Background: Ewing sarcoma (EWS) is a rare, aggressive bone and soft tissue malignancy predominantly affecting children and young adults. Despite advances in multimodal therapy, treatment options remain limited for refractory or relapsed EWS (1). Chimeric antigen receptor (CAR) T-cell therapy has shown marked efficacy in hematologic malignancies and is being developed for solid tumors. AZD0754 is a STEAP2-targeted CAR T-cell therapy incorporating a dominant-negative TGFbetaRII (dnTGFbetaRII) armoring strategy and is in Phase 1 clinical development for prostate cancer. Recent published proteomic analyses identified STEAP2 expression in EWS patient samples and cell line-derived xenografts, supporting STEAP2 as a potential candidate immunotherapeutic target (2). Methods: STEAP2 surface expression was evaluated across different human EWS cell lines by flow cytometry. AZD0754-mediated cytotoxicity was assessed in vitro using the xCELLigence real-time cell analysis platform. Based on in vitro results, A673, RD-ES, and SK-NEP-1 xenograft models were selected for in vivo efficacy studies. Mice received varying doses of AZD0754, and tumor growth and overall survival were monitored. Blood was collected at different time points for serum cytokine analysis to assess pharmacodynamic activity. Results: EWS cell lines expressed detectable cell surface expression of STEAP2 at receptor densities much lower than workhorse prostate cancer cell lines. Despite this, AZD0754 was capable of inducing antigen-dependent cytotoxicity in vitro and suppressing tumor growth in EWS xenograft models. AZD0754 exhibited dose-dependent antitumor activity that correlated with serum IFNgamma levels, indicating on-target immune engagement. Conclusions: AZD0754 demonstrates robust preclinical anti-tumor activity against EWS in vitro and in vivo , with dose-dependent efficacy and corresponding cytokine levels. These findings support STEAP2 as a potential therapeutic target in EWS. STEAP2 targeting has the potential to address a critical unmet need in EWS by expanding treatment options to biologic and immune-based therapies. References: 1. National Center for Biotechnology Information (2021). Ewing Sarcoma. NCBI Bookshelf.2. Mooney B, Negri GL, Shyp T, Delaidelli A, et al. Surface and global proteome analyses identify ENPP1 and other surface proteins as actionable immunotherapeutic targets in Ewing sarcoma. Clin Cancer Res. 2024;30(5):1022-1037. doi:10.1158/1078-0432.CCR-23-2187.