Agbuduwe, Charles Eromosele; (2024) Investigating a Novel Gamma Delta (γδ) T Cell-Based Chimeric Antigen Receptor Therapy for Solid Tumours. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Despite impressive outcomes for Chimeric Antigen Receptor (CAR) therapy in haematological cancers, little progress has so far been made for solid tumours. This is due to the hostile solid tumour microenvironment (TME) and the lack of ideal solid cancer targets. B7-H3, an immune checkpoint molecule which is overexpressed by a wide range of solid tumours with minimal expression in healthy tissues, was the cancer-associated antigen target selected for evaluation in this project. The aims of this research were to develop an expansion protocol from peripheral blood for the potent Vδ1 subset of gamma delta (γδ) T cells which are naturally tissue-resident and thus more adapted towards tumour immune surveillance, the evaluation of a B7-H3 specific CAR and the development of a hypoxia-sensing CAR construct. Expansion of Vδ1 γδ T cells was achieved by stimulation of healthy donor peripheral mononuclear blood cells (PBMCs) with a CD3 antibody (OKT3 clone) and Interleukin-15. The Vδ1-enriched γδ T cells demonstrated potent anti-cancer properties which were further enhanced by the B7-H3 targeting CAR based on a novel scFv binder. In vitro co-culture experiments with cancer cell lines demonstrated enhanced cytotoxicity, cytokine secretion and proliferation against cancer cell lines but not against allogeneic PBMCs. Furthermore, proof-of-concept demonstration of a B7-H3 targeting hypoxiCAR encoding an oxygen degradation domain (ODD) and hypoxia responsive elements (HRE) showed dual oxygen sensing with reversible induction of CAR expression under hypoxic conditions and subsequent CAR degradation in normoxia. To enhance persistence within the TME, transduction of a constitutively active STAT5B mutant construct conferred enhanced cytokine-independent survival and proliferation to γδ T cells. In summary, this project has demonstrated successful expansion of the Vδ1 subset of γδ T cells using a technique that is permissive for retroviral transduction. A novel B7-H3 targeting CAR construct enhanced γδ T cell function and demonstrated proofof-principle for hypoxia-induced finetuning of CAR expression.

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