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Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance

Department of Dermatology, Universtitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Hartmannstraße 14, 91052 Erlangen, Germany
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Author to whom correspondence should be addressed.
Beatrice Schuler-Thurner and Niels Schaft contributed equally.
Cancers 2019, 11(8), 1198; https://doi.org/10.3390/cancers11081198
Received: 20 May 2019 / Revised: 14 July 2019 / Accepted: 14 August 2019 / Published: 16 August 2019
(This article belongs to the Special Issue CAR-T Cell Therapy-Novel Approaches and Challenges)
Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward. View Full-Text
Keywords: CAR-T cell; melanoma; CSPG4; clinical scale production; full GMP compliance; clinical study; consistency runs CAR-T cell; melanoma; CSPG4; clinical scale production; full GMP compliance; clinical study; consistency runs
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Wiesinger, M.; März, J.; Kummer, M.; Schuler, G.; Dörrie, J.; Schuler-Thurner, B.; Schaft, N. Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance. Cancers 2019, 11, 1198.

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