George, Vaques; (2023) The Bioprocessing of T cells and T cell Derived Extracellular Vesicles. Doctoral thesis (Ph.D), UCL (University College London).

Text Doctoral Thesis by Vaques George.pdf - Other Access restricted to UCL open access staff until 1 April 2026. Download (10MB)

Abstract

Cell and gene therapies have been gaining increasing interest over the years as a result of positive clinical outcomes. Most recently, genetically engineered T cells expressing CD19- chrimeric antigen receptor (CD19 CAR-T cells) have been hailed as ‘the cure for cancer’ following the 10-year remission of patients. However, the use of ‘living drugs’ for adoptive cell-based therapies warrants the risk of difficult to treat side-effects. Alternatively, γδT cells are extensively being studied in parallel for their unique innate/adaptive qualities in mitigating against the risk of off-target toxicities. Nevertheless, several researchers have sought to explore the potential of immune cell-derived extracellular vesicles (EVs) as replacement upon the discovery of their ability to function as parent-cell mimics in absence of their producer cell. Both γδT cell and CAR-T cell derived EVs have previously demonstrated in vivo cytotoxicity against target cells and at reduced toxicities when compared to their respective parent cell. Although, challenges associated with the bioprocessing of T cells and T cell products has contributed to the delay of their clinical use. This doctoral thesis sets out to explore and highlight the bioprocessing considerations required in producing T cells and T cell derived EVs for potential adoption within cancer immunotherapy. This was attempted by assessing the ability to culture donor derived PBMC in serum-free conditions for the expansion of T cells. Furthermore, PBMC derived EVs were also characterised and assessed for their cytotoxic potential against Nalm-6 (pre-B cell leukaemia) cell line. EVs harvested from day 6 of expanding PBMC in serum-free media showed significant cytotoxic capabilities over EVs harvested at later timepoints. This was found to be in agreement with the data obtained from CD19 CAR-T cell derived EVs whereby day 3 harvest contained significantly higher populations of cytotoxic proteins when compared to EVs harvested at day 7 of culture. However, in order to influence the production of cytotoxic EVs, γδT cells were exploited for their innate-like capabilities by developing LPS-coated beads for their activation and production cytotoxic EVs. This was proven to be successful when compared with / without the use of CD3/CD28-coated beads. This thesis provides novel insight into methods of bioprocessing T cells and T cell derived EVs towards advancing the field of cancer immunotherapy.

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