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Shortened ex vivo manufacturing time of EGFRvIII-specific chimeric antigen receptor (CAR) T cells reduces immune exhaustion and enhances antiglioma therapeutic function

  • Hillary G. Caruso
  • Ryuma Tanaka
  • Jiyong Liang
  • Xiaoyang Ling
  • Aria Sabbagh
  • Verlene K. Henry
  • Tiara L. Collier
  • Amy B. HeimbergerEmail author
Laboratory Investigation

Abstract

Background

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.

Method

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).

Results

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.

Conclusion

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.

Keywords

Chimeric antigen receptor T cell Exhaustion markers PD-1 PD-L1 LAG-3 TIM-3 

Notes

Acknowledgements

We thank David M. Wildrick, Ph.D., for editorial assistance and Audria Patrick for assistance in preparing the manuscript.

Authors’ contributions

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.

Funding

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.

Ethical approval

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.

Supplementary material

11060_2019_3311_MOESM1_ESM.docx (14 kb)
Supplementary file1 (DOCX 13 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hillary G. Caruso
    • 2
  • Ryuma Tanaka
    • 1
  • Jiyong Liang
    • 2
  • Xiaoyang Ling
    • 2
  • Aria Sabbagh
    • 2
  • Verlene K. Henry
    • 2
  • Tiara L. Collier
    • 2
  • Amy B. Heimberger
    • 2
    Email author
  1. 1.Division of PediatricsThe University of Texas MD Anderson Cancer CenterHoustonUSA
  2. 2.Department of NeurosurgeryThe University of Texas MD Anderson Cancer CenterHoustonUSA

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