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A genetically engineered cell for treating tumors

A genetic engineering and cell technology, applied in the field of genetic engineering cells for the treatment of tumors, which can solve the problems of CAR-T cell failure and loss of precious time for tumor treatment.

Active Publication Date: 2021-05-18
SINOSHENG SHENZHEN GENE IND DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, patients also face the possibility of failure in the production process of their own CAR-T cells, and face the loss of precious time for tumor treatment

Method used

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  • A genetically engineered cell for treating tumors
  • A genetically engineered cell for treating tumors
  • A genetically engineered cell for treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of anti-CD19-positive tumor CD19-CAR-EGFRt chimeric antigen receptor and safety kill switch factor gene

[0057] The designed CD19-CAR is based on the structure of the second-generation CAR, using the anti-CD19 tumor antigen scFv and CD28 derived from the FMC63 mouse hybridoma as co-stimulatory molecules linked to the intracellular signaling domain of CD3zeta. Choosing CD28 as a co-stimulatory molecule can rapidly activate CAR-NK cells in vivo and eliminate tumor cells in a short period of time.

[0058]In order to improve the flexibility and flexibility of the CAR protein structure, this application added a hub domain from the CD8 alpha chain between the anti-CD19 tumor antigen scFv and the CD28 co-stimulatory molecule, and its nucleotide sequence is shown in SEQ ID NO.5 . In order to facilitate the detection of anti-CD19-CAR expression on the cell surface after gene transduction by flow cytometry, a gene expressing a T2A cleavable polypeptide (...

Embodiment 2

[0060] Example 2 Preparation of CD19-CAR-EGFRt recombinant lentivirus

[0061] The CD19-CAR-EGFRt chimeric antigen receptor and safety kill switch factor synthesized in Example 1 were cloned into the vector plasmid (Carnova, USA) for the production of lentivirus to obtain the CD19-CAR-EGFRt lentiviral vector plasmid 98708 . figure 2 A schematic diagram of the structure of the 98708 vector plasmid is shown. The 98708 vector plasmid DNA was amplified by the usual molecular biology experiment method. Using 98708 vector plasmid DNA and four plasmid DNAs encoding lentivirus gag / pol, rev, and VSV-G virus envelope respectively, co-precipitate and infect HEK293T cells cultured in a culture dish to produce CD19-CAR-EGFRt recombinant lentivirus , named LV-CD19-CAR-EGFRt. Culture fluid containing LV-CD19-CAR-EGFRt was collected two days after DNA infection. Centrifuge at a speed of 500g for 15 minutes to remove cell debris, and then store the supernatant containing LV-CD19-CAR-EGFRt...

Embodiment 3

[0062] Example 3 LV-CD19-CAR-EGFRt recombinant lentivirus transduction natural killer cell line

[0063] NK-SBN is a genetically engineered natural killer cell line developed by Shenzhen Saibainuo Gene Technology Co., Ltd. It has been disclosed in the Chinese patent (application number 201810392435.3) that it can grow without the addition of IL-2. Remove and thaw cells from liquid nitrogen tank. Use RPMI + 10% FBS medium at 37°C, 5% CO 2 NK-SBN cells were cultured and expanded in a cell culture incubator. The LV-CD19-CAR-EGFRt virus prepared in Example 2 was used to transduce NK-SBN cells in logarithmic growth phase at different MOIs. On the second day after viral transduction, cell samples were taken and treated with fluorophore-labeled anti-EGFR monoclonal antibody (Human EGFR (Cetuximab) Alexa 488-conjugated Antibody, R&D Systems, Cat#FAB9577G-100) stained cells. The gene transduction efficiency of cells was checked by flow cytometry. The detection was carried out acc...

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Abstract

The invention provides a genetically engineered cell for treating tumors, the genetically engineered cell can simultaneously express a chimeric antigen receptor (CAR) of a tumor antigen and a safety kill switch factor, the cell is an allogeneic human immune cell, and The safe kill switch factor is a non-immunogenic truncated human epidermal growth factor receptor (EGFR) polypeptide. The genetically engineered cells provided by the present invention have good stability and anti-tumor effects. If necessary, the genetically engineered cells injected into the patient's body can be eliminated by injecting anti-EGFR monoclonal antibody drugs, which improves the use of the genetically engineered cells in tumors. The safety of treatment. The genetically engineered cell of the present invention is a kind of allogeneic cell, which can be prepared into a clinical-grade cell therapy product for treating and preventing various human malignant tumors, and is suitable for large-scale GMP production.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a chimeric antigen receptor (CAR) capable of expressing tumor antigens and genetically engineered cells for treating tumors. Background technique [0002] Tumor biological therapy is the fourth major treatment method after surgery, radiotherapy and chemotherapy. Since the development of traditional surgery, radiotherapy and chemotherapy has entered a plateau, more and more people are focusing on the biological treatment of tumors. Gene and cell immunotherapy for cancer is the cutting-edge technology of tumor biotherapy, which has the advantages of relatively less toxic and side effects and significant curative effect. People in the industry expect that gene cell immunotherapy has the potential to become the fifth largest tumor treatment method. To achieve cellular immunotherapy for cancer, the immune system's containment of malignant cells must first ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/62C12N15/12A61K39/00A61P35/00
CPCA61K2039/5156A61P35/00A61K39/001112C07K14/7051C07K14/70517C07K14/70521C07K14/71C07K16/2803C07K2317/622C07K2319/00C07K2319/33C07K2319/74C12N5/0646C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 徐卫
Owner SINOSHENG SHENZHEN GENE IND DEV CO LTD
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