Abstract

Chimeric antigen receptor (CAR) is a type of immunotherapy that relies on adoptive transfer of genetically modified T-cells. In brief, a patient’s T-cells are isolated and engineered to express a CAR targeting a designated tumour antigen. The resulting CAR T-cells (CAR T) are injected into the patient, where they recognize and cytotoxically engage antigen positive tumour cells. The development of CAR T-cells has revolutionized the treatment of haematological malignancies. However, such success is currently not observed in solid tumours. A major factor contributing to this failure is the immunosuppressive function of the tumour microenvironment. HLA-G is a tumour‑associated antigen (TAA) expressed in various solid tumours, including clear cell renal cell carcinoma (ccRCC). The research project aims to study the function of anti-HLA-G CAR T on tumour cells and the wider TME. We show that anti-HLA-G CAR T show antigen-specific cytotoxicity and cytokine production against tumour cells. Then, we demonstrate the ability of anti‑HLA-G CAR T to infiltrate and control tumour growth in vivo using various xenograft murine models. In order to evaluate the effects of anti-HLA-G CAR T on the TME, we utilise a novel preclinical model based on the ex vivo culture of fresh human tumour slices. We show that CAR T can efficiently recognize HLA-G-expressing tumour cells in ccRCC tumour slices through analysis of the calcium response. Then, we show that CAR T can efficiently reprogram the TME of ccRCC slices through induction of an IFNγ response. This IFNγ response leads to induction of a pSTAT1 response and upregulates many IFNγ‑induced factors including chemokines, adhesion molecules and immune checkpoints. Notably, this CAR T-IFNγ response is not observed in all ccRCC samples, highlighting interpatient TME heterogeneity. Lastly, using single cell technologies, we show that tumour cells with ccRCC show extensive heterogeneity of response to the CAR T-IFNγ response.