Abstract 4822: Efficacy of 4-1BB-costimulated NKG2D CAR-T cells for synovial sarcoma in comparison with CAR-T cells targeting other antigens

Abstract Although the development of multimodal therapy consisting of surgery, radiotherapy, and chemotherapy has significantly improved the survival rate for patients with localized synovial sarcoma (SS), the prognosis of metastatic or recurrent SS remains poor. This has not changed over the past three decades, and a novel approach including immunotherapy is urgently needed. Clinical studies of CAR-T cell therapies against sarcomas have been performed. However, the clinical outcomes for patients with sarcomas are still limited. We have previously reported cytotoxicity of HER2-targeted CAR-T cells against SS in vitro. Here, we demonstrated that NK2GD-based CAR-T cells showed significantly higher cytotoxicity than that of HER2-targeted CAR-T cells for SS cell lines.Surface expressions of HER2 and NKG2D ligands (NKG2DLs) on three SS cell lines (SYO-I, HS-SY-II, and Yamato-SS) were evaluated by flow cytometry. Gene expression level of HER2 and NK2GDLs of SS patients were also evaluated by RNA-sequencing using public data base. Then, we generated 4-1BB-type second generation CAR-T cells targeting HER2 or NKG2DLs and examined their cytotoxicity and specific responses against SS cells. Furthermore, we compared the differences between HER2-targeted CAR-T cells and NKG2D-based CAR-T cells by co-culturing with SS cells using short-term assay (WST-8 assay), long-term assay (Real-time Cell Analysis), intracellular cytokine assay, and CD107a assay. Finaly, animal experiments were also performed to evaluate antitumor effect of CAR-T cells against SS. CAR-T cells were injected one week after the implantation of human SS cell lines.Flow cytometry revealed that surface expression HER2 and NKG2DLs were observed in all three SS cell lines. In vitro, NKG2D-based CAR-T cells showed antitumor effects against SS cells with degranulation and cytokine production. Furthermore, NKG2D-based CAR-T cells showed much stronger cytotoxicity than HER2-targeted CAR-T cells in both short- and long-term assay. Higer expression level of cytokines was found in NK2GD-based CAR-T cells than that of HER2-targeted CAR-T cells.In this study, NKG2D-based CAR-T cells demonstrated much stronger antitumor effects than HER2-targeted CAR-T cells. Although CAR-T cell therapy has been shown to be promising in blood malignancies, the efficacy in solid tumors has been disappointing due to problems such as low penetrance to tumors, the immunosuppressive microenvironment, and immune evasion of tumor cells. While further validation of clinical application of CAR-T cells against SS is necessary, we demonstrated that CAR-T cell therapy would be promising immunotherapy for SS patients. Citation Format: Naoki Oike, Tomohiro Miyazaki, Yudai Murayama, Akira Ogose, Takashi Ariizumi, Hiroyuki Kawashima, Chihaya Imai. Efficacy of 4-1BB-costimulated NKG2D CAR-T cells for synovial sarcoma in comparison with CAR-T cells targeting other antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4822.