Oporto Espuelas, Macarena; (2025) Chimeric antigen receptor target discovery for paediatric acute myeloid leukaemia stem cells with multimodal single cell RNA sequencing. Doctoral thesis (Ph.D), UCL (University College London).

Text Oporto Espuelas_10207629_Thesis.pdf Access restricted to UCL open access staff until 1 May 2027. Download (45MB)

Abstract

CAR T cell therapy in paediatric acute myeloid leukaemia (AML) is significantly limited by the absence of an ideal target. AML displays significant inter-patient and intra-tumour heterogeneity, with leukaemic stem cells (LSC) as drivers of relapse –the main cause of treatment failure. Therapeutic targets often overlap with normal haematopoietic stem and progenitor cells (HSPC). This overlap contributes to the limited efficacy and substantial toxicity of CAR T cell therapies for AML, including the need for haematopoietic stem cell transplantation (HSCT). To address these issues, we used CITEseq (Cellular indexing of transcriptomes and epitopes by sequencing) to investigate optimal targets and target combinations for paediatric AML LSC. We set up and validated CITEseq technology in our lab and built a custom CITEseq antibody panel targeting 79 potential LSC surface markers. We generated a unique scRNAseq dataset of paediatric AML (pAML) with 22 samples from 13 high- risk patients and 5 healthy controls, which is unique in that it is 1) enriched for LSC, 2) is carefully annotated including a robust malignant/non-malignant annotation, as well as LSC annotation based on functionally validated gene signatures and 3) integrates surface protein data for >70 candidates. Systematic profiling of the cell surface proteome revealed that no single target would cover all LSC whilst sparing normal HSPC. We identified CLL1, IL1RAP, CD84 and CD32 as top targets that each targeted ~70% of LSC whilst sparing normal HSC. Further analysis revealed that combining CLL1 and CD84 would target >90% of LSC across the cohort with limited non-haematological toxicity. Further, we confirmed known group-specific targets and identified novel candidate LSC targets through our unbiased analysis of the transcriptome. In conclusion, this work enabled the design of rational CAR T cell target combinations to enhance efficacy and limit toxicity in pAML and found CLL1/CD84 as an optimal LSC target combination.

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