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A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

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TCR Gene Therapy for Cancer.

Simone Rhein¹, Neşe Çakmak-Görür²

¹Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. simone.rhein@mdc-berlin.de.

Methods in Molecular Biology (Clifton, N.J.)

|June 22, 2022

Summary

This study details methods for generating antigen-specific T cells and identifying their T cell receptors (TCRs) for adoptive T cell therapy. These techniques enable the development of targeted T cell therapies for various diseases.

Keywords:

Antigen-specific T cells RACE PCR T cell sort T cell stimulation TCR Viral transduction

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Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Published on: January 3, 2025

Area of Science:

Immunology
Cell Therapy
Molecular Biology

Background:

Adoptive T cell therapy requires generating antigen-specific T cells.
Identifying the T cell receptor (TCR) is crucial for T cell function.

Purpose of the Study:

To describe a protocol for generating antigen-specific T cells.
To outline a method for T cell receptor (TCR) identification for adoptive T cell therapy.

Main Methods:

Two methods for T cell stimulation: autologous dendritic cells with peptide or in vivo stimulation via gene gun.
T cell sorting using fluorescence-activated cell sorting (FACS) for CD107α or IFNγ.
TCR identification using 5' rapid amplification of cDNA ends (RACE)-PCR and retroviral cloning.

Main Results:

Successful generation of antigen-specific T cells through described methods.
Identification and isolation of TCRs from stimulated T cells.
Demonstration of TCR re-expression on human T cells for therapeutic potential.

Conclusions:

The described protocol provides a robust method for generating antigen-specific T cells and identifying their TCRs.
This approach is applicable for developing T cell therapies.
The identified TCRs can be re-expressed in human T cells for therapeutic applications.