGFRt chimeric antigen receptor and safe kill switch factor.
  • NK-SBN cells can be transduced with this genome to obtain SBN-BCMA-CAR-NK cells for the treatment of BCMA-positive multiple myeloma.
  • the BCMA-CAR-EGFRt chimeric antigen receptor and a fragment that safely kills the switch factor gene were synthesized by ordering genes from the US IDT company.
  • nucleotide 1263-2030 is the scFv sequence of the anti-BCMA tumor antigen, including the linker sequences of the VH and VL genes (nucleotides 1596-1649).
  • SEQ ID No. 19 still uses the human elongation factor-1 ⁇ promoter to drive the antibody BCMA-CAR and HER1t gene expression.
  • Nucleotide 1-1178 is the human elongation factor-1 ⁇ promoter gene sequence, SEQ ID NO.2.
  • Nucleotide 1197-1262 is the human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, SEQ ID NO3. The GMCSFR ⁇ signal sequence was added before the anti-BCMA-CAR sequence to guide the expression of the anti-BCMA-CAR protein on the cell surface.
  • Nucleotides 2064-2198 are the pivot domain sequences of the CD8alpha chain, SEQ ID NO.5.
  • Nucleotides 2199-2519 are sequences of the CD28 transmembrane region and cytoplasmic co-stimulatory domain, SEQ ID NO.6.
  • Nucleotides 2520-2855 are the cytoplasmic signal domain sequence of CD3zeta, SEQ ID NO.7.
  • Nucleotide 2871-2942 is the sequence of T2A cleavable polypeptide, SEQ ID NO.8. T2A cleavable peptides play an equivalent role in expressing anti-BCMA-CAR protein and EGFRt polypeptide.
  • Nucleotides 2886-2951 are human granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFR ⁇ ) signal sequence, please refer to the gene sequence table 3. The GMCFSR ⁇ signal sequence was added before the anti-EGFRt sequence to guide the expression of the anti-EGFRt polypeptide on the cell surface.
  • Nucleotide 3009-4019 is the sequence of the EGFRt polypeptide, SEQ ID NO.9.
  • Nucleotides 4020-4250 are the sequence of bovine growth hormone BGH-polyA, SEQ ID NO.10.
  • BCMA-CAR-EGFRt recombinant lentivirus was produced by the method described above and used to transduce NK-SBN cells to produce SBN-BCMA-CAR-NK cells.
  • RTCA real-time cytotoxicity analysis
  • the RTCA detection system continuously measures the impedance caused by the cells growing between the microelectronic biosensing electrodes through a microelectronic biosensor. Impedance increases as the cell grows. Conversely, cell death results in a decrease in impedance.
  • H929 and MRC-5 cells were seeded into a 92-well culture plate with a microelectronic biosensor for the RTCA detection system.
  • the cell ratio used includes 1: 1 5: 1 and 10: 1 (ratio of effector cells to cancer cells).
  • NK-SBN cells and SBN-BCMA-CAR-NK cells that do not express BCMA-CAR at different ratios to MRC-5 cells are added to the wells of MRC-5 cell culture plates.
  • the cell ratio used includes 1: 1. 5: 1 and 10: 1.
  • Figures 15 and 16 show the results of RTCA detection of the effects of SBN-BCMA-CAR-NK cells on cancer cells and human normal cells, respectively.
  • Fig. 17 and Fig. 18 respectively show the cell killing kinetic curves converted according to the results of RTCA detection.
  • the results show that SBN-BCMA-CAR-NK cells have a clear and powerful killing effect on cancer cells. After co-culturing SBN-BCMA-CAR-NK cells and H929 cancer cells for 24 hours, the cancer cell death rate exceeded 90%.
  • SBN-BCMA-CAR-NK cells The outstanding anti-cancer effect of SBN-BCMA-CAR-NK cells is related to the anti-cancer efficacy of their parental NK-SBN cells.
  • Gene transduction BCMA-CAR further enhanced the anti-cancer effect of NK cells.
  • SBN-BCMA-CAR-NK cells showed a high killing rate for cancer cells, but did not show obvious toxic effects on human normal cells. After 24 hours of co-cultivation, all human normal cells maintained good growth activity.
  • the experimental results confirm that the anti-cancer effect of SBN-BCMA-CAR-NK cells is safe and specific.
  • the invention provides a genetically engineered cell for treating a tumor.
  • the genetically engineered cell can simultaneously express a chimeric antigen receptor (CAR) of a tumor antigen and a safe killing switch factor.
  • the cell is an allogeneic human immune cell.
  • the safety kill switch factor is a non-immunogenic truncated human epidermal growth factor receptor (EGFR) polypeptide.
  • the genetically engineered cells provided by the invention have good stability and anti-tumor effects. When necessary, by injecting anti-EGFR monoclonal antibody drugs, the genetically engineered cells injected into the patient can be eliminated, and the genetically engineered cells can be used in tumors. Safety of treatment.
  • the genetically engineered cell of the invention is an allogeneic cell, which can be prepared into a clinical-grade cell therapy product, used to treat and prevent various malignant tumors in humans, and suitable for large-scale GMP production.

Landscapes

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

Abstract

L'invention concerne une cellule génétiquement modifiée utilisée pour traiter une tumeur, la cellule étant une cellule immunitaire humaine allogénique, qui exprime simultanément un récepteur d'antigène chimérique (CAR) pour un antigène tumoral et un facteur kill switch de sécurité, le facteur kill switch de sécurité étant un polypeptide de récepteur de facteur de croissance épidermique (EGFR) humain tronqué non immunogène. La cellule génétiquement modifiée est une cellule allogénique, qui augmente la sécurité du traitement des tumeurs, et est appropriée pour une production GMP à grande échelle.
PCT/CN2019/095482 2018-07-27 2019-07-10 Cellule génétiquement modifiée utilisée pour traiter une tumeur WO2020019983A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201810846001.6 2018-07-27
CN201810846001 2018-07-27
CN201910527599.7 2019-06-18
CN201910527599.7A CN110305847B (zh) 2018-07-27 2019-06-18 一种用于治疗肿瘤的基因工程细胞

Publications (1)

Publication Number Publication Date
WO2020019983A1 true WO2020019983A1 (fr) 2020-01-30

Family

ID=68076044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/095482 WO2020019983A1 (fr) 2018-07-27 2019-07-10 Cellule génétiquement modifiée utilisée pour traiter une tumeur

Country Status (2)

Country Link
CN (1) CN110305847B (fr)
WO (1) WO2020019983A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11661459B2 (en) 2020-12-03 2023-05-30 Century Therapeutics, Inc. Artificial cell death polypeptide for chimeric antigen receptor and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110904048A (zh) * 2019-12-04 2020-03-24 合肥中科干细胞再生医学有限公司 一种car-cd123t2嵌合抗原受体t细胞及其应用
CN111925448B (zh) * 2020-08-03 2022-06-21 山东大学 在体生成car-巨噬细胞的制备方法及肿瘤免疫治疗中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016123143A1 (fr) * 2015-01-26 2016-08-04 The University Of Chicago Lymphocytes t à récepteur d'antigène chimérique (car) reconnaissant le récepteur il 13rα2 spécifique au cancer
WO2017053889A2 (fr) * 2015-09-23 2017-03-30 Precision Immunotherapy, Inc. Cellules car dirigée contre flt3 pour l'immunothérapie
CN107849112A (zh) * 2015-06-25 2018-03-27 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016123143A1 (fr) * 2015-01-26 2016-08-04 The University Of Chicago Lymphocytes t à récepteur d'antigène chimérique (car) reconnaissant le récepteur il 13rα2 spécifique au cancer
CN107849112A (zh) * 2015-06-25 2018-03-27 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法
WO2017053889A2 (fr) * 2015-09-23 2017-03-30 Precision Immunotherapy, Inc. Cellules car dirigée contre flt3 pour l'immunothérapie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MEHTA, R. S. ET AL.: "Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer", FRONTIERS IN IMMUNOLOGY, vol. 9, 15 February 2018 (2018-02-15), pages 283, XP055681692, ISSN: 1664-3224, DOI: 10.3389/fimmu.2018.00283 *
SUN, SHANGJUN ET AL.: "Immunotherapy with CAR-Modified T Cells: Toxicities and Overcoming Strategies", JOURNAL OF IMMUNOLOGY RESEARCH, vol. 2018, 17 April 2018 (2018-04-17), pages 1 - 10, XP055619319, ISSN: 2314-7156 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11661459B2 (en) 2020-12-03 2023-05-30 Century Therapeutics, Inc. Artificial cell death polypeptide for chimeric antigen receptor and uses thereof

Also Published As

Publication number Publication date
CN110305847A (zh) 2019-10-08
CN110305847B (zh) 2021-05-18

Similar Documents

Publication Publication Date Title
JP6997620B2 (ja) Ｂ細胞成熟抗原を標的化するキメラ抗原受容体およびその使用
JP2023107931A (ja) 免疫療法のための組成物および方法
CN105683214B (zh) 触发针对多种肿瘤的抗体依赖细胞毒性的嵌合受体
CN108715859B (zh) 靶向cd22的嵌合抗原受体及其应用
JP2024040376A (ja) Ｆｃ受容体様５を標的とするキメラ抗原受容体およびその使用
CN109593721B (zh) 具有自杀基因开关的靶向人间皮素的工程化免疫细胞
CN112142854B (zh) 免疫调节特异性嵌合抗原受体细胞及制备方法和应用
US20140302608A1 (en) Modified Effector Cell (or Chimeric Receptor) for Treating Disialoganglioside GD2-Expressing Neoplasia
US20180369285A1 (en) Specific Chimeric Antigen Receptor T Cells Targeting To NKG2DL, Its Preparation Method and Application Thereof
CN108409840B (zh) 抗cd123单链抗体及其组合的嵌合抗原受体和应用
US20210221906A1 (en) Carbonic anhydrase ix specific chimeric antigen receptors and methods of use thereof
CN109266667B (zh) 靶向cd5的嵌合抗原受体及其应用
WO2022007795A1 (fr) Récepteur antigénique chimérique et son utilisation
JP7475088B2 (ja) ヒトメソセリンを特異的に認識する細胞表面分子、ｉｌ－７、及びｃｃｌ１９を発現する免疫担当細胞
KR20210013013A (ko) 종양 치료 방법 및 조성물
WO2020019983A1 (fr) Cellule génétiquement modifiée utilisée pour traiter une tumeur
CN113717288B (zh) 逆转肿瘤微环境的融合蛋白及其应用
CN113913379A (zh) T淋巴细胞及其应用
CN113416260B (zh) 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
WO2023109257A1 (fr) Anticorps anti-bcma humanisé et cellule bcma-car-t/bcma-car-dnt
CN113896801A (zh) 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用
CN112500497A (zh) Cltx-nkg2d双特异性嵌合抗原受体细胞及其制备方法和应用
CN111138548A (zh) 一种egfr靶向性的嵌合抗原受体、car-nk细胞及其制备方法、应用
CN107164412B (zh) 一种安全型抗cea嵌合抗原受体修饰t细胞的制备方法及其应用
CN108699163B (zh) 一种多基因重组嵌合抗原受体分子及其应用

Legal Events

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

Ref document number: 19841361

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 09/06/2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19841361

Country of ref document: EP

Kind code of ref document: A1