Shah, Rivani Rohin; (2024) Genetic modifications to enhance CAR T-cell stemness and persistence in brain tumours. Doctoral thesis (Ph.D), UCL (University College London).

Text R Shah PhD 2024.pdf - Accepted Version Access restricted to UCL open access staff until 1 January 2026. Download (13MB)

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has shown incredible success against haematological malignancies, but its efficacy against solid tumours remains limited. Key challenges include impaired trafficking, a hostile tumour microenvironment, and subsequent CAR T-cell exhaustion, leading to reduced persistence and loss of tumour control. This thesis explores the potential of enhancing CAR T-cell memory phenotypes by overexpressing specific transcription factors (TFs) to promote a stem-like state characterised by reduced exhaustion and increased persistence. Initially, four IL13 mutein CAR constructs were evaluated against IL13Rα2-expressing cell lines in vitro to identify the construct with the highest activation and exhaustion profile. The IL13KR.28z construct emerged as the lead candidate due to its heightened sensitivity to low IL13Rα2 expression, enhanced cytotoxicity, proliferation, and increased expression of terminal exhaustion markers, making it ideal for further exhaustion mitigation modifications. Given the Wnt signalling pathway’s role in regulating T-cell stemness, I investigated the effect of overexpressing key family members – TCF1, LEF1, and β-catenin – on CAR T-cell stemness during manufacture and repeated antigen exposure. Traditional lentiviral methods initially resulted in suboptimal CAR and TF expression. This was addressed by developing a dual vector approach that separated the CAR and TF constructs, which successfully maintained high CAR expression and TF overexpression, confirmed using RFP expression as a surrogate marker for the TF. Phenotypic assessment of IL13KR.28z CARs overexpressing TCF1, LEF1, or β-catenin through repeat stimulation assays revealed that β-catenin overexpression significantly reduced proliferation and increased the proportion of naïve and central memory T-cells, consistent with a more stem-like phenotype and suggestive of enhanced long-term persistence. Conversely, TCF1 and LEF1 overexpression had minimal impact, likely due to variable baseline expression or the presence of co-repressors. These findings underscore β-catenin’s potential as a therapeutic target to enhance CAR T-cell persistence and reduce exhaustion in both solid and liquid tumours.

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