Endocrine Abstracts

Background: Adrenocortical carcinoma (ACC) is a very rare and aggressive, endocrine malignancy with still limited treatment options. Approximately 60% of patients with ACC show endogenous glucocorticoid excess which could be one potential cause, why first clinical trials with immunotherapies, like immune checkpoint inhibitors, showed only modest results. Due to the lack of an ACC-specific antigen structure, other immunotherapeutic approaches, like specialized cancer treatments using chimeric antigen receptor (CAR) therapy in ACC, have not been tested so far. In this study, we evaluated the expression of a new enticing tumor antigen (TA*) structure and investigated the effect of TA-specific CAR-T cells in vitro.

Methods: TA expression has been initially evaluated at mRNA level by qRT-PCR and at the protein level by qFACS analysis in five human ACC cell lines. TA mRNA expression has been investigated in 54 adrenocortical tissues (13 normal adrenal glands (nAG) and 41 ACC samples). Chromogenic immunohistochemistry (IHC) was assessed in 135 paraffin-embedded ACC tissues. In addition, RNAscope single cell analyses in an IHC subcohort of 30 ACC tumor samples was performed. At last, functional testing was conducted for four different TA-specific CAR-T cell modifications, including proliferation, tumor killing and cytokine release.

Results: All five ACC cell lines express the investigated TA. TA expression at mRNA level was present in 60.92% and at protein level at 75.56% of all ACC samples. TA expression was significantly higher in ACC compared to nAG (0.00 vs 0.0015, P=0.0146) and in metastasis more than primary tumors (0,0021 vs 0,0060, P=0.0020). High TA mRNA levels were also strongly associated with glucocorticoid excess, (0.0066 vs 0.0010, P=0.0341) ENSAT tumor stage (0.0032 vs 0.0005, P=0.0091) and sex (0.0012 vs 0.0039, P=0.0237). Furthermore, RNAscope single cell analysis revealed fairly homogenous TA expression in ACC. In vitro treatment showed effective cytotoxic effects of TA-specific CAR-T cells in cell lines (74.9% specific lysis of NCI-H295R cells, E-T 1:1) and primary tumor cells as well as CAR-T cell proliferation upon antigen contact and cytokine release in all five TA positive cell lines.

Conclusions: Our preliminary results showed that the investigated TA is sufficiently and fairly homogenous expressed in human ACC specimens and that TA-specific CAR-T cells exert effective antitumor efficacy under immunosuppressive conditions on ACC cells in vitro.

Keywords: CAR-T cell therapy, adrenocortical carcinoma, glucocorticoids, immunotherapy*The evaluated ACC-specific tumor antigen is kept confidential for the time being for patent law reasons