Investigating the functional biology of chimeric antigen receptor T cells

© 2016 Dr Alexander DavenportDespite the success of autologous chimeric antigen receptor (CAR) T cells to treat patients with refractory B cell acute lymphoblastic leukaemia, (ALL) and lymphoma, there are many aspects of CAR T cell biology that remains unknown. For this reason, this thesis explored whether the recognition of antigen via either antigen receptor (CAR vs endogenous T cell receptor (TCR)) affected the CAR-T cell immune synapse, receptor signalling and tumour target killing kinetics. By addressing this issue we aim to translate this new knowledge to the clinic and broaden the CAR T therapy success to patients with a wider range of cancer subtypes. To explore the above questions, a novel transgenic mouse (designated CAR.OT-I) was developed, in which CD8+ T cells co-expressed the OVA257-specific T cell receptor (TCR) and a second generation CAR with an scFv specific for human HER2. Chapter 3 of this thesis validated the model system and compared CTL activation from CAR.OT-I and OT-I mice. Chapter 4 used time-lapse and confocal microscopy to explore whether the killing kinetics of CAR.OTI CTL was different when stimulated via with OVA257-pulsed (TCR) or HER2-expressing tumour cells (CAR). This thesis showed for the first time, individual CAR.OT-I CTL killed multiple tumour cells (‘serial killing’) and detached faster from dying targets after CAR ligation. Furthermore, in chapter 5, the CAR immune synapse gross molecular structure was described for the first time. This disrupted immune synapse had Lck micro-clusters, poor actin clearance and no peripheral LFA-1 clustering. Finally, phosphoprotein signalling and Ca2+ flux studies revealed faster, stronger signalling initiated via CAR compared to TCR ligation. This observation was also correlated with faster recruitment of cytotoxic granules to the target cell after CAR ligation. Taken together, the chapter 5 data reveals the mechanisms whereby CAR ligation initiates rapid tumour killing and detachment (Chapter 4). Information from this body of work can be used to inform on the next generation of CAR designs and provides a baseline for comparing CAR and TCR killing events