Ripoll Fiol, Carme; (2024) Development of a scalable and robust upstream process for the chemically-defined production of lentiviral vectors for gene-modified cellular immunotherapies. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Ex vivo gene-modified cellular immunotherapy, including chimeric antigen receptor (CAR)-T and CAR-NK cell therapy, is a promising therapeutic modality for the treatment of various diseases and has demonstrated remarkable clinical outcomes for indications such as haematological malignancies. Lentiviral vectors (LVV) are commonly used to genetically modify the immune cells before infusing them back into the patient to target the malignant cells. However, robust and large-scale LVV manufacturing processes are needed to address this increased demand. This thesis focuses on the development of a scalable and robust upstream process for the chemically-defined production of fully functional lentiviral vectors for gene-modified cellular immunotherapies. The initial studies focused on the optimisation of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0, delaying the transient transfection, reducing plasmid DNA and adding 5 mM sodium butyrate 6 h post-transfection improved functional LVV titres by 180-fold. The optimised animal component-free production process was then transferred to the iCELLis® Nano bioreactor, a fixed-bed bioreactor, where titres of 1.2x10⁶ TU/cm2 were achieved when it was operated in perfusion. Notably, comparable functional LVV titres were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco’s Modified Eagle Medium supplemented with animal-derived serum both at small- and large-scale. Finally, to demonstrate not only high process yields but also the function and quality of the produced LVV, the LVV were used to generate clinically relevant CAR-T and CAR-NK cells. Undifferentiated, highly functional CAR-T cells capable of triggering cytotoxicity against target cells with specific lysis rates up to 97% were produced. The transduction process did not impact cell growth kinetics or phenotype when compared to non-transduced T cells. Additionally, the generation of CAR-NK cells was also assessed as an emerging alternative to CAR-T cells using the same LVV, where high specific lysis rates were also achieved. Altogether, the work hereby presented demonstrates the feasibility of producing high-quality LVV production yields in a fixed-bed bioreactor under chemically-defined conditions, which can then be used to generate fully functional relevant immune cells such as CAR-T and CAR-NK cell therapies.

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