TY - JOUR T1 - Automated, scaled, transposon-based production of CAR T cells JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1136/jitc-2022-005189 VL - 10 IS - 9 SP - e005189 AU - Dominik Lock AU - Razieh Monjezi AU - Caroline Brandes AU - Stephan Bates AU - Simon Lennartz AU - Karin Teppert AU - Leon Gehrke AU - Rafailla Karasakalidou-Seidt AU - Teodora Lukic AU - Marco Schmeer AU - Martin Schleef AU - Niels Werchau AU - Matthias Eyrich AU - Mario Assenmacher AU - Andrew Kaiser AU - Sabrina Prommersberger AU - Thomas Schaser AU - Michael Hudecek Y1 - 2022/09/01 UR - http://jitc.bmj.com/content/10/9/e005189.abstract N2 - Background There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.Methods We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.Results We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.Conclusions We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.Data are available on reasonable request. All data directly relevant to the study are included in the article. Further data are available on reasonable request. ER -