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A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor

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Abstract

Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 108 CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.

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Acknowledgments

We would like to thank Katrin Birkholz, Christian Hofmann, and Stefanie Hoyer for excellent practical advisory and interesting discussions; Verena Wellner, Stefanie Baumann, and Ina Müller for excellent technical assistance; Stefanie Gross for evaluation of flow cytometry data and providing antibodies; Kris Thielemans for providing the pGEM4Z RNA-production vector; Saskia Santegroets and Tanja de Gruijl for providing the KATO III cell line; Miltenyi Biotech for kindly providing the anti-CD28 antibody; Caroline Bosch-Voskens for providing antibodies and donor cells; and Michael Aigner and Özcan Met for fruitful discussion on elutriation-derived T cells. This project was financed by the Wilhelm Sander-Stiftung (2010.001.1) and BayImmuNet (F5121.7.1.1/10/).

Conflict of interest

The authors declare no conflict of interest.

Ethical standards

The manuscript does not contain clinical studies or patient data. Human blood was used after informed consent approved by the institutional review board.

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Authors and Affiliations

  1. Department of Dermatology, Universtitätsklinikum Erlangen, Hartmannstraße 14, 91052, Erlangen, Germany

    Christian Krug, Manuel Wiesinger, Beatrice Schuler-Thurner, Gerold Schuler, Jan Dörrie & Niels Schaft

  2. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Hinrich Abken

  3. Department I Internal Medicine, University Hospital Cologne, Cologne, Germany

    Hinrich Abken

  4. Chair of Genetics, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany

    Christian Krug

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  1. Christian Krug
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Correspondence to Niels Schaft.

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Krug, C., Wiesinger, M., Abken, H. et al. A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor. Cancer Immunol Immunother 63, 999–1008 (2014). https://doi.org/10.1007/s00262-014-1572-5

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  • DOI: https://doi.org/10.1007/s00262-014-1572-5

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