转移性横纹肌肉瘤与生存率低和治疗结果不令人满意有关。因此，迫切需要新的免疫治疗方法。成纤维细胞生长因子受体4（FGFR4）是横纹肌肉瘤的新治疗靶点，在其发生和发展中发挥着至关重要的作用。本研究旨在生成基于 FGFR4 单链可变片段的嵌合抗原受体 (CAR) T 细胞，不会引起明显的毒性，并纳入诱导型 caspase-9 (iCasp9) 自杀基因系统以增强其安全性。在正常鼠组织、正常人体组织和横纹肌肉瘤患者标本中评估了 FGFR4 抗原的表达。结合 4-1BB 共刺激结构域、CD3ze 信号结构域和 iCasp9 自杀基因，开发了具有 FGFR4 特异性单链可变片段的 CAR-T 细胞。在体外和体内研究了 FGFR4 CAR-T 细胞的特异性细胞毒性作用、T 细胞增殖、细胞因子分泌、化学二聚化 (AP20187) 诱导细胞凋亡和毒性。 FGFR4 CAR-T细胞产生多种免疫促进细胞因子，包括肿瘤坏死因子α、白细胞介素2和干扰素γ，并在体外对FGFR4过表达的横纹肌肉瘤细胞表现出有效的细胞毒活性。 FGFR4 CAR-T 细胞对 FGFR4 过表达的横纹肌肉瘤相对有效，在皮下异种移植模型中肿瘤消退且存活率较差。 iCasp9基因被整合到FGFR4 CAR-T细胞中，并证明有效且可靠的自杀基因活性取决于AP20187的施用。本研究通过利用FGFR4 CAR-T细胞与小鼠FGFR4在同基因肿瘤模型中的交叉反应，发现FGFR4 CAR-T细胞可以调节肿瘤的生长，且没有明显的毒性。我们的研究表明，FGFR4 是横​​纹肌肉瘤 CAR-T 细胞治疗的前瞻性靶标，且没有严重的靶向脱瘤毒性。带有 iCasp9 自杀基因系统的 FGFR4 CAR-T 细胞作为限制毒性的安全开关可能会拓宽细胞疗法的临床应用。© 2024。作者。

Metastatic rhabdomyosarcoma is associated with poor survival and unsatisfactory treatment outcomes. Therefore, new immunotherapeutic methods are urgently required. Fibroblast growth factor receptor 4 (FGFR4), a new therapeutic target for rhabdomyosarcoma, plays a crucial role in its onset and development. This study aimed to generate FGFR4 single-chain variable fragment-based chimeric antigen receptor (CAR) T cells without causing evident toxicity and incorporating an inducible caspase-9 (iCasp9) suicide gene system to enhance their safety. FGFR4 antigen expression was evaluated in normal murine tissues, normal human tissues, and specimens from patients with rhabdomyosarcoma. Combined with a 4-1BB co-stimulatory domain, a CD3ζ signaling domain, and an iCasp9 suicide gene, CAR-T cells with an FGFR4-specific single-chain variable fragment were developed. The specific cytotoxic effects, T-cell proliferation, cytokine secretion, apoptosis induction by chemical dimerization (AP20187), and toxicity of FGFR4 CAR-T cells were investigated in vitro and in vivo. FGFR4 CAR-T cells generated a variety of immune-promoting cytokines, including tumor necrosis factor α, interleukin 2, and interferon γ, and displayed effective cytotoxic activity against FGFR4-overexpressing rhabdomyosarcoma cells in vitro. FGFR4 CAR-T cells were relatively effective against FGFR4-overexpressing rhabdomyosarcoma, with tumor regression and poor survival in a subcutaneous xenograft model. The iCasp9 gene was incorporated into FGFR4 CAR-T cells and it was demonstrated that effective and reliable suicide gene activity depends on the administration of AP20187. By making use of the cross-reaction of FGFR4 CAR-T cells with murine FGFR4 in a syngeneic tumor model, this study found that FGFR4 CAR-T cells could regulate the growth of tumors without evident toxicity. Our study demonstrates that FGFR4 is a prospective target for CAR-T cell therapy in rhabdomyosarcoma without serious on-target off-tumor toxicity. FGFR4 CAR-T cells with the iCasp9 suicide gene system as a safety switch to limit toxicity may broaden the clinical applications of cellular therapy.© 2024. The Author(s).