Enhancing CAR T cell therapy
AffiliationSir Peter MacCallum Department of Oncology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-09-25.
© 2020 Jessica Michie
Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has shown remarkable efficacy in the treatment of haematological malignancies, with complete remission rates of 90% reported in early clinical trials. Following this success, two CD19 targeting CAR T cell products have been approved in Australia for the treatment of Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, acquired resistance to this therapy through CD19 antigen loss is an emerging problem. Moreover, such promising results have not been recapitulated against solid tumours. This is thought to be due in part to poor CAR T cell trafficking and infiltration, the immunosuppressive tumour microenvironment and antigen heterogeneity in solid tumours, as well as intrinsic and acquired tumour resistance. Therefore, it is necessary to uncover mechanisms of resistance to CAR T cell therapy in both the haematological and solid tumour settings, in order to improve patient outcomes. In this thesis, we sought to uncover the tumour-intrinsic mechanisms of resistance to CAR T cell therapy. Whole-genome loss-of-function CRISPR-Cas9 screening has recently emerged as a powerful tool to screen all protein-coding genes in the genome for conferring resistance to various immune and drug pressures. Herein, we utilised a genome-wide mouse pooled-sgRNA library to investigate genes that protect MC38 mouse adenocarcinoma cells from CAR T cell killing. Here, we identified that loss of cytokine response pathways, in particular, TNF and IFN-y signalling were critical for the effector function of CAR T cells. Additionally, we report for the first time that loss of the transcriptional co-binding partner Cbfb protects tumour cells against T cell killing. Investigation in to the mechanisms of Cbfb-mediated resistance uncovered that loss of tumour cell Cbfb protected tumour cells against T cell-derived TNF. Importantly, we report that mice bearing Cbfb-deficient MC38 tumours did not respond to anti-PD-1 in vivo, and analysis of clinical trial data suggests that in melanoma cohorts, low expression of CBFB or the transcriptional co-binding partner RUNX1 was correlated with a poorer prognosis following immune checkpoint blockade or adoptive cell therapy. Next, we sought to design a rational combination therapy to overcome some of the challenges associated with CAR T cell therapy against solid tumours and enhance CAR T cell therapy in this context. Based on data from the CRISPR screen which highlighted the role of TNF in CAR T cell-mediated cytotoxicity, we sought to enhance TNF-mediated killing using a small-molecule smac-mimetic. We demonstrated that antagonism of the Inhibitor of Apoptosis Proteins with the smac-mimetic birinapant, significantly enhanced CAR T cell killing in a TNF-dependent manner. Using a syngeneic HER2+ self-antigen model, we report that birinapant significantly enhanced CAR T cell-mediated tumour clearance in a combination therapy approach, and we demonstrated enhanced human CAR T cell killing in patient biopsy-derived tumouroids. Critically, we report that birinapant significantly enhanced TNF-mediated “bystander killing”, which occurred in the absence of target antigen expression, which may be a strategy to overcome the challenge of tumour antigen heterogeneity associated with solid tumours. Finally, we sought to uncover mechanisms by which B cells lose CD19 antigen expression following CAR T cell attack. By utilising a second genome-wide CRISPR screening approach, we applied a human pooled sgRNA library to the CD19+ B-ALL cell line MHH-CALL4, and sorted 3 times the cells with the lowest 20% CD19 surface expression by flow cytometry, in order to uncover genes or pathways of interest that are implicated in down-regulation of CD19 surface expression. Along with several known factors, such as CD81 trafficking and N-linked glycosylation, we also identified several novel processes including histone methylation, aryl hydrocarbon receptor processing and E3 ubiquitin ligase activity, which will be the focus of future investigations. In summary, insights gained from the work in this thesis elucidate some of the resistance pathways utilised by tumour cells in order to evade immune pressure from CAR T cells, and highlight the cytotoxic potential of TNF in CAR T cell therapy. In addition, we herein present a novel strategy to enhance CAR T cell efficacy in solid tumours, and demonstrate the potential for combination therapy approaches to overcome the challenges associated with CAR T cell therapy against solid tumours. Taken together, this work significantly addresses several of the existing limitation of CAR T cell therapy in both B cell leukaemias and against solid malignancies, and the novel combination therapy we describe is poised to be rapidly translated in to the clinic.
KeywordsCAR T cell therapy; TNF; Smac-mimetic; Inhibitor of Apoptosis Proteins; Bystander killing; CRISPR-Cas9; Genome-wide CRISPR screening; CD19
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