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

Abstract

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.

CAR-T cell therapy in multiple myeloma: Current limitations and potential strategies

“…Due to the decreased complexity of manufacturing processes and the better cargo capacity, transposon systems reduce the manufacturing costs and are more suitable for multi-targeted CAR-T cell manufacturing compared with viral vectors. Furthermore, transposon systems can be utilized on an automated process platform to generate clinical therapeutic doses of CAR-T cells, which will further promote the scale-up manufacturing of CAR-T cells and increase R/R patient access to CAR-T cell therapy ( 128 , 130 ). In addition, transposon-based CAR-T cells exhibit early memory T cell phenotype ( 128 ).…”

“…Furthermore, transposon systems can be utilized on an automated process platform to generate clinical therapeutic doses of CAR-T cells, which will further promote the scale-up manufacturing of CAR-T cells and increase R/R patient access to CAR-T cell therapy ( 128 , 130 ). In addition, transposon-based CAR-T cells exhibit early memory T cell phenotype ( 128 ). Encouragingly, a recent study demonstrated that CRISPR-Cas9-mediated non-viral specifically targeted CAR-T cells were safe and effective in patients with R/R non-Hodgkin lymphoma (NHL) ( 56 ), indicating that CRISPR-Cas9 is a new tool for precise genome editing in CAR-T cell manufacturing and will facilitate the development of more gene-specific targeted CAR-T cells in the future ( 56 , 131 ).…”

An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR‐T and TCR‐T Cellular Therapies Development

“…Different molecular (genotypic) assays, such as qPCR/dPCR, Nanostring, or next‐generation sequencing (NGS) DNA or RNA‐seq, could be used to monitor CAR‐T CK in cell therapy studies. Both Nanostring and RNA‐seq can be multiplexed for the quantitation of several gene targets in a single run 9,10 and RNA‐seq provides complete sequence information which could be useful in the discovery space. Overall, due to higher sensitivity, easy sampling, reproducibility, robustness, and cost/ease of handling a small number of targets, PCR‐based methods are widely used to monitor CAR‐T CK and preferred over NGS methodologies 11,12 .…”

Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells

“…However, these systems require well-trained staff and rigorous hygiene monitoring to avoid contaminations. Therefore, with the clinical successes of CAR-T cell therapies, the production has shifted not only in industrial facilities but also in academic institutions more and more to closed and semi- or fully-automated platforms ( 22 – 25 ). Most advances to the manufacturing platforms for CAR-T cells are likely transferable for the production of TCR-T cells with some modifications to the protocols.…”

“…SB transposition has already been used successfully for the manufacturing of CD19 or SLAMF7 CAR-T cells in early clinical trials without severe toxicity ( 62 – 65 ). In addition, automation of SB transposition was feasible and could be very attractive for large-scale manufacturing ( 25 ). Although PB transposition was also successful for the manufacturing of CAR-T cells and even showed an inclination to promote the generation of desired TSCM CAR-T cells ( 66 , 67 ), a recent clinical trial observed the formation of CAR-T cell lymphoma in two out of ten patients ( 68 , 69 ).…”