Sir Peter MacCallum Department of Oncology - Theses
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ItemAvenues to enhance chimeric antigen receptor (CAR) T cell stemness and therapeutic efficacy against solid tumours( 2023-02)Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable activity in B cell malignancies leading to multiple Food and Drug Administration (FDA) approvals. However, in solid tumours, CAR T cells have only yielded limited efficacy. This is thought to be due to several factors including poor persistence of CAR T cells and terminal differentiation. In the present thesis, two approaches for improving the persistence and efficacy of CAR T cells against solid tumours were explored. The first strategy involved overexpressing T cell memory-associated transcriptional regulators in CAR T cells. Clinical data has shown that CAR T cell persistence and therapeutic outcomes are associated with the adoptive transfer of less differentiated, “stem-like” CAR T cells that assume a similar phenotype to circulating memory T cell subsets. I hypothesised that the overexpression of the transcriptional regulators associated with memory T cells would reduce CAR T cell differentiation, thereby leading to their improved persistence and enhanced anti-tumour efficacy. FOXO1 was identified as a primary candidate, and the impact of overexpressing a constitutively active form of Foxo1 (Foxo1-ADA) in HER2 directed murine CAR T cells and wild type FOXO1 in human Lewis Y directed CAR T cells was evaluated. Foxo1-ADA overexpression improved the therapeutic efficacy of CAR T cells in breast and colon carcinoma tumour models. Additionally, Foxo1-ADA overexpression enhanced CAR T cell polyfunctionality and mitochondrial health in vivo. I found that the overexpression of wild type FOXO1 similarly improved the stem-like characteristics of human CAR T cells, enhancing overall respiratory capacity and therapeutic responses in a human ovarian cancer model. Our data indicates that reinforcement of a stem-like program through FOXO1 overexpression significantly enhances CAR T cell responses against solid tumours. The second approach involved the use of a dual-specific CAR T cell that expressed two distinct CARs against a solid tumour and B cell antigen. The success of CD19 directed CAR T cells may be attributed to engaging antigens outside of a solid tumour microenvironment (TME). Therefore, this approach aimed to co-opt these benefits in the solid tumour setting by co-transducing a unique T cell designated as a ‘dual CAR T cell’ with two CARs having specificity for the human HER2 and murine CD19 antigens. I hypothesised that by first engaging the CD19 antigen in sites away from the immunosuppressive TME, dual CAR T cells would be primed to better mount responses against solid tumours. Dual CAR T cells demonstrated higher polyfunctionality in vitro against HER2 expressing tumour cells when primed through the CD19 directed CAR and mediated enhanced therapeutic efficacy against orthotopic breast tumours in vivo. Notably, dual CAR T cells demonstrated improved persistence with a similar differentiation phenotype relative to control T cells. These results indicate that maintenance of a less differentiated CAR T cell phenotype and engagement of target antigens outside of the TME are instrumental for maintaining CAR T cell persistence. These investigations contribute to the growing body of work that aims to enhance CAR T cell stemness. As these studies have been conducted in both syngeneic and human CAR T cell models, I believe that this work carries high translational potential.