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WO2020019983A1 - 一种用于治疗肿瘤的基因工程细胞 - Google Patents

一种用于治疗肿瘤的基因工程细胞 Download PDF

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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
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cells
car
genetically engineered
engineered cell
cell
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French (fr)
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张书元
徐卫
林鸿
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赛诺生（深圳）基因产业发展有限公司
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    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
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    • 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)
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    • 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
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    • 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.

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Abstract

提供了一种用于治疗肿瘤的基因工程细胞，所述细胞为异体人免疫细胞，同时表达肿瘤抗原的嵌合抗原受体（CAR）和安全杀死开关因子，所述安全杀死开关因子是无免疫原性的截短的人表皮生长因子受体（EGFR）多肽。该基因工程细胞是一种同种异体细胞，可以提高肿瘤治疗的安全性，并适合大规模GMP生产。

Description

[根据细则26改正23.07.2019]　一种用于治疗肿瘤的基因工程细胞
交叉引用
本申请引用于2018年07月27日提交的专利名称为“一种用于治疗肿瘤的基因工程细胞”的第2018108460016号中国专利申请，以及于2019年06月18日提交的专利名称为“一种用于治疗肿瘤的基因工程细胞”的第2019105275997号中国专利申请，上述两件专利申请通过引用被全部并入本申请。
技术领域
本发明属于生物基因工程技术领域，具体地说，涉及一种能够表达肿瘤抗原的嵌合抗原受体(CAR)、用于治疗肿瘤的基因工程细胞。
背景技术
肿瘤的生物治疗是继手术、放疗、化疗之后的第四大治疗方法。由于传统的手术、放化疗的发展已进入平台期，人们把越来越多地目光投到肿瘤的生物治疗上。基因和细胞免疫治疗癌症是肿瘤生物治疗的前沿技术，具有相对毒副作用小、疗效显著等优势。行业界人士预期基因细胞免疫治疗有潜力成为第五大肿瘤治疗方法。要实现细胞免疫治疗癌症，首先要克服免疫系统对恶性细胞的容纳。随着基因治疗技术和靶向精准单抗生物技术的发展和成熟，基因治疗技术和靶向精准单抗生物技术为解决免疫系统细胞对癌症细胞容纳的难题提供了一个有效的，从前没有过的解决方法。这种方法涉及到用基因转导修饰自体T细胞来重新编程T细胞以识别肿瘤特异性抗原，促使T细胞激活消除恶性癌细胞。这就是嵌合抗原受体(CAR)修饰T细胞(CAR-T)免疫治疗的原理。
嵌合抗原受体(CAR)嵌合蛋白有两个重要的功能区。第一个是表达在T细胞表面的单克隆抗体片段(scFv)肿瘤抗原靶结合域，它允许CAR-T细胞特异性的识别癌细胞表面上存在的癌抗原靶，实现精准治疗的目的。单克隆抗体片段是由柔性接头连接的单克隆抗体的轻链和重链的可变区组成。第二个是表达在T细胞内的T细胞激活因子。当CAR-T细胞表面的 抗体和癌抗原靶相接后，可以以主要组织相容性复合物(MHC)无关的方式激活T细胞，实现细胞扩增并分泌大量的抗肿瘤细胞因子，达到高效的抗癌疗效。
最初CAR的设计是直接将单克隆抗体片段(scFv)肿瘤抗原靶结合域通过铰链和T细胞受体跨膜区连接到CD3zeta的细胞内信号结构域。尽管这第一代CAR-T在临床前实验室实验和早期临床实验上显示了有限的效果，但是第一代CAR-T在受免疫抑制的肿瘤微环境内不能充分的激活T细胞，缺乏T细胞扩增。经过多年的反复研究，研究人员发现当在CAR的细胞内域区CD3zeta前加入一个共刺激域信号分子时，可以明显的提高CAR-T细胞的激活，扩增和持续性，大大地提高了抗肿瘤效果。实现了CAR-T细胞领域的一个重大突破。这就是通常所说的第二代CAR。目前全球CAR-T细胞临床试验绝大多数都是采用的第二代CAR。CD28或4-1BB(CD137)是第二代CAR中最常用的共刺激分子。
用于CAR-T细胞治疗的肿瘤，应具有肿瘤特异性的靶向抗原，此抗原在肿瘤细胞上有着普遍的表达，但在正常细胞上没有表达。B细胞恶性肿瘤细胞表面表达的CD19糖蛋白是相对理想的肿瘤抗原，CD19是在整个B细胞发育过程中一直表达的B细胞表面蛋白。几乎所有的B细胞恶性肿瘤细胞表面都表达CD19，包括慢性淋巴细胞性白血病(CLL)，急性淋巴细胞性白血病ALL和许多非霍奇金淋巴瘤(NHL)。重要的是，CD19抗原只是在B细胞上表达，从而会避免对非血液组织的损伤毒性，而对正常B细胞在预料之内的损伤毒性所造成的发育不全症状是可控的。因此，CD19阳性肿瘤成为普遍用于CAR-T细胞免疫治疗临床研究的对象。临床治疗晚期慢性和急性B淋巴细胞白血病，以及成年人复发性或难治性非霍奇金淋巴瘤，达到了杰出的80％-90％的完全缓解率。目前两个CAR-T细胞免疫治疗产品，Novartis公司的Kymriah TM(tisagenlecleucel)和Kite Pharma公司的Yescarta TM(axicabtagene ciloleucel)，已先后获得美国FDA的批准上市。
除了CD19阳性肿瘤外，B细胞成熟抗原(BCMA)在绝大多数情况下只在浆细胞骨髓瘤癌细胞上表达，在非浆细胞上不出现表达，也是一个适合于CAR-T细胞治疗的肿瘤抗原。实验结果证实注入BCMA-CART细胞到植入人多发性骨髓瘤的小鼠中可以有效的根除癌细胞，到达完全缓解。用BCMA-CART细胞治疗多发性骨髓癌已相继进入了临床实验。另外，CD33和CD123糖蛋白在骨髓性白血病癌细胞表面有着明显高的表达，也可以作为肿瘤特异抗原用于CART细胞的治疗。近期已用于临床实验治疗复发性或难治性急性骨髓性白血病。
鉴于T细胞能够主动的传走到几乎身体的每个部位，并且有克服肿瘤逃避的能力，CAR-T细胞除了用于治疗血液癌症外，也具有消除实体瘤的独特潜力。因为大多数潜在的实体肿瘤抗原靶标是非特异性的，在健康组织上也有不同程度的表达，选择适当的实体肿瘤靶标是相对困难和具有挑战性的。迄今为止，CAR-T细胞治疗的实体肿瘤靶标包括有GD2,IL13Ra2,间皮素(mesothelin),EGFRvIII，HER2等。有的已相继进入了人临床I期实验用于治疗多形性胶质母细胞瘤(GBM)。
虽然自体CAR-T细胞免疫治疗恶性白血病表现出了空前的疗效，此类产品也带来了其独特的严重副作用。接受自体CAR-T细胞免疫治疗肿瘤患者所经历最多的副作用是细胞因子释放综合征(Cytokine release syndrome,CRS)。细胞因子释放综合征(CRS)是一种明显的由CAR-T细胞在体内激活和指数性扩增所引发的大量多种细胞因子的急剧释放而导致的全身性炎症反应。通常发生在初次CAR-T细胞输注的几天之内。初步的CRS临床症状包括发热和流感样症状，严重的CRS症状包括血管扩张,休克性毛细血管渗漏和呼吸困难，导致需要重症监护室护理。极个别情况下会造成患者脑水肿，甚至死亡。另外，在接受了CAR-T细胞治疗的患者中也报道了不同程度的神经毒性反应，包括谵妄，脑病和癫痫发作。深入的临床监测表明患者CRS的严重性和很多因素有关。IL6是所释放的多种细胞因子中的一个最明细的因子，可能在CRS所引发的病理生理学中 起着中枢作用。虽然有报道给患者注射抗IL6受体拮抗剂(托美珠单抗,tocilizumab)可以快速改善大多数患者的CRS症状，为了进一步提高CAR-T细胞治疗肿瘤的安全性，有必要开发带有无免疫原性安全杀死开关因子的CAR-T细胞产品，在必要的时候，可以给患者注射相关的药物，启动安全杀死开关因子来从体内清除CAR-T细胞，消除安全隐患。
自然杀伤(NK)细胞是人外周淋巴细胞中的一小部分(约占10％)有特异先天免疫功能的细胞，在不需要预先启发的条件下，可以自发的介导“天然”细胞毒性消除某些肿瘤和病毒感染的细胞。进而被取名为自然杀伤(NK)细胞。是人免疫系统的一个重要组成部分。由于几乎所有的健康细胞都在细胞表面表达I类MHC，NK细胞在受到自身Ig样受体(KIR)抑制受体的控制下，对正常表达I类MHC的健康自身细胞没有损伤作用。下调I类MHC的表达是肿瘤和病毒感染细胞逃避溶解性T细胞TCR识别和被杀伤的相当常见的机制。恰好NK细胞的主要杀伤的靶标细胞是那些下调了或没有I类MHC表达的病变细胞，也就是所谓的“丢失自我”的原则。NK细胞的免疫机制克服了这种潜在的T细胞免疫性作用机制的空缺，起到了重要的互补作用。NK细胞杀伤靶细胞的机制是由溶细胞颗粒的定向胞吐介导释放穿孔素和其穿透靶细胞膜到细胞浆内引发细胞凋亡。
NK细胞在体内抗癌的活性受激活受体信号和抑制受体信号强度平衡的控制。和T细胞的激活不同，NK细胞不具有单一激活受体，而是多个的共激活受体，包括自然细胞毒性受体(NCR)，NKp30，NKp44，和NKp46，NKG2D,CD16,2B4等。这些激活受体通过链接信号转接蛋白，如DAP10，DAP12和CD3ζ，启动释放含有穿孔素和颗粒酶的溶细胞颗粒，和介导产生释放细胞因子和趋化因子如IFN-γ和TNF-α等。NK细胞的激活活性受到严格的反控抑制作用。NK细胞具有许多抑制受体，包括杀伤细胞Ig样受体(KIR),CD94-NKG2A,和LILR等。最终NK细胞的激活程度权衡于激活受体和抑制受体的平衡整合。这是和杀伤T细胞激活的一个原则上的不同。
目前已上市和正在临床研发的CAR-T细胞产品基本都是自体(autologous)细胞产品。自体CAR-T细胞产品生产过程十分复杂，生产周期长需要2-3个星期。由于自体CAR-T细胞产品是患者特异性的，不适合大规模GMP生产，产品价格昂贵。对一些晚期的癌症患者，用2-3个星期等待自己的CAR-T细胞将是一个很大的医疗决定，因为在此期间患者的病情会有加重的可能性。除此之外，患者还要面对自己的CAR-T细胞在生产过程中有失败的可能性，面临丧失肿瘤治疗的宝贵时间。因此，有必要开发适合GMP大规模产业化，价格合适的“现成的”非自体(异体)CAR细胞产品。
发明内容
本发明的目的是提供一种适合GMP大规模产业化的用于治疗肿瘤的异体基因工程细胞。
本发明提供的用于治疗肿瘤的基因工程细胞，其为能够同时表达肿瘤抗原的嵌合抗原受体CAR和安全杀死开关因子的免疫细胞。
所述免疫细胞为异体人免疫细胞。
优选地，所述免疫细胞为异体人自然杀伤细胞NK。因此，优选本申请的基因工程细胞为CAR-NK细胞。
本发明的用于治疗肿瘤的基因工程细胞中，添加了没有免疫反应的安全杀死开关因子，所采用的安全杀死开关因子为无免疫原性的截短的人表皮生长因子受体多肽(EGFRt)。由于人表皮生长因子受体(EGFR)在造血系统和淋巴系统细胞上没有表达，用EGFRt作为安全杀死开关因子是特异性的，对正常的血液系统和淋巴系统细胞没有伤害副作用。此多肽缺少膜远端表皮生长因子结合结构域和细胞质尾巴信号域，因此不但失去了和人表皮生长因子配体结合的能力，也丧失了细胞质内激活信号活性的功能。只保留了和抗EGFR单抗相结合的胞外区域。当和市场现有的抗EGFR单抗药品(例如西妥昔单抗)结合后，仍然能通过抗体依赖性细胞毒性(ADCC)途径作用可以杀死CAR-NK细胞。
优选地，本发明所述截短的人表皮生长因子受体多肽的核苷酸序列如SEQ ID NO.9所示。
所述肿瘤抗原的嵌合抗原受体CAR与安全杀死开关因子通过C端T2A可切割链相结合，所述C端T2A可切割链的核苷酸序列如SEQ ID NO.8所示。
另一方面，本发明基因工程细胞内，肿瘤抗原的嵌合抗原受体CAR的分子结构包括：a)细胞表面的单克隆抗体片段scFv肿瘤抗原靶结合域，b)铰链和跨细胞膜域，和(c)T细胞受体CD3ζ域和共刺激信号组成的细胞内质信号传递域。
本发明的优选实施例的基因工程细胞中，CAR修饰的NK细胞(CAR-NK)可通过将细胞表面的单克隆抗体片段(scFv)结构域与细胞内CD3zeta激活域(NK细胞激活的必需细胞内信号分子)连接而增强激活信号强度。当CAR-NK细胞和肿瘤抗原相接合时，所产生的激活信号会超越抑制信号，达到抗癌效果。
所述单克隆抗体为CD19、CD20,BCMA、CD22、CD33、CD47,CD123、CD133,CD138,ROR1(Receptor Tyrosine Kinase Like Orphan Receptor 1),GD2,Mesothelin,Muc1和Muc 16,CEA(carcinoembryonic antigen)。优选地，单克隆抗体片段scFv的核苷酸序列如SEQ ID NO.4、16或19所示。
本发明基因工程细胞内嵌合抗原受体CAR的分子结构中，所述铰链来自DC8 alpha链，其核苷酸序列如SEQ ID NO.5所示。
嵌合抗原受体CAR的分子结构中，所述跨细胞膜域和共刺激信号来自CD28，其核苷酸序列如SEQ ID NO.6所示；或T细胞受体CD3ζ信号传递域，其核苷酸序列如SEQ ID NO.7所示。
更优选地，该基因工程细胞含有能够同时表达CD19嵌合抗原受体、CD33嵌合抗原受体或BCMA嵌合抗原受体(核苷酸序列分别如SEQ ID NO.11、17、20所示)和截短的EGFR多肽的基因构件(核苷酸序列如SEQ ID NO.9所示)，该基因构件核苷酸序列如SEQ ID NO.1、15、18所示。
本发明还提供了上述的基因工程细胞的制备方法，将含有CAR编码基因和EGFRt编码基因的T2A融合体的重组载体转入NK细胞中，或将CAR编码基因和EGFRt编码基因的T2A融合体导入NK细胞中，使NK细胞能够同时表达CAR和EGFRt；所述EGFRt为截短的人表皮生长因子受体多肽。
所述重组载体为重组慢病毒载体、重组逆转录病毒载体或电穿孔DNA质粒载体。
所述CAR编码基因的核苷酸序列如SEQ ID NO.11,17或20所示，所述EGFRt编码基因的核苷酸序列如SEQ ID NO.9所示。
本发明的上述基因工程细胞的制备方法中，在获得基因工程细胞后，可以将获得的基因工程细胞在GMP条件下采用悬浮培养技术大规模培养。
优选地，本发明制备方法中，所述CAR编码基因和EGFRt编码基因的T2A融合体的核苷酸序列如SEQ ID NO.1，15或18所示。
本发明提供了上述基因工程细胞或上述的制备方法制得的基因工程细胞在制备肿瘤治疗药物中的应用。
进一步地，所述的肿瘤治疗药物的剂型为注射剂，所述注射剂可用于血管内注射、瘤内注射、皮下注射、器官注射、胸水内注射或腹水内注射。
本领域技术人员基于本发明的基因工程细胞，可以合理地预期，将本发明的基因工程细胞与药学上可接受的载体和辅料进行配合，可以制备药物，因此含有本发明上述基因工程细胞的药物属于本发明的保护范围。
进一步地，所述药物为治疗肿瘤的药物。
更进一步地，所述药物为注射剂，所述注射剂可用于血管内注射、瘤内注射、皮下注射、器官注射、胸水内注射或腹水内注射。
NK细胞在受体内引发GvHD的可能性和严重程度要远远轻于T细胞。这和NK细胞独特的细胞生物学和免疫学特征有关。NK细胞扩增受到多个抑制性受体如杀伤免疫球蛋白样受体(KIR)，CD94/自然杀伤细胞组2A(NKG2A)和其他抑制性受体的严格控制。NK细胞在体内寿命较 短。通常不攻击诸如肝，肾，肌肉和肺等非造血组织。这是本发明开发异体NK-CAR细胞产品的免疫学基础。本发明提供的基因工程细胞具有良好的稳定性和抗肿瘤效应，并且细胞内携带安全杀死开关因子，在必要的情况下通过注入抗EGFR单抗药物，可以消除注入患者体内的该基因工程细胞，提高了该基因工程细胞细胞用于肿瘤治疗的安全性。本发明的基因工程细胞是一种同种异体细胞，异体细胞不受自体细胞产品生产的限制，适合于大规模GMP产业化生产，可制备成临床级细胞治疗制品，用于治疗和预防人类各种恶性肿瘤，广泛地应用于临床癌症治疗。
附图说明
图1为CD19-CAR-EGFRt嵌合抗原受体和杀伤开关基因构件示意图。
图2为CD19-CAR-EGFRt慢病毒载体质粒98708结构示意图。
图3为流式细胞仪检测NK-SBN细胞的基因转导率结果。
图4为免疫印迹检测CD19-CAR蛋白分子在SBN-C19-CAR-NK细胞内的表达。
图5为免疫印迹检测EGFRt蛋白分子在SBN-CD19-CAR-NK细胞内的表达。
图6为RTCA检测SBN-CD19-CAR-NK细胞杀伤癌细胞HeLa-CD19的结果。
图7为RTCA检测SBN-CD19-CAR-NK细胞和人正常细胞MRC-5共同培养的结果。
图8为SBN-CD19-CAR-NK细胞杀伤癌细胞HeLa-CD19动力学曲线。
图9为SBN-CD19-CAR-NK细胞和人正常细胞MRC-5共同培养的动力学曲线。
图10为SBN-CD19-CAR-NK细胞对西妥昔单抗的敏感性结果。
图11为RTCA检测SBN-CD33-CAR-NK细胞杀伤癌细胞MOLM-13的结果。
图12为RTCA检测SBN-CD33-CAR-NK细胞和人正常细胞MRC-5共同培养的结果。
图13为SBN-CD33-CAR-NK细胞杀伤癌细胞MOLM-13动力学曲线。
图14为SBN-CD33-CAR-NK细胞和人正常细胞MRC-5共同培养的动力学曲线。
图15为RTCA检测SBN-BCMA-CAR-NK细胞杀伤癌细胞H929结果。
图16为RTCA检测SBN-BCMA-CAR-NK细胞和人正常细胞MRC-5共同培养的结果。
图17为SBN-BCMA-CAR-NK细胞杀伤癌细胞H929的动力学曲线。
图18为SBN-BCMA-CAR-NK细胞和人正常细胞MRC-5共同培养的动力学曲线。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，实施例中所用的生化试剂、材料均为市售可得。
实施例1抗CD19阳性肿瘤的CD19-CAR-EGFRt嵌合抗原受体和安全杀死开关因子基因的构建
所设计的CD19-CAR基于第二代CAR的结构，采用来源于FMC63小鼠杂交瘤的抗CD19肿瘤抗原的scFv和CD28作为共刺激分子连接到CD3zeta的细胞内信号结构域。选择CD28作为共刺激分子可以在较短的时间在体内快速激活CAR-NK细胞，消除肿瘤细胞。
为了提高CAR蛋白结构的灵活和伸缩性，本申请在抗CD19肿瘤抗原的scFv和CD28共刺激分子之间加入了来自CD8 alpha链的枢纽域，其核苷酸序列如SEQ ID NO.5所示。为了方便用流式细胞仪检测抗CD19-CAR在基因转导后细胞表面的表达，在抗CD19-CAR基因构件的3’端添加了表达T2A可裂解的多肽的基因(序列如SEQ ID NO.8所示)和截短的人表皮生长因子受体多肽(EGFRt)的基因片段(序列如SEQ ID NO.9所示)。所 表达的截短的人表皮生长因子受体多肽去掉了和人表皮生长因子配体结合的能力，也丧失了细胞质内激活信号活性的功能，但保留了和抗EGFR单抗药物相结合的胞外区域。T2A和截短的人表皮生长因子受体的基因片段是按基因转录框架顺序克隆到抗CD19-CAR基因构件内，在同一个驱动因子下，实现等当量的抗CD19-CAR和截短的人表皮生长因子受体多肽在细胞表面的表达。这意味着可以用荧光团标记后的市场现有的抗EGFR单抗药品-西妥昔单抗(cetuximab)，来用流式细胞仪检测截短的人表皮生长因子受体多肽和抗CD19-CAR蛋白在细胞表面的表达。
图1显示了CD19-CAR-EGFRt嵌合抗原受体和杀伤开关基因构件的示意图。基因构件长度为4193bp。从美国IDT公司订购基因合成了CD19-CAR-EGFRt嵌合抗原受体和杀伤开关基因的片段。基因测序验证了基因序列的正确性，基因序列如SEQ ID NO.1所示。采用了人延伸因子-1α启动因子驱动抗CD19-CAR和EGFRt的基因表达。核苷酸1-1178是人延伸因子-1α启动因子基因序列，如SEQ ID NO.2所示。核苷酸1197-1262是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列,如SEQ ID NO.3所示。在抗CD19-CAR序列前加入GMCSFRα信号序列是为了导向抗CD19-CAR蛋白在细胞表面的表达。核苷酸1263-1997是抗CD19肿瘤抗原的鼠单抗FMC63V H和V L的基因片段，包括V H和V L基因的接头序列(核苷酸1584-1637)，如SEQ ID NO.4所示。核苷酸2007-2141是CD8alpha链的枢纽域序列，如SEQ ID NO.5所示。核苷酸2142-2462是CD28跨膜区和细胞质共刺激域序列，如SEQ ID NO.6所示。核苷酸2463-2798是CD3zeta的细胞质信号域序列，如SEQ ID NO.7所示。核苷酸2814-2885是T2A可裂解多肽的序列，其核苷酸序列如SEQ ID NO.8所示。T2A可裂解多肽起到等当量表达抗CD19-CAR蛋白和EGFRt多肽的作用。核苷酸2886-2951是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列，如SEQ ID NO.3所示。在抗EGFRt序列前加入GMCFSRα信号序列是为了导向抗EGFRt多肽在细胞表面的表达。核苷酸 2952-3962是EGFRt多肽的序列，如SEQ ID NO.9所示。核苷酸3963-4193是牛生长激素BGH-polyA的序列，如SEQ ID NO.10所示。
实施例2 CD19-CAR-EGFRt重组慢病毒的制备
将实施例1合成的CD19-CAR-EGFRt嵌合抗原受体和安全杀死开关因子克隆到用于生产慢病毒的载体质粒(美国Carnova公司)中，获得CD19-CAR-EGFRt慢病毒载体质粒98708。图2显示了98708载体质粒的结构示意图。用通常的分子生物学实验方法扩增98708载体质粒DNA。用98708载体质粒DNA和分别编码慢病毒gag/pol，rev，和VSV-G病毒膜壳的四个质粒DNA，共同沉淀感染培养在培养皿内的HEK293T细胞来生产CD19-CAR-EGFRt重组慢病毒，命名为LV-CD19-CAR-EGFRt。在DNA感染两天后收集含有LV-CD19-CAR-EGFRt的培养液。在500g速度下离心15分钟去掉细胞碎片，再将含有LV-CD19-CAR-EGFRt的上清液保存在-80℃下备用。
实施例3 LV-CD19-CAR-EGFRt重组慢病毒转导自然杀伤细胞系
NK-SBN是深圳赛百诺基因技术公司开发的基因工程自然杀伤细胞系已在中国专利(申请号201810392435.3)中公开可以在不添加IL-2的条件下生长。从液氮罐取出并融冻细胞。用RPMI+10％FBS培养液在37℃,5％CO 2细胞培养箱内培养扩增NK-SBN细胞。用实施例2制得的LV-CD19-CAR-EGFRt病毒在不同的MOI下转导对数生长期内的NK-SBN细胞。在病毒转导后的第二天，取细胞样品，用荧光团标记的抗EGFR单抗(Human EGFR(Cetuximab)Alexa
Figure PCTCN2019095482-appb-000001
488-conjugated Antibody，R&D Systems,Cat#FAB9577G-100)染色细胞。用流式细胞仪检细胞基因转导效率。检测参照R&D Systems推荐的方法进行。图3显示了流式细胞仪检测结果和NK-SBN细胞的基因转导率。
表1 LV-CD19-CAR-EGFRt重组慢病毒转导NK-SBN自然杀伤细胞系基因转导率
MOI(TU/细胞) ％EGFRt+NK-SBN细胞
20 60.9％
40 71.1％
结果显示用LV-CD19-CAR-EGFRt重组慢病毒转导NK-SBN自然杀伤细胞系可以达到相当高的基因转导率。
采用同样的荧光团标记的抗EGFR单抗(Human EGFR(Cetuximab)Alexa
Figure PCTCN2019095482-appb-000002
488-conjugated Antibody，R&D Systems,Cat#FAB9577G-100)染色细胞，用流式细胞仪筛选纯化基因转导了的NK-SBN细胞。用RPMI+10％FBS培养液在37℃，5％CO 2细胞培养箱内继续培养扩增筛选的LV-CD19-CAR-EGFRt重组慢病毒转导了的NK-SBN细胞。将细胞取名为SBN-CD19-CAR-NK。
实施例4 SBN-CD19-CAR-NK自然杀伤细胞系CD19-CAR和EGFRt蛋白表达的分析
采用免疫印迹(Western blot)检测方法分析CD19-CAR蛋白在SBN-CD19-CAR-NK自然杀伤细胞表面的表达。取1×10 7SBN-CD19-CAR-NK，NK-SBN(没有转导，阴性对照)细胞，和人T细胞作为阳性对照。依照生产厂家(Roche Applied Science)的说明，用含有蛋白酶抑制剂的RIPA缓冲液(50mM Tris,pH7.5,150mM NaCl,0.1％SDS,0.5％Sodium Deoxycholate,1％TritonX100,1mM PMSF)制备细胞裂解物。用BCA试剂盒测定蛋白质浓度。
取10mg蛋白质在变性的条件下在4-20％的凝胶上分离蛋白质。将分离的蛋白质转移到PVDF膜上。先用5％的奶液阻碍，再用抗CD3zeta的抗体(Santa Cruz,SC-70617)来结合表达的CD19-CAR蛋白分子中的CD3zeta激活信号域。所结合的抗CD3zeta的抗体再用羊抗鼠IgG Fc HRP(Sigma-Aldrich)来显色。
图4显示了CD19-CAR蛋白分子在SBN-CD19-CAR-NK细胞内的表达。图中A泳道是人T细胞(显示了内源性的CD3zeta蛋白)(阳性对照)；B泳道是SBN-CD19-CAR-NK细胞(显示了CD19-CAR蛋白)；C泳道是 NK-SBN(NK细胞不表达CD3zeta蛋白，也不表达CD19-CAR蛋白)(阴性对照)。结果证实了所表达的CD19-CAR蛋白的分子量的正确性，约73kDa。作为阳性对照的人T细胞作显示了分子量正确的内源性CD3zeta蛋白，约16kDa。
采用类似的免疫印迹(Western blot)检测方法分析EGFRt蛋白在SBN-CD19-CAR-NK自然杀伤细胞表面的表达。取1×10 7SBN-CD19-CAR-NK,NK-SBN(没有转导，阴性对照)细胞，和人表皮样癌细胞A431细胞
Figure PCTCN2019095482-appb-000003
作为阳性对照。依照生产厂家(Roche Applied Science)的说明，用含有蛋白酶抑制剂的RIPA缓冲液(50mM Tris,pH7.5,150mM NaCl,0.1％SDS,0.5％Sodium Deoxycholate,1％Triton×100,1mM PMSF)制备细胞裂解物。用BCA试剂盒测定蛋白质浓度。取10mg蛋白质在变性的条件下在4-20％的凝胶上分离蛋白质。将分离的蛋白质转移到PVDF膜上。先用5％的奶液阻碍，再用抗EGFR的抗体(R&D Systems,#MAB8336)来结合表达的EGFRt蛋白。所结合的抗EGFRt的抗体再用羊抗鼠IgG Fc HRP(Sigma-Aldrich)来显色。
图5显示了EGFRt蛋白分子在SBN-CD19-CAR-NK细胞内的表达。图中1泳道是SBN-CD19-CAR-NK细胞(显示了EGFRt蛋白)(泳道加了10ug细胞蛋白)；2泳道是SBN-CD19-CAR-NK细胞(显示了EGFRt蛋白)(泳道加了2ug细胞蛋白)；3泳道是NK-SBN细胞(NK细胞不表达EGFRt蛋白)(阴性对照)(泳道加了2ug细胞蛋白)；4泳道是人表皮样癌细胞A431细胞(表达EGFR蛋白)(阳性对照)(泳道加了2ug细胞蛋白)；5泳道是标准分子量。结果证实了所表达的EGFRt蛋白的分子量的正确性，约40kDa。作为阳性对照的人表皮样癌细胞A431细胞显示了分子量正确的内源性EGFR蛋白，约170kDa。结果证实了正确分子量的CD19-CAR和EGFRt蛋白同时在SBN-CD19-CAR-NK细胞表面的表达。
实施例5 SBN-CD19-CAR-NK细胞的抗癌效应
用基因工程处理的稳定表达CD19肿瘤抗原的HeLa癌细胞HeLa-CD19(美国Carnova公司)作为模型癌细胞(阳性)，人正常的成纤维细胞MRC-5作为阴性对照。使用实时细胞毒性分析(RTCA)方法检测了SBN-CD19-CAR-NK细胞的抗癌效应。该RTCA检测系统通过微电子生物传感器连续测量由生长在微电子生物传感电极之间的细胞所引起的阻抗。阻抗随着细胞的生长而增加，相反，细胞的死亡会导致阻抗的降低。首先将HeLa-CD19和MRC-5细胞接种到RTCA检测系统用的，带有微电子生物传感器的92孔培养板内。培养约26小时后，向培养板孔内加入MOI 20和40条件下获得的SBN-CD19-CAR-NK细胞。SBN-CD19-CAR-NK细胞和HeLa-CD19癌细胞的比率是10:1。同样的，SBN-CD19-CAR-NK细胞和MRC-5细胞的比率也是10:1。继续培养到80小时来检测细胞生长的变化。图6和图7分别显示了SBN-CD19-CAR-NK细胞对癌细胞和人正常细胞作用的RTCA检测结果。图8和图9分别显示了依照RTCA检测结果转换来的细胞杀伤动力学曲线。SBN-CD19-CAR-NK细胞对癌细胞有着快速和强有力的杀伤作用。和HeLa-CD19癌细胞共同培养5个小时后，95％的癌细胞已经死亡，到24小时，癌细胞的死亡率已达到99％。
SBN-CD19-CAR-NK细胞对癌细胞的杀伤作用强于文献报道(Berahovich,R.et al.FLAG-tagged CD19-specific CAR-T cells eliminate CD19-bearing solid tumor cells in vitro and in vivo,Frontiers In Bioscience,Landmark,22,1644-1654,June 1,2017)的CD19-CART细胞对癌细胞的杀伤作用。使用同样的RTCA检测方法，CD19-CART细胞和HeLa-CD19癌细胞共同培养8个小时后，癌细胞的死亡率只达到了50％-70％。
SBN-CD19-CAR-NK细胞杰出的抗癌细胞效应和其母系的NK-SBN细胞自身所表现的抗癌细胞功效是有关系的。基因转导CD19-CAR进一步提高了NK细胞的抗癌细胞效应。这是SBN-CD19-CAR-NK细胞相对于一般的CD19-CAR-T细胞产品的一个突出优点。SBN-CD19-CAR-NK细胞在对癌细胞显示出高杀伤率的同时，对人正常细胞没有表现出明显的毒性作 用。在共同培养24个小时后，绝大多数细胞依然健存。实验结果证实，SBN-CD19-CAR-NK细胞的抗癌作用是有安全性和特异性的。
实施例6 SBN-CD19-CAR-NK细胞对西妥昔单抗的敏感性
SBN-CD19-CAR-NK细胞基因构件中的EGFRt序列不但可以方便用于流式细胞仪来检测抗CD19嵌合抗原受体(CAR)在细胞表面的表达，它还会起着安全杀死开关因子的作用。当和抗EGFR单抗药品(西妥昔单抗)结合后，能通过抗体依赖性细胞毒性(ADCC)途径作用杀死SBN-CD19-CAR-NK细胞。由于人表皮生长因子受体(EGFR)在造血系统和淋巴系统细胞上没有表达，用EGFRt作为安全杀死开关因子是特异性的，对正常的血液系统和淋巴系统细胞没有伤害副作用。这意味着，在必要的，严重的临床副作用情况下，可以给患者注入市场现有的抗EGFR单抗药品——西妥昔单抗，来清除体内的SBN-CD19-CAR-NK细胞，缓解治疗的副作用。这对本发明开发的可广泛应用的异体SBN-CD19-CAR-NK细胞产品来说，是一个十分重要的安全保障措施。
取在RPMI培养液培养的SBN-CD19-CAR-NK细胞(选MOI 20的细胞)，分三组，第一组不添加单抗(对照组),第二组分别添加了1,5,和10μg/mL的西妥昔单抗，第三组分别添加了1,5,和10μg/mL的抗CD20的利妥昔单抗(对照组)。培养90分钟。离心细胞，并用PBS清洗细胞。采用DELFIA细胞毒性检测试剂盒(AD0116,PerkinElmer)，来检测西妥昔单抗对SBN-CD19-CAR-NK细胞的杀伤作用。按照试剂盒的指示，首先用DELFIA BATDA试剂标签三组的细胞，在37℃下培养30分钟。再用PBS清洗标签后的细胞，用RPMI培养液稀释到1×10 5/mL。放5000个细胞在96孔板的孔内(50微升)。向每个孔内加入5×10 5个新筛选的人正常PBMC(外周血单核细胞)。将96孔板放在37℃/5％CO2细胞培养箱内培养3个小时。用多道移液器混合每个孔的液体5-10次。在500g下离心96孔板5分钟。从每个孔内取出20微升，放入一个新的用于荧光检测的96孔板内。向每个孔内加入200微升的DELFIA Europium检测液。将96孔板用 铝膜包裹，放在轨道摇床上摇15分钟。用荧光酶标仪(Synergy 2,Biotek)在不同的时间间隙下测量荧光强度。测量设置条件为，激发波长：330nm，发出波长：615nm，时间滞后：400μs，测量间隙：1000μs，测量高度：365毫米。用试剂盒提供的裂解液直接裂解在96孔板内的SBN-CD19-CAR-NK细胞作为阳性对照。
西妥昔单抗对SBN-CD19-CAR-NK细胞的杀伤率(％)＝处理过的SBN-CD19-CAR-NK细胞荧光强度/阳性对照细胞荧光强度。
图10显示了SBN-CD19-CAR-NK细胞对西妥昔单抗的敏感性结果。随着西妥昔单抗浓度的提升，SBN-CD19-CAR-NK细胞的死亡率有着明显的提高。SBN-CD19-CAR-NK细胞的死亡对西妥昔单抗是有特异性的。利妥昔单抗对SBN-CD19-CAR-NK细胞没有明显的作用。实验结果证实了SBN-CD19-CAR-NK细胞内EGFRt基因和蛋白的活性，可以起到安全杀死开关因子的作用。
实施例7 SBN-CD19-CAR-NK细胞整合基因的拷贝数和稳定性
在连续培养SBN-CD19-CAR-NK细胞时，用常规的实验室方法从不同传代数的SBN-CD19-CAR-NK细胞中提取基因组DNA。用定量PCR方法检测和确定整合的CD19-CAR基因拷贝数。用RNase P基因作为定量PCR基因拷贝数计算的标准。左引物序列如SEQ ID NO.12所示，右引物序列如SEQ ID NO.13所示，探针序列如SEQ ID NO.14所示。
PCR循环方法：变性/激活：95℃，5分钟，一次；变性：95℃，15秒；退火/延伸：60℃，1分钟；循环40次。
表2 SBN-CD19-CAR-NK细胞CAR整合基因的拷贝数和稳定性
SBN-CD19-CAR-NK细胞培养代数 细胞培养时间(天) CAR整合基因的拷贝数
2 7 1.1
10 40 0.9
20 75 1.2
SBN-CD19-CAR-NK细胞内有约1个拷贝的整合的CAR基因。在连续长时间的细胞培养过程中，细胞内整合的CAR基因保持了良好稳定性。
实施例8抗CD33阳性肿瘤的CD33-CAR-EGFRt嵌合抗原受体和杀伤开关基因的构建
在实施例1的CD19-CAR-EGFRt嵌合抗原受体和安全杀死开关因子设计的基础上，将抗CD19肿瘤抗原的scFv的基因序列更换为抗CD33肿瘤抗原的scFv的基因，构建了CD33-CAR-EGFRt嵌合抗原受体和杀伤开关的基因组。用类似的方法，用此基因组转导NK-SBN细胞可以获得SBN-CD33-CAR-NK细胞用于CD33阳性的急性骨髓性白血病的治疗。从美国IDT公司订购基因合成了CD33-CAR-EGFRt嵌合抗原受体和安全杀死开关因子的片段。基因测序验证了基因序列的正确性，基因序列见SEQ ID NO.15。其中核苷酸1263-2054是抗CD33肿瘤抗原的scFv序列,包括VH和VL基因的接头序列(核苷酸1662-1706)基因序列见SEQ ID NO.16。
依然采用了人延伸因子-1α启动因子驱动抗CD33-CAR和HER1t的基因表达。核苷酸1-1178是人延伸因子-1α启动因子基因序列，SEQ ID NO.2。核苷酸1197-1262是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列,SEQ ID NO.3。在抗CD33-CAR序列前加入GMCSFRα信号序列是为了导向抗CD33-CAR蛋白在细胞表面的表达。核苷酸2064-2198是CD8alpha链的枢纽域序列，SEQ ID NO.5。核苷酸2199-2519是CD28跨膜区和细胞质共刺激域序列，SEQ ID NO.6。核苷酸2520-2855是CD3zeta的细胞质信号域序列，SEQ ID NO.7。核苷酸2871-2942是T2A可裂解多肽的序列，SEQ ID NO.8。T2A可裂解多肽起到等当量表达抗CD33-CAR蛋白和EGFRt多肽的作用。核苷酸2886-2951是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列,请参阅基因SEQ ID NO.3。在抗EGFRt序列前加入GMCSFRα信号序列是为了导向抗EGFRt多肽在细胞表面的表达。核苷酸3009-4019是EGFRt多肽的序列，SEQ ID NO.9。核苷酸4020-4250是牛生长激素BGH-polyA的序列，SEQ ID NO.10。
实施例9 SBN-CD33-CAR-NK细胞的抗癌效应
用以上描述的方法生产CD33-CAR-EGFRt重组慢病毒，并用于转导NK-SBN细胞，产生SBN-CD33-CAR-NK细胞。
用CD33阳性的MOLM-13急性髓性白血病细胞(德国DSMZ公司)作为模型癌细胞(阳性)，人正常的成纤维细胞MRC-5作为阴性对照。使用实时细胞毒性分析(RTCA)方法检测了SBN-CD33-CAR-NK细胞的抗癌效应。该RTCA检测系统通过微电子生物传感器连续测量由生长在微电子生物传感电极之间的细胞所引起的阻抗。阻抗随着细胞的生长而增加，相反，细胞的死亡会导致阻抗的降低。首先将MOLM-13和MRC-5细胞接种到RTCA检测系统用的，带有微电子生物传感器的92孔培养板内。
培养约25小时后，向MOLM-13癌细胞培养板孔内加入和MOLM-13癌细胞不同比率的不表达CD33-CAR的NK-SBN细胞和SBN-CD33-CAR-NK细胞，采用的细胞比率包括1:1和5:1(效应细胞和癌细胞的比率)。
同样的，向MRC-5细胞培养板孔内加入和MRC-5细胞不同比率的不表达CD33-CAR的NK-SBN细胞和SBN-CD33-CAR-NK细胞，采用的细胞比率包括1:1，5:1和10:1。继续培养到50小时来检测细胞生长的变化。图11和图12分别显示了SBN-CD33-CAR-NK细胞对癌细胞和人正常细胞作用的RTCA检测结果。图13和图14分别显示了依照RTCA检测结果转换来的细胞杀伤动力学曲线。结果显示SBN-CD33-CAR-NK细胞对癌细胞有着快速和强有力的杀伤作用。SBN-CD33-CAR-NK细胞和MOLM-13癌细胞共同培养5个小时后，超过90％的癌细胞已经死亡。
SBN-CD33-CAR-NK细胞杰出的抗癌细胞效应和其母系的NK-SBN细胞自身所表现的抗癌细胞功效是有关系的。基因转导CD33-CAR进一步提高了NK细胞的抗癌细胞效应。这是SBN-CD33-CAR-NK细胞相对于一般的CD33-CAR-T细胞产品的一个突出优点。SBN-CD33-CAR-NK细胞在对癌细胞显示出高杀伤率的同时，对人正常细胞没有表现出明显的毒性作 用。在共同培养24个小时后，绝大多数细胞依然健存。实验结果证实，SBN-CD33-CAR-NK细胞的抗癌作用安全性高和特异性强。
实施例10抗BCMA阳性肿瘤的BCMA-CAR-EGFRt嵌合抗原受体和杀伤开关基因的构建
在实施例1的CD19-CAR-EGFRt嵌合抗原受体和安全杀死开关因子基因设计的基础上，将抗CD19肿瘤抗原的scFv的基因序列更换为抗BCMA(B细胞成熟抗原)肿瘤抗原的scFv的基因，构建了BCMA-CAR-EGFRt嵌合抗原受体和安全杀死开关因子的基因组。用类似的方法，用此基因组转导NK-SBN细胞可以获得SBN-BCMA-CAR-NK细胞用于BCMA阳性的多发性骨髓瘤的治疗。从美国IDT公司订购基因合成了BCMA-CAR-EGFRt嵌合抗原受体和安全杀死开关因子基因的片段。基因测序验证了基因序列的正确性，基因序列见SEQ ID NO.18。其中核苷酸1263-2030是抗BCMA肿瘤抗原的scFv序列,包括VH和VL基因的接头序列(核苷酸1596-1649)见SEQ ID NO.19依然采用了人延伸因子-1α启动因子驱动抗BCMA-CAR和HER1t的基因表达。核苷酸1-1178是人延伸因子-1α启动因子基因序列，SEQ ID NO.2。核苷酸1197-1262是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列,SEQ ID NO3。在抗BCMA-CAR序列前加入GMCSFRα信号序列是为了导向抗BCMA-CAR蛋白在细胞表面的表达。核苷酸2064-2198是CD8alpha链的枢纽域序列，SEQ ID NO.5。核苷酸2199-2519是CD28跨膜区和细胞质共刺激域序列，SEQ ID NO.6。核苷酸2520-2855是CD3zeta的细胞质信号域序列，SEQ ID NO.7。核苷酸2871-2942是T2A可裂解多肽的序列，SEQ ID NO.8。T2A可裂解多肽起到等当量表达抗BCMA-CAR蛋白和EGFRt多肽的作用。核苷酸2886-2951是人粒细胞-巨噬细胞集落刺激因子受体α(GMCSFRα)信号序列,请参阅基因序列表3。在抗EGFRt序列前加入GMCFSRα信号序列是为了导向抗EGFRt多肽在细胞表面的表达。核苷酸3009-4019是EGFRt多肽的序列，SEQ ID NO.9。核苷酸4020-4250是牛生长激素 BGH-polyA的序列，SEQ ID NO.10。
实施例11 SBN-BCMA-CAR-NK细胞的抗癌效应
用以上描述的方法生产BCMA-CAR-EGFRt重组慢病毒，并用于转导NK-SBN细胞，产生SBN-BCMA-CAR-NK细胞。
用BCMA阳性的H929骨髓瘤细胞(美国ATCC,CRL-9068)作为模型癌细胞(阳性)，人正常的成纤维细胞MRC-5作为阴性对照。使用实时细胞毒性分析(RTCA)方法检测了SBN-BCMA-CAR-NK细胞的抗癌效应。该RTCA检测系统通过微电子生物传感器连续测量由生长在微电子生物传感电极之间的细胞所引起的阻抗。阻抗随着细胞的生长而增加，相反，细胞的死亡会导致阻抗的降低。首先将H929和MRC-5细胞接种到RTCA检测系统用的，带有微电子生物传感器的92孔培养板内。
培养约25小时后，向H929癌细胞培养板孔内加入和H929癌细胞不同比率的不表达BCMA-CAR的NK-SBN细胞和SBN-BCMA-CAR-NK细胞，采用的细胞比率包括1:1,5:1和10:1(效应细胞和癌细胞的比率)。
同样的，向MRC-5细胞培养板孔内加入和MRC-5细胞不同比率的不表达BCMA-CAR的NK-SBN细胞和SBN-BCMA-CAR-NK细胞，采用的细胞比率包括1:1，5:1和10:1。继续培养到50小时来检测细胞生长的变化。图15和图16分别显示了SBN-BCMA-CAR-NK细胞对癌细胞和人正常细胞作用的RTCA检测结果。图17和图18分别显示了依照RTCA检测结果转换来的细胞杀伤动力学曲线。结果显示SBN-BCMA-CAR-NK细胞对癌细胞有着明显和强有力的杀伤作用。SBN-BCMA-CAR-NK细胞和H929癌细胞共同培养24小时后，癌细胞的死亡率超过了90％。
SBN-BCMA-CAR-NK细胞杰出的抗癌细胞效应和其母系的NK-SBN细胞自身所表现的抗癌细胞功效是有关系的。基因转导BCMA-CAR进一步提高了NK细胞的抗癌细胞效应。这是SBN-BCMA-CAR-NK细胞相对于一般的BCMA-CAR-T细胞产品的一个突出优点。SBN-BCMA-CAR-NK细胞在对癌细胞显示出高杀伤率的同时，对人正常细胞没有表现出明显的 毒性作用。在共同培养24个小时后，所有的人正常细胞保持了良好的生长活性。实验结果证实，SBN-BCMA-CAR-NK细胞的抗癌作用是有安全性和特异性的。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供了一种用于治疗肿瘤的基因工程细胞，该基因工程细胞能够同时表达肿瘤抗原的嵌合抗原受体(CAR)和安全杀死开关因子，所述细胞为异体人免疫细胞，所述安全杀死开关因子是无免疫原性的截短的人表皮生长因子受体(EGFR)多肽。本发明提供的基因工程细胞具有良好的稳定性和抗肿瘤效应，在必要的情况下通过注入抗EGFR单抗药物，可以消除注入患者体内的该基因工程细胞，提高了该基因工程细胞用于肿瘤治疗的安全性。本发明的基因工程细胞是一种同种异体细胞，可制备成临床级细胞治疗制品，用于治疗和预防人类各种恶性肿瘤，并且适合于大规模GMP生产。

Claims (23)

  1. 一种用于治疗肿瘤的基因工程细胞，其特征在于，其能够同时表达肿瘤抗原的嵌合抗原受体CAR和安全杀死开关因子。
  2. 如权利要求1所述的基因工程细胞，其特征在于，所述免疫细胞为异体人免疫细胞。
  3. 如权利要求1所述的基因工程细胞，其特征在于，所述免疫细胞为异体人自然杀伤细胞。
  4. 如权利要求1所述的基因工程细胞，其特征在于，所述安全杀死开关因子为截短的人表皮生长因子受体多肽。
  5. 如权利要求4所述的基因工程细胞，其特征在于，所述多肽缺少膜远端表皮生长因子结合结构域和细胞质尾巴信号域，但保留了和抗EGFR单抗相结合的胞外区域。
  6. 如权利要求5所述的基因工程细胞，其特征在于，所述多肽的核苷酸序列如SEQ ID NO.9所示。
  7. 如权利要求1-6任一所述的基因工程细胞，其特征在于，所述肿瘤抗原的嵌合抗原受体CAR与安全杀死开关因子通过C端T2A可切割链相结合，所述C端T2A可切割链的核苷酸序列如SEQ ID NO.8所示。
  8. 如权利要求1-6任一所述的基因工程细胞，其特征在于，所述肿瘤抗原的嵌合抗原受体CAR的分子结构包括：a)细胞表面的单克隆抗体片段scFv肿瘤抗原靶结合域，b)铰链和跨细胞膜域，和c)T细胞受体CD3ζ域和共刺激信号组成的细胞内质信号传递域。
  9. 如权利要求8所述的基因工程细胞，其特征在于，所述单克隆抗体为抗CD19、BCMA、CD 20、CD22、CD33、CD47,CD123，CD133,CD138,ROR1,GD2,Mesothelin,Muc1和Muc 16或CEA。
  10. 如权利要求9所述的基因工程细胞，其特征在于，单克隆抗体片段scFv的核苷酸序列如SEQ ID NO.4、16或19所示。
  11. 如权利要求8所述的基因工程细胞，其特征在于，所述铰链来自CD8alpha链，其核苷酸序列如SEQ ID NO.5所示。
  12. 如权利要求8所述的基因工程细胞，其特征在于，所述跨细胞膜域和共刺激信号来自CD28，其核苷酸序列如SEQ ID NO.6所示；或T细胞受体CD3ζ信号传递域，其核苷酸序列如SEQ ID NO.7所示。
  13. 如权利要求1-6任一所述的基因工程细胞，其特征在于，该基因工程细胞含有能够同时表达CD19嵌合抗原受体、CD33嵌合抗原受体或BCMA嵌合抗原受体和截短的EGFR多肽的基因构件，该基因构件的核苷酸序列分别如SEQ ID NO.1、15、18所示。
  14. 如权利要求1-13任一所述的基因工程细胞的制备方法，其特征在于，将含有CAR编码基因和EGFRt编码基因的T2A融合体的重组载体转入NK细胞中，或将CAR编码基因和EGFRt编码基因的T2A融合体导入NK细胞中，使NK细胞能够同时表达CAR和EGFRt；所述EGFRt为截短的人表皮生长因子受体多肽。
  15. 如权利要求14所述的制备方法，其特征在于，所述重组载体为重组慢病毒载体、重组逆转录病毒载体,或电穿孔DNA质粒载体。
  16. 如权利要求14所述的制备方法，其特征在于，所述CAR编码基因的核苷酸序列如SEQ ID NO.11,17或20所示，所述EGFRt编码基因的核苷酸序列如SEQ ID NO.9所示。
  17. 如权利要求14所述的制备方法，还包括将获得的基因工程细胞在GMP条件下采用悬浮培养技术大规模培养。
  18. 如权利要求14-17任一所述的制备方法，所述CAR编码基因和EGFRt编码基因的T2A融合体的核苷酸序列如SEQ ID NO.1,15或18所示。
  19. 权利要求1-13任一所述的基因工程细胞或权利要求14-18 任一所述的制备方法制得的基因工程细胞在制备肿瘤治疗药物中的应用。
  20. 如权利要求19所述的应用，其特征在于，所述的肿瘤治疗药物的剂型为注射剂，所述注射剂可用于血管内注射、瘤内注射、皮下注射、器官注射、胸水内注射或腹水内注射。
  21. 一种药物，其特征在于，含有权利要求1-13任一所述的基因工程细胞或权利要求14-18任一所述的制备方法制得的基因工程细胞。
  22. 如权利要求21所述的药物，其特征在于，所述药物为治疗肿瘤的药物。
  23. 如权利要求21或22所述的药物，其特征在于，所述药物为注射剂，所述注射剂可用于血管内注射、瘤内注射、皮下注射、器官注射、胸水内注射或腹水内注射。
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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 (en) * 2015-01-26 2016-08-04 The University Of Chicago CAR T-CELLS RECOGNIZING CANCER-SPECIFIC IL 13Rα2
WO2017053889A2 (en) * 2015-09-23 2017-03-30 Precision Immunotherapy, Inc. Flt3 directed car cells for immunotherapy
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 (en) * 2015-01-26 2016-08-04 The University Of Chicago CAR T-CELLS RECOGNIZING CANCER-SPECIFIC IL 13Rα2
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 (en) * 2015-09-23 2017-03-30 Precision Immunotherapy, Inc. Flt3 directed car cells for immunotherapy

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

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