Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function
Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4 weeks to generate CAR T cells of presumed sufficient quantity and function for clinical applications.
We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population).
While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2 weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2 weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2 weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas.
These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.
KeywordsChimeric antigen receptor T cell Exhaustion markers PD-1 PD-L1 LAG-3 TIM-3
We thank David M. Wildrick, Ph.D., for editorial assistance and Audria Patrick for assistance in preparing the manuscript.
HC, RT, XL, AS, and ABH conceived and designed the experiments, HC, RT, and XL developed the methodology, HC, RT, JL, XL, AS, VKT, and TLC carried out the experiment and data acquisition, HC, JL, and ABH wrote, reviewed, and revised the manuscript, ABH provide the administrative, technical, and material support, and all authors read and approved the final manuscript.
This study was funded by the Brooks Foundation for Cancer Research.
Compliance with ethical standards
Conflict of interest
H.C. has licensed intellectual property to Ziopharm and Precigen Therapeutics on CAR therapeutics. The other authors have no competing interests related to the subject of this manuscript.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In vivo experiments involving mice were approved by Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center.
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