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Target cell U87MG-EGRL for evaluating targeting EGFRvIII antigen and construction method and application of target cell U87MG-EGRL

A technology of U87MG-EGRL and its construction method, which is applied in the field of target cell U87MG-EGRL and its construction, can solve the problems of inaccurate evaluation of the killing effect of target cells, and achieve the effect of fast and sensitive operation

Pending Publication Date: 2019-10-08
焦顺昌 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current evaluation methods of in vitro killing efficiency include LDH method, flow cytometry method, etc., but none of them can accurately evaluate the killing effect on target cells

Method used

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  • Target cell U87MG-EGRL for evaluating targeting EGFRvIII antigen and construction method and application of target cell U87MG-EGRL
  • Target cell U87MG-EGRL for evaluating targeting EGFRvIII antigen and construction method and application of target cell U87MG-EGRL
  • Target cell U87MG-EGRL for evaluating targeting EGFRvIII antigen and construction method and application of target cell U87MG-EGRL

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of fusion protein gene expression vector of GFP and Red Luciferase

[0043] 1. Materials

[0044] 1. The lentiviral backbone plasmid pZX (the lentiviral vector structure is as follows: figure 1 shown), lentiviral packaging plasmids PSPAX2 and pMD2.G, HEK293T cells;

[0045] 2. The nucleotide sequence (SEQ ID NO.1) of the human EGFRvIII gene, the nucleotide sequence (SEQ ID NO.2) of the GFP gene, the nucleotide sequence (SEQ ID NO.3) of the Red Luciferase gene, and the connecting peptide The nucleotide sequence (SEQ ID NO.4), the nucleotide sequence (SEQ ID NO.5) of the GFP+ connecting peptide+Red Luciferase, the nucleotide sequence (SEQ ID NO.6) of the EGFRvIII target antigen used to connect the carrier );

[0046] 3. The DNA sequences shown in SEQ ID NO.5 and SEQ ID NO.6 were synthesized by Jinweizhi Biotechnology Co., Ltd. and preserved in the form of plasmids;

[0047] 4. The tool enzymes Nhe I and Not I were purchased from Thermo Company;

[0048] ...

Embodiment 2

[0072] Construction and screening of target cell U87MG-EFRL

[0073] The specific method for infecting cells with lentivirus comprises the following steps:

[0074] (1) Infection of U87MG cells with lentivirus pZX-GFP-T2A-LUC

[0075] (1) Dilute the cultured cells to be infected to 1×10 after digesting and counting 5 cells / mL, take 2mL and add it to a 6-well culture dish, shake well and place in an incubator for cultivation.

[0076] (2) The next day, add the packaged virus to the culture dish, shake it well after labeling, and place it in an incubator for culture. After 2-3 days, the culture can be expanded according to the cell density.

[0077] (2) Sorting GFP-positive cell populations by flow cytometry

[0078] (1) Collect the cells in the T175 culture flask, centrifuge at 200×g for 5 min, and collect uninfected cells as a control.

[0079] (2) Add 1mL PBS to each tube to wash the cells once, and centrifuge at 200×g for 5min.

[0080](3) Add 500μL PBS to each tube to ...

Embodiment 3

[0112] Detection of target cell killing efficiency of U87MG-EFRL cells

[0113] (1) Collect target cells U87MG-EFRL and U87MG-FRL, inoculate them into 96-well plates, and inoculate 5000 target cells per well;

[0114] (2) Collect effector cells (CAR-T cells targeting EGFRvIII antigen), wash twice with PBS, and resuspend cells with 10% FBS-1640;

[0115] (3) The effect-to-target ratio is set at 20:1, 10:1, 5:1, 2.5:1;

[0116] (4) 24 hours after inoculation, add CAR-T cells according to the corresponding effect-to-target ratio;

[0117] (5) Check the chemiluminescence value after killing for 4 hours.

[0118] (6) Calculate the killing efficiency according to the chemiluminescence value.

[0119] Using CAR-EGFRvIII cells as effector cells, using U87MG-GRL and U87MG-EGRL as target cells, the change curve of killing rate is as follows Figure 8 As shown, CAR-EGFRvIII cells have a strong killing effect on the target cell U87MG-EGRL, but a weak killing effect on the U87MG-GRL ce...

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Abstract

The invention provides a target cell U87MG-EGRL for evaluating a targeting EGFRvIII antigen and a construction method and application of the target cell U87MG-EGRL, and belongs to the technical fieldof cellular immunity. The target cell U87MG-EGRL is a glioma cell which can express fusion protein genes of CAR-T cell target antigen EGFRvIII genes, GFP and Red Luciferase. The target cell can efficiently express the EGFRvIII antigen, both GFP genes and luciferase genes are expressed, and the killing effect of CAR-T cells can be evaluated by the luciferase reaction, therefore, the target cell canbe applied to the pharmacodynamic evaluation of CAR-T in vivo and in vitro.

Description

technical field [0001] The invention belongs to the technical field of cellular immunity, and in particular relates to a target cell U87MG-EGRL for evaluating targeting EGFRvIII antigen and its construction method and application. Background technique [0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T immunotherapy) is a new type of cellular immunotherapy technology that has developed rapidly in recent years. It uses genetic engineering technology to integrate target-specific recognition (single-chain antibody scFv), activate immune activity and attack tumor cells into a gene, and transfer it into the patient's own T lymphocytes and infuse it back into the patient's body , so that patients regain the ability to specifically recognize tumor cells, activate their own T cells, and attack and kill the identified tumor cells in a targeted manner. [0003] Glioma is one of the most malignant tumors and develops rapidly. According to statistics, the average survival pe...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/65
CPCC07K14/71C12N5/0693C12N15/65C12N15/86C12N2740/15043
Inventor 焦顺昌张嵘宋玉洁
Owner 焦顺昌
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