Abstract B058: Clusterin overexpression in therapeutic T cell induces resistance to exhaustion

Abstract Ewing Sarcoma (EwS) is characterized by an immunosuppressive tumor microenvironment (TME), which promotes T cell exhaustion, among other factors by chronic inflammation and hypoxia. This results in decreased cytotoxicity and reduced functional capabilities of the immune effector cells. In previous studies, we demonstrated that CHM1319-327-specific transgenic T-cell receptor (TCR)-T cells, named ChoTCR-T cells, exhibit notable cytotoxic effects against EwS both in vitro and in vivo. As GD2 is an epitope commonly expressed in EwS, CAR-T cells targeting GD2 are an alternative approach for the treatment of EwS. Since T cell exhaustion is a major challenge in therapeutic T cells, especially in solid tumors, current research is focused on developing T cells that resist exhaustion. Clusterin (CLU) plays a crucial role in metastasis formation, as it prevents apoptosis and enhances stress tolerance in tumor cells. Therefore, we considered CLU a promising candidate protein for protecting therapeutic T cells. In this study, we wanted to assess the effects of CLU overexpression on exhaustion in therapeutic T cells. Our findings revealed decreased CLU expression across various therapeutic T-cell models upon stimulation with tumor cells. After overexpressing CLU in ChoTCR-T cells and Anti-GD2 CAR-T cells, we conducted co-culture studies with tumor cells and T cells over a period of up to 10 days to evaluate CLU's impact on exhaustion. Notably, engineered therapeutic T cells with CLU overexpression demonstrated decreased expression of the exhaustion markers PD-1, LAG-3, TIM-3, and CD39 during prolonged co-cultures of up to 10d. Additionally, they exhibited increased long-term cytotoxicity against diverse target expressing tumor cell lines and increased cytokine production. We also investigated CLU's influence on spheroid infiltration using an EwS spheroid model. CLU-overexpressing T cells demonstrated enhanced infiltration into the hypoxic core of the spheroids. Moreover, mass spectrometric analysis suggested potential mechanisms underlying these findings, including a resistance to hypoxia, and a reduced translational pathway signature in proteomic analysis. Consistently, under hypoxic conditions (1.5% O2), CLU-overexpressing T cells displayed better functionality and activation. Furthermore, the impact of CLU on the expression of exhaustion markers was increased under hypoxic culture conditions. In conclusion, CLU protects tumor-specific T-cells from both exhaustion and hypoxia in vitro, possibly enhancing their endurance within the TME. Thus, this protein represents an intriguing prospect for protecting therapeutic T cells from exhaustion, to overcome diminished therapeutic efficacy in both TCR- and CAR-T cells in solid tumors. Citation Format: Constantin Segner, Alisa Kolesnikova, Mansour Poorebrahim, Julia Höbart, Miriam Schulz, Busheng Xue, Christian Brückner, Alissia Fernandes Madeira, Julia Hauer, Jürgen Ruland, Poul H. Sorensen, Stefan EG Burdach. Clusterin overexpression in therapeutic T cell induces resistance to exhaustion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B058.