Mehra, Vedika; (2023) Modulating Akt signalling to enhance CAR T-cell therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

CD19 targeting chimeric antigen receptor (CAR) T-cells deliver excellent clinical responses in relapsed/refractory acute lymphoblastic leukaemia (B-ALL) and high-grade B cell lymphoma. However, many patients do not respond or relapse post treatment because of poor CAR T-cell expansion and persistence. Adoptive cell transfer studies have shown that stem-cell memory/naïve/central memory (Tscm/Tn/Tcm) T-cell subsets compared to effector/terminal (Te/Tte) subsets, deliver superior T-cell expansion, persistence, and anti-tumour efficacy in-vitro and in-vivo. In patients, the proportion of Tn/Tcm subsets can vary considerably, and current manufacturing protocols prioritising mass cell expansion often give rise to Te/Tte skewed products. Studies have demonstrated that the ex-vivo inhibition of Akt signalling, an important mediator of T-cell activation and differentiation, can enrich such Tn/Tcm populations. A novel CD19 targeting single chain fragment variable (scFv) was developed at UCL and named CAT. The CAT scFv was found to have lower affinity for the CD19 target over the FMC63 scFv, utilised in current FDA approved KymriahTM, YescartaTM and TecartusTM CAR therapies. This CAT CAR has demonstrated durable responses against paediatric/adult B-ALL and was used as a model to explore the effects of ex-vivo Akt inhibition. We describe a genetic engineering approach of Akt inhibition through the expression of a dominant negative Akt1 molecule and highlight the benefits of pharmacological Akt inhibition. We assessed the incorporation of Akt inhibitor VIII (further referred to as VIII), originally developed by Merck Research Laboratories, Pennsylvania, USA (Compound 16g) into the CliniMACS Prodigy® manufacture process, utilised in current UCL based CAR T-cell trials, in both heathy/B-ALL patients at small/large scale. We show that ex-vivo Akt inhibition can promote enrichment of less differentiated Tscm/Tcm subsets with improved expansion and cytotoxicity against antigen expressing targets in-vitro and superior anti-tumour activity in-vivo. Inhibition using VIII supported enrichment of CD4 Th1 and Th17 subsets with no skew towards Th2 and Tregs, as previously reported resulting in increased T-cell polyfunctionality. Transcriptome and metabolic analysis revealed a signature for autophagy in CAR CD8 T-cells validated by an increase in autophagic vesicles, not previously described in the context of ex-vivo Akt inhibition in CAR T-cells. VIII-treated CAR CD8 T-cells were found to have a unique metabolic profile with high mitochondrial membrane potential (ΔΨm) and increased fatty acid oxidation to support energy demands of enhanced expansion and cytotoxicity. Lastly, we demonstrate that Akt inhibition can be incorporated into large scale automated manufacturing processes with the potential to generate functionally superior products compared to standard untreated products. This ultimately may help to improve clinical responses by rescuing patients/products at risk of CD19+ relapse.

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