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Method for rapidly detecting activity of CD19/CD20-CAR-T cells

A technology of CD20-CAR-T and cell activity, which is applied in the field of immune cell and its activity measurement, can solve the problems of time-consuming and unclear guidance

Pending Publication Date: 2022-04-01
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional anti-tumor methods include blood indicators, tumor size, cell component analysis, etc., but these methods are often time-consuming, or are qualitative results and the guiding role is not clear

Method used

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  • Method for rapidly detecting activity of CD19/CD20-CAR-T cells
  • Method for rapidly detecting activity of CD19/CD20-CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction of NFAT-RE-luciferase-K562 stably transfected cell lines

[0044] NFAT-RE-luciferase-K562 effector cells are K562 cells driven with NFAT-RE for luciferase expression, responsive to CD3 but not CD28 stimulation.

[0045] 1) Construction and packaging of NFAT-RE-luciferase lentiviral plasmid

[0046] The sequence of the CD19 / CD20 antibody in NFAT-RE was obtained by us through phage screening technology. It was expressed in the lentiviral plasmid pMDL through genetic recombination based on engineering and luciferase luciferase disclosed in the UNIPROT database, and 293 cells were used for viral isolation. Pack and collect.

[0047] Amino acid sequence of CD19 antibody:

[0048] LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRD...

Embodiment 2

[0058] Example 2. NFAT-RE-luciferase-K562 stably transfected cell line is used to evaluate the activity of CD19 / CD20 CAR-T receptor

[0059] 1) Raji cell culture

[0060]Raji is a tumor cell that highly expresses CD19 antigen. Resuscitate Raji cells from a liquid nitrogen tank with 10% newborn calf serum, 100 U / ml penicillin, 100 U / ml streptomycin, 2.5 g / L glucose, 0.11 g / L sodium pyruvate, 1.5 g / L Raji cells were cultured in RPMI 1640 medium with sodium bicarbonate, pH 7.2, 5% CO2, 37 °C, and 90% relative humidity, and passaged once every 2 days.

[0061] 2) Activity detection of NFAT-RE-luciferase-K562 stably transfected cell line

[0062] Raji cells and NFAT-RE-luciferase-K562 were mixed and cultured according to different ratios. After co-cultivation for 4 hours, the fluorescence signal intensity of K562 cells was detected by a microplate reader, and the affinity of the antigen was judged according to the difference in the fluorescence intensity produced by K562 cells. ...

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Abstract

The invention belongs to the field of biological medicine, and provides an immune cell and a method for determining the activity of the immune cell. The invention provides an engineering cell for detecting the activity of a CD19 / CD20-CAR-T cell, and the cell is a tumor cell strain containing an NFAT-RE-luciferase carrier, and contains a coding sequence of a CD19 antibody and / or a coding sequence of a CD20 antibody. When the engineering cell and a cell expressing a target antigen are co-cultured, the engineering cell stimulates fluorescent gene expression in the cell to generate fluorescence with different intensities, so that the cell activation condition after CD19 / 20 CAR-T transfection in different proportions is judged. The method disclosed by the invention is simple and convenient to operate, efficient and rapid, the condition of CD19 / CD20 in the CD19 / CD20 CAR-T cells can be rapidly and intuitively obtained, and the construction condition and cell viability of the CD19 / CD20 CAR-T cells can be evaluated.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to an immune cell and a method for measuring its activity. Specifically, the present invention provides a method for rapidly detecting the activity of CAR-T (CD19 / CD20-CAR-T cells) cells that recognize CD19 and CD20 and its use. Background technique [0002] Immunotherapy aims to strengthen the patient's own immune system to fight against diseases, and can be divided into two categories: active immunity and passive immunity. In recent years, various immunotherapeutic strategies aimed at inducing, boosting or engineering T cell responses have emerged as promising approaches for the treatment of cancer and autoimmune diseases. Immunotherapy plays an important role in the occurrence and development of tumors. It can not only prevent the occurrence of cancer, but also alleviate or even treat cancer by enhancing the body's immunity. [0003] Adoptive cell therapy is a major research direction o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/09C12N15/13G01N21/64C12Q1/02
Inventor 崔大祥梁辉沈琦张文桦田静彭家伟李雪玲王丹刘泽熙陈晓敏崔明青
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH