Grothier, Thomas I; (2023) Engineering agnostic modules for the tuning of CAR T cell function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Chimeric antigen receptor (CAR) T cells constitute an effective cancer immunotherapy, yet several challenges remain. For example, although CD22 is a promising B-ALL target, selective pressure from CAR T cells reduces CD22 expression resulting in patient relapse. To circumvent this hurdle, I aimed to develop a module to improve CAR T cell sensitivity. CSK is an inhibitory kinase that negatively regulates T cell signalling. I envisioned that a dominant-negative CSK might improve CAR T cell sensitivity. Therefore, I engineered a number of dominant-negative iterations of CSK (dnCSK). The co-expression of dnCSK modules in different CAR T cell platforms was seen to enhance sensitivity, improving cytotoxicity and cytokine release (IFN-γ and IL-2) against low antigen density target cells. Another challenge for CAR T cell therapies is on-target off-tumour toxicity. The second aim was to develop a module enabling tuneable control of CAR T cell function. To do so, I employed a CSK mutant (CSKAS) that is inhibited by a PP1 analogue, 3-IBPP1. Co-expression of CSKAS in CD22 and GD2 targeting CAR T cells dampened CAR function in the absence of 3-IB-PP1. However, after the addition of 3-IB-PP1, both CD22 and GD2 targeting CAR T cells displayed improved function compared to control CAR T cells. An advantage of this approach is the lack of CAR architecture reengineering, permitting simple implementation into existing CAR platforms.

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