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Method of detecting chimeric antigen receptor or genetically-modified T-cell receptor expression and application of method

A chimeric antigen receptor and gene modification technology, applied in the fields of immunology and molecular biology, can solve the problems of not being able to prove the specific binding of CAR or TCR tumor antigens, and the inability to accurately predict the tumor killing efficacy, so as to improve the accuracy. Effect

Active Publication Date: 2017-10-17
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method can calculate the efficiency of virus infection, neither of the above two methods can prove whether the expressed CAR or TCR can specifically bind to the corresponding tumor antigen, and the actual killing effect on the tumor cannot be accurately predicted.

Method used

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  • Method of detecting chimeric antigen receptor or genetically-modified T-cell receptor expression and application of method
  • Method of detecting chimeric antigen receptor or genetically-modified T-cell receptor expression and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0048] The verification of the lentivirus (pCDH-CAR19) carrying the CAR gene of the CD19 molecule was selected, and the corresponding tumor cells were selected as B lymphoma cells expressing CD19——Raji cells.

[0049] (1) Infect 293T cells with pCDH-CAR19 to obtain stably transfected 293T cells expressing chimeric antigen receptors;

[0050] (2) Plate the stably transfected 293T cells, add the first culture medium (the mixed solution of DMEM medium and 10% fetal bovine serum by mass fraction), in the concentration of 5% CO 2 Conditions, 37 ℃ constant temperature culture;

[0051] (3) When the fusion degree of stably transfected 293T cells reaches 80%, remove the first culture medium and add the second culture medium (mixed solution of 1640 medium and fetal bovine serum) containing Raji cells to obtain a cell mixture ; The initial concentration of Raji cells was 1.0 × 10 6 ;

[0052] (4) After culturing the cell mixture for 1 h, 2 h, 4 h, and 6 h, collect the supernatant;

...

Embodiment 2

[0055] Compared with Example 1, except step (3) the fusion degree of 293T cells is 60%, the rest is the same as Example 1.

Embodiment 3

[0057] Compared with Example 1, except step (3) the fusion degree of 293T cells is 70%, the rest is the same as Example 1.

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Abstract

The invention relates to the field of immunology and molecular biology, in particular to a method of detecting chimeric antigen receptor or genetically-modified T-cell receptor expression and application of the method; the method mainly comprises the steps of (1) infecting adherent cells or suspension cells with packaged CAR (chimeric antigen receptor) or TCR (T-cell receptor) lentivirus to obtain stably-infected adherent cells or suspension cells for CAR or TCR expression; (2) spreading the adherent cells, and adding the corresponding suspension cells when fusion degree of the adherent cells reaches 70-90%; (3) continuing culturing, collecting supernate, counting the cells suspended in the supernate, and observing binding of the suspension cells and the adherent cells. The method of the invention not only can judge whether CAR or TCR is correctly expressed, but also verify whether the expressed CAR or TCR can specifically bind with corresponding tumor antigens; CAR or TCR expression detection accuracy can be further improved, and the immunological therapy of tumors is further guaranteed.

Description

technical field [0001] The invention relates to the fields of immunology and molecular biology, in particular to a method for detecting the correct expression of receptors, in particular to a method and application for detecting the expression of chimeric antigen receptors or gene-modified T cell receptors. Background technique [0002] Tumor immunotherapy is divided into two categories: one is to release the tumor's immune tolerance or shielding effect, allowing immune cells to "recognize" tumor cells, and then attack the tumor, such as PD-1 antibody, PD-L1 antibody , CTLA-4 antibody, etc.; the other is to "tell" the immune cells the characteristics of the tumor, and then the immune cells search and locate to kill the tumor, such as chimeric antigen receptor T cells (CAR-T), T cell receptor Genetically modified T cells (TCR-T), tumor infiltrating lymphocytes (TIL), tumor antigen-loaded dendritic cells induced specific cytotoxic T lymphocytes (DC-CTL), etc. [0003] Among t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C07K14/705A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C12N15/86C12N2740/15043
Inventor 韦丹付苏雷连杰赵礼军李德志
Owner HENAN HUALONG BIOLOGICAL TECH