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  • SCALABLE MANUFACTURING OF C...
    Couto, P. Silva; Stibbs, D.; Springuel, P.; Rafiq, Q.A.

    Cytotherapy (Oxford, England), June 2024, 2024-06-00, Letnik: 26, Številka: 6
    Journal Article

    CAR-T cell therapies have emerged as a revolutionary approach in the field of haematological malignancies, offering unprecedented clinical efficacy when patients have exhausted any other treatment option. Six CAR-T products are available worldwide, but many of the manufacturing challenges are yet to be addressed. A significant fraction of the cost is related to manufacturing the viral vector required to introduce the clinically relevant CAR construct. As such, non-viral gene delivery platforms such as electroporation or mechanoporation can potentially decrease the costs of manufacturing associated with CAR-T products. This study focused on establishing a scalable manufacturing method for CAR-T cell therapies using electroporation and a transposon-transposase system employed to ensure stable integration of the CAR transgene. Several stages were undertaken to establish the scalable manufacturing process: (1) electroporation optimisation, (2) identification of suitable cell viability recovery strategy, (3) small-scale expansion studies followed by (4) scalable expansion (ongoing). The workflow used featured a screening across electroporation conditions, followed by a study focused on changing the feeding strategy to maximize cell viability recovery post-electroporation. Once these were identified, the non-viral CAR-T product was characterized using growth rate, metabolic profile, CD4:CD8 ratios, CD8 subsets and CAR expression. This work was conducted using cryopreserved CD3+ cells, RPMI supplemented with 10% FBS, 2 mM of L-glutamine and 50 IU/mL of IL-2 as expansion medium and TransAct as activation method. Here, suitable electroporation programs that minimised cell viability loss after electroporation were identified. In addition, a DNA-to-cell ratio study demonstrated that 1 µg of each minicircle per million cells transfected is sufficient to generate CD3+ populations with viability levels above 90% with CAR expression levels reaching 30%, 48 hours past electroporation. On harvesting day, our optimized process led to a CAR-T product with a CD4:CD8 ratio of 3, with 95% of the CD8 in the central memory phenotype. CAR expression levels were shown to be above 25% on harvesting day. A total of 20-fold increase was reported during the 7-day expansion period. The next stage of this study will include the integration of the electroporation with stirred tank bioreactors to demonstrate the feasibility of modular manufacturing in the non-viral CAR-T space.