Endocrine Abstracts

Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy that produces glucocorticoids in 60% of cases causing clinically relevant hypercortisolism. Since, we observed in another study, that shaping these immunosuppressive and life-threatining properties with corticosteroid inhibitors not only led to a glucocorticoid receptor-related downregulation of ROR1 but also to the upregulation of another promising membrane bound oncogenic antigen* (AG), we sought to investigate the role of a dual targeting approach using bispecific CAR-T cells in ACC. We initially evaluated AG-expression in a representative cohort of ACC samples (n=62) using qRT-PCR and immunohistochemistry (IHC) and show that the AG is sufficiently and homogenously overexpressed in human ACC tissues (P<0.001). Moreover, it is strongly associated with a poor median overal survival (Chi²=6.207, P=0.013) and disease progression (Chi²=5.485, P=0.019). We further demonstrate high levels of AG-expression in four different ACC cell lines using qRT-PCR and qFACS-analysis. To evaluate the interplay between ROR1 and AG we used Duolink technology, RNA-Nanostring nCounter sequencing, qRT-PCR and IHC. While observing a strong downregulation of ROR1 after corticosteroid inhibitor treatment on the one hand, we demonstrate a strong increase of AG-expression after different doses of metyrapone (21-fold change, P=0.003) and ketoconazole (35-fold change, P<0.001) on the other (values are shown for NCI-H295R cell line, respectively). Next, we used non-viral sleeping beauty transposons and CRISPR/Cas9 to generate different mono- and bispecific CAR-T cells targeting ROR1 and AG and evaluated their antitumor functionality in vitro. We observed potent antitumor efficacy, cytokine release and proliferation upon antigen contact of all different CAR-T cell modifications. To maintain T cell functionality, to prevent CAR-T cell tonic signalling and to reduce on-target off-tumor toxicities (OTOT) by aiming two antigens, we also attempted to apply Boolean logic gating to our CAR-T cells to prevent toxicity in healthy tissues that express very low levels of one of our two antigens. We exploited the approach of a new way of CAR engineering using co-opted intracellular proximal T cell signalling molecules that can be repurposed into surface receptors demonstrating no OTOTs of CAR-T cell function in single antigen positive tissue in vitro. Our preliminary data demonstrate an interplay between two CAR-T cell targets which can be used for a combined treatment approach with steroid inhibitors. We also show Boolean logic gating as novel and potentially safer approach of CAR-T cell engineering in advanced ACC. *The investigated antigen will be kept confidential because of patent law reasons.