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.