Endocrine Abstracts

Background: Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine disease in which 60% of patients show endogenous glucocorticoid (GC) secretion that potentially contributes to a lack of immune cell infiltration and limited efficacy of immunotherapeutic approaches. In another study, we already demonstrated potent antitumor efficacy or ROR1 CAR-T cells in preclinical models of ACC. Nevertheless, solid tumors often show cell intrinsic resistance mechanisms to CAR-T cell cytotoxicity which recently could be attributed to a lack of IFNγR-signalling and immunogenic pathway activation. Here, we report of a new potent small molecule inhibitor* (SMI) that is able to modulate innate antitumor immune-response pathways and potently enhances CAR-T cell functionality by activating the IFNγR-pathway in ACC.

Methods: We evaluated target gene expression in 62 ACC patients at mRNA and protein level. We evaluated efficacy of our SMI in five ACC cell lines and activation of immunogenic proinflammatory tumor signalling pathways using RNA Nanostring nCounter analysis, qRT-PCR and Western Blot. Cytokine secretion was measured by Enzyme-linked Immunosorbent Assay (ELISA). We evaluated antitumour functionality alone and in combination with several CAR-T cell modifications using different preclinical models of ACC.

Results: We show significantly higher expression of our required target gene in five ACC cell lines and ACC tissues when compared to normal adrenal glands (0.073 vs 0.713; P=0.004). Moreover, target expression strongly correlates with glucocorticoid secretion and other clinicopathological parameters including Ki67, Weiss-score and survival supporting its potential utility as biomarker in ACC. The SMI shows potent antitumor functionality alone (NCI-H295R: IC50 195 nM, respectively) by reshaping cell autonomous and immune-stimulating activity in ACC cell lines. We demonstrate strong intratumoral upregulation of PD-L1 (11.5-fold change; P=0.003) and IDO1 (17-fold change; P=0.033) as well as strong secretion of immunogenic upstream signals and a significant increase of IFNγR expression (2.9-fold change, P=0.019) in all five ACC cell lines after co-culture for 48 hours (NCI-H295R cells shown respectively). Consistent with these findings, after selectively activating the IFNγR-pathway when combined with the SMI, we show enhanced immune cell adhesion and responsiveness of different CAR-T cell modifications in ACC upon antigen contact in preclinical models using subtherapeutic effector-to-target-ratios.

Conclusion: Our results illustrate an enhanced immunotherapeutic approach using the combination of a new potent SMI and CAR-T cells by activating immunogenic intratumoral signalling and the IFNγR pathway in adrenocortical carcinoma.*The small molecule inhibitor will be kept confidential for patent law reasons