Related Experiment Video
Updated: Mar 21, 2026
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor
Hillary G Caruso1, Hiroki Torikai, Ling Zhang
1*Division of Pediatrics Departments of ‡Biostatistics ∥Neurosurgery §Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center †The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX.
Abstract:
Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.
Insights
Transiently modified T cells expressing chimeric antigen receptors (CARs) target tumors while minimizing off-tissue toxicity. This method enhances safety for CAR T-cell therapy by controlling CAR expression in T cells.
Area of Science:
- Immunology
- Cell Therapy
- Molecular Biology
Background:
- Chimeric antigen receptor (CAR) T-cell therapy faces limitations due to on-target, off-tissue toxicity, especially when targeting antigens like epidermal growth factor receptor (EGFR) present in normal tissues.
- Transiently modifying T cells using mRNA offers a strategy to mitigate this toxicity by controlling CAR expression duration.
Purpose of the Study:
- To evaluate the expression and efficacy of an EGFR-specific CAR delivered via mRNA in human T cells expanded ex vivo.
- To assess the impact of T cell expansion methods on CAR modification efficiency and T cell phenotype.
- To determine the therapeutic potential of transient CAR expression for targeting EGFR-expressing tumors while minimizing off-tissue effects.
Main Methods:
- Human T cells were expanded ex vivo using activating and propagating cells (AaPC) derived from K562 cells preloaded with anti-CD3 antibody.
- T cell phenotype was modulated by adjusting the AaPC density, optimizing for mRNA electrotransfer.
- EGFR-specific CAR was introduced into T cells using in vitro-transcribed mRNA, and expression/functionality was assessed.
Main Results:
- Reduced AaPC ratios yielded T cells with phenotypes more suitable for mRNA electrotransfer, characterized by higher CD8 and central memory T cell populations.
- RNA-modified CAR T cells exhibited comparable cytolytic activity against EGFR-expressing glioblastoma cells as DNA-modified CAR T cells, but produced less cytokine.
- CAR expression was transient and accelerated by cytokine and antigen stimulation, with loss of CAR abrogating T-cell function against EGFR-expressing cells.
Conclusions:
- A clinically applicable method for propagating and modifying T cells to transiently express EGFR-specific CARs was established.
- This transient expression approach holds promise for targeting EGFR-expressing tumor cells while reducing on-target, off-tissue toxicity to normal tissues.
- The transient nature of mRNA-mediated CAR expression provides a controllable therapeutic window for CAR T-cell applications.
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