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.

Manuel Wiesinger,Niels Schaft,Beatrice Schuler-Thurner,Johannes März,Gerold Schuler,Jan Dörrie,Mirko Kummer

doi:10.3390/cancers11081198

Manuel Wiesinger, Niels Schaft + Show 5 more

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https://doi.org/10.3390/cancers11081198

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Abstract

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.

Highlights

Autologous T cells, reprogrammed to target malignant cells via the expression of a chimeric antigen receptor (CAR-T cells) represent a promising tool in the adoptive cellular therapy of cancer.Impressive clinical regressions of leukemias or lymphomas have been achieved using CD19-specificChimeric antigen receptor (CAR)-T cells in several clinical trials
To circumvent concerns about potential on-target/off-tumor toxicities, we have previously demonstrated that transient transfection of T cells with chondroitin sulfate proteoglycan 4 (CSPG4)-CARs using mRNA electroporation might be an effective and safe tool in cancer immunotherapy [51,52,53]
This study describes the establishment of the clinical-scale production of CAR-T cells for the treatment on the generation of such cells are focusing on virally transduced CAR-T cells

Summary

Introduction

Autologous T cells, reprogrammed to target malignant cells via the expression of a chimeric antigen receptor (CAR-T cells) represent a promising tool in the adoptive cellular therapy of cancer.Impressive clinical regressions of leukemias or lymphomas have been achieved using CD19-specificCAR-T cells in several clinical trials. Most clinical trials focus on the elimination of these so-called liquid tumors; the development of CAR-T cells against solid tumors lags behind (reviewed in [2,3,4,5]). This is Cancers 2019, 11, 1198; doi:10.3390/cancers11081198 www.mdpi.com/journal/cancers. The expression of the target antigen on healthy tissue always bears the risk of severe side effects due to tissue toxicity This is probably the reason that very few CAR-T cells against different antigens expressed on melanoma (e.g., VEGFR2, CD70, GD2, c-Met) were tested in clinical trials (NCT03060356, NCT01218867, NCT02107963, NCT02830724)

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