Automated, scaled, transposon-based production of CAR T cells

• Published in: Journal for immunotherapy of cancer 2022-09, Vol.10 (9), p.e005189
• Main Authors: Lock, Dominik, Monjezi, Razieh, Brandes, Caroline, Bates, Stephan, Lennartz, Simon, Teppert, Karin, Gehrke, Leon, Karasakalidou-Seidt, Rafailla, Lukic, Teodora, Schmeer, Marco, Schleef, Martin, Werchau, Niels, Eyrich, Matthias, Assenmacher, Mario, Kaiser, Andrew, Prommersberger, Sabrina, Schaser, Thomas, Hudecek, Michael
• Format: Article
• Language: English
• Subjects: Antigens; Automation; Cell Engineering; Cytokines; Immune Cell Therapies and Immune Cell Engineering; Immunotherapy; Lymphocytes; Manufacturers; Manufacturing; Prodigies; Receptors, Chimeric Antigen; Translational Medical Research; Vectors (Biology)
• ISSN: 2051-1426; 2051-1426
• DOI: 10.1136/jitc-2022-005189

BackgroundThere 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.