A method for tracking and quantifying activated CAR-T cells based on a two-color fluorescence system
A two-color fluorescence and cellular technology, applied in the field of tracing and quantification of activated CAR-T cells
Active Publication Date: 2022-02-11
NANJING DRUM TOWER HOSPITAL
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[0005] The purpose of the present invention is to provide a CAR-T cell-based activation system based on a multi-color fluorescence system in view of the lack of detection methods that can directly assess the activation state, activation ratio, and exhaustion degree of immunocompetent cells in the existing CAR-T cell therapy and clinical research process. Tracing and Quantification Methods of CAR-T Cells
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[0032] The present invention will be further described below in conjunction with drawings and embodiments. If specific conditions are not indicated in the examples, it is carried out according to the conventional conditions well known in the art or the conditions suggested by the manufacturer, and the instruments or reagents used are not indicated by the manufacturer, all are conventional products that can be purchased commercially.
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The invention discloses a method for tracing and quantifying activated CAR-T cells based on a multi-color fluorescence system. The first carrier is used to package lentivirus and screen stably transfected cells, which can display the total number and location of CAR-T cells in the body. On this basis, the lentivirus packaged with the second vector is transfected. After the CAR-T cells bind to the surface antigen of the target cell and are successfully activated, the NFAT transcription factor enters the nucleus, binds to the NFAT transcription factor-specific enhancer sequence and initiates the second Fluorescence expression shows the location of activated CAR-T cells in the body, and the ratio of activated CAR-T cells to the total infused cells can be obtained by calculation, indicating the activation status, activation ratio and exhaustion degree of immune active cells. The present invention can not only track the location of activated cells in real time, but also calculate the proportion of activated CAR-T cells, so as to understand the activation degree and survival time of immune adoptive cells in vivo, which is of great significance for the maintenance and adjustment of subsequent treatment plans.
Description
technical field [0001] The invention relates to the field of cellular immunotherapy, in particular to a method for tracing and quantifying activated CAR-T cells based on a multicolor fluorescence system. Background technique [0002] In recent years, immunotherapy has played an increasingly important role in tumor treatment. Among them, Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) has been widely used in hematological tumors and is gradually developing into the field of solid tumors. CAR-T cells transfect the gene encoding the fusion protein formed by the antigen-binding part of the antibody corresponding to the tumor antigen and the intracellular segment of CD3-ζ chain or FcεRIγ into the patient's T cells, so that the patient's own T cells can Express tumor-specific chimeric antigen receptors, thereby eliminating tumor cells. [0003] The efficacy of CAR-T cells in vivo is affected by many factors, including the patient's disease state, whether he has received c...
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IPC IPC(8): C12N15/867C12N15/65C12N5/10
CPCC12N15/86C12N15/65C12N5/0636C12N2740/15043C12N2800/107C12N2510/00
Inventor 许佩佩欧阳建陈兵柳旭程书文彭苗新
Owner NANJING DRUM TOWER HOSPITAL