The dynamics of anti-tumour responses generated by chimeric antigen receptor-modified immune effector cells

Abstract

A patient’s own T cells can be genetically modified and amplified in the laboratory to target antigens expressed on tumour cells through the introduction of chimeric antigen receptor (CAR) genes. Despite considerable advances in the treatment of B cell leukaemia using chimeric antigen receptor T (CART) cells targeting the CD19 antigen, some patients have not responded well, particularly those with solid tumours. Understanding the biology of CART cell effector function may explain treatment failure, and lead to more effective cell therapy products. Population-based assays such as flow cytometry give a snapshot of these complex cellular systems, but do not allow one to study the fate of individual cells over time. Therefore, the aim of this thesis was to apply flow cytometry, time-lapse imaging and singlecell tracking to characterise the dynamics of CART and tumour cell interactions in vitro. The utility of time-lapse imaging and single-cell tracking was demonstrated by quantifying the cytotoxicity of CART cells targeting CD19+ leukaemia cell lines. This thesis also addresses the problem of minimal CART cell effector function against solid tumours by studying the potency of anti-GD2 re-directed Natural Killer (GD2NK) and T cells (GD2T) targeting neuroblastoma spheroids with time-lapse imaging. Flow cytometry studies show that at least 90% of CD19+ leukaemia cells were killed at high effector to target ratios (E:T=10:1), however only 10% killing was achieved at lower ratios (E:T=1:1). Cooperative killing by CART cells was observed by time-lapse imaging without serial killing. In the clinical context, localisation of effector cells through chemotaxis and proliferation at tumour sites may be required for tumour elimination. NK and GD2NK cell penetration of solid neuroblastoma spheroids was superior to T or CART cells. NK but not T cells were able to destroy tumour spheroids. However, coculture of neuroblastoma spheroids with NK and T cells resulted in loss of NK-mediated tumour killing. In conclusion, this thesis provides methodological advances to the study of anti-tumour response generated by innate and CAR-enhanced effector cells against haematological malignancy and solid tumours by application of time-lapse imaging. This thesis also provides insights into the detailed mechanism of effector cell killing by direct observation of the dynamics of effector cell migration, infiltration, conjugate formation and cytotoxicity using single-cell tracking.