Kadirkamanathan, Renuka; (2024) Base Edited Universal CAR T Cells Against Childhood Acute Myeloid Leukaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Childhood acute myeloid leukaemia (AML) is an aggressive, life-threatening disease with a devastatingly limited arsenal of approved curative treatment options. Despite accounting for only one fifth of all cases per year, it remains the leading cause of childhood leukaemia-related deaths due to the unique challenges it poses against the development of efficacious and clinically safe therapies. Target antigens include CD7 and CD33, but recent detailed phenotypic profiling has also revealed C-type lectin- like molecule-1 (CLL-1) as a promising candidate due to its frequently overexpression on blasts and coverage on disease-sustaining leukaemic stem cells (LSCs). Several anti-CLL-1 chimeric antigen receptor (CAR) T cells are now under clinical investigation for use as a monotherapy, or in combination to address disease heterogeneity. These are however relatively confined to the autologous setting which carries with it several caveats. The aim of this project has therefore been to develop a universal alternative, available in an “off-the-shelf” manner, for incorporation into a pre-existing CAR T cell AML pipeline. Base-editing offers a “seamless” approach to the multiplexed genome engineering of therapeutically relevant targets in the absence of genotoxic double stranded DNA breaks. The cytidine base editor (CBE) BE3 was therefore employed for the development of universal anti-CLL-1 CAR T cells harbouring phenotypic knockouts of the alloreactive a/b T cell receptor, the Alemtuzumab antigen CD52, and the shared T-cell antigen CD7. In vitro studies confirmed the cytotoxicity of base-edited universal (BE-u)CLL-1CAR T cells, including against patient derived xenograft (PDX) sample, whilst human xenograft murine models of AML were used to evaluate their anti-leukaemic effects against established disease in vivo. The Molm-14 AML cell line appeared unsuitable for this assessment, whilst results were most promising against the HL-60 AML cell line. The latter was taken forward to generate a human xenograft murine model representative of heterogenous disease, against which a combinatorial dosing strategy with BE- uCLL-1CAR and BE-uCD33CAR T cells was investigated. Finally, targeting of neutrophil and haematopoietic stem and progenitor cell (HSPC) populations by BE- uCLL-1CAR T cells was assessed to determine the compatibility of this product with proposed strategies to help mitigate periods of treatment-related neutropoenia, as well as a bridge-to-transplant approach to accelerate immune recovery.

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