The Combined Treatment Efficacy of Anti-CD123 CAR T cells with Azacitidine for the Treatment of Acute Myeloid Leukaemia

Abstract

Chimeric antigen receptor (CAR) T cells have yielded impressive remission rates in treatment-refractory B cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in CAR T cell therapies entering into clinical practice. However, the utility of CAR T cells for acute myeloid leukaemia (AML) remains a challenge. CAR T cells against AML-associated antigens are typically hampered by cytotoxic effects against normal haematopoietic progenitor cells and by CAR T cell exhaustion. Current clinical trials using CAR T cells that target various antigens in AML have resulted in either transient leukaemia cell clearance, or complete clearance of leukaemia at the expense of severe on-target off-tumour toxicities. In this thesis, third-generation anti-CD123 CAR T cells were developed with a humanised binding moiety for CD123 incorporating two intracellular signalling domains. The CAR developed in this project demonstrated strong anti- AML activity without elimination of the healthy haematopoietic system or epithelial tissue damage in mouse xenograft models. However, a sustained and long-term tumour eradication was not observed in the mice. In the clinical setting, this would mean that patients have suboptimal responses to the CAR T cells and may relapse. The AML microenvironment is immunosuppressive by employing a variety of mechanisms to escape the host immune surveillance, which may hamper the efficacy of CAR T cell therapy. To improve the long-term efficacy of the CAR T cells, combination therapy with DNA methyltransferase inhibitors, such as azacitidine (AZA), was explored. AZA has previously been shown to upregulate the expression of leukaemia-associated antigens on AML thereby inducing more effective T cell responses. AZA was therefore combined with CD123 CAR T cells and evaluated in AML xenograft models. Priming of AML cells with AZA increased the expression of the target antigen, CD123, on the cell surface. CD123 CAR T cells were more effective at eliminating AML cells in vivo and induced long term eradication. Interestingly, the combined treatment strategy induced a CTLA-4negative CD123 CAR T cell population. Functionally, these CTLA-4negative CD123 CAR T cells exhibited superior cytotoxicity against AML cells with sustained tumour necrosis factor (TNF) production and higher proliferative capacity compared to CTLA-4positive CD123 CAR T cells. Furthermore, AML xenograft mice treated with CTLA-4negative CD123 CAR T cells survived longer than CTLA-4positive CD123 CAR T cell treated mice, and demonstrated recall immunity in secondary AML xenograft recipients. Mechanistically, when AML cells were primed with AZA, the CAR T cells demonstrated increased intracellular retention of CTLA-4 and reduced extracellular expression upon exposure to the AML cells. The decreased expression of extracellular CTLA-4 was associated with decreased numbers of regulatory CAR T cells. Normally, high extracellular CTLA-4 expression prevents the phosphorylation of Lck and Zap70; intracellular molecules required for effective T cell induction and function. In this case, a higher phosphorylation level of these molecules was observed in the CAR T cells exposed to AML cells previously primed with AZA compared to without priming. The findings in this thesis project indicate that AZA increases the target antigen, CD123, on AML cells, allowing enhanced recognition and elimination by cytotoxic CTLA-4negative CD123 CAR T cells. These novel findings pave the way for a clinical trial combining AZA and CD123 CAR T cells for AML treatment.