横纹肌肉瘤（RMS）是儿童时期最常见的软组织肉瘤，其预后仍然较差，尤其是转移性、高级别和复发性横纹肌肉瘤。迫切需要新的治疗方法，特别是全身治疗。嵌合抗原受体T细胞（CAR T）对血液恶性肿瘤非常有效，但其对实体瘤的疗效还有待提高。 CD276 (B7-H3) 是 RMS 中上调的靶标，在正常组织中检测到水平较低。 FGFR4 是 RMS 的一个非常具体的目标。在这里，我们针对这两个靶标单独或组合优化了 CAR T，并在体外和体内测试了它们的抗肿瘤活性。使用四种不同的单域抗体来选择最特异的 FGFR4-CAR 构建体。体外测试了表达 CD8α 或 CD28 HD/TM 结构域的 CD276-和 FGFR4-CAR T 与 4-1BB 和/或 CD28 共刺激结构域组合对 RMS 细胞的杀伤和细胞因子的产生。最有效的 CD276- 和 FGFR4-CAR T 用于生成双 CAR T。在三种原位 RMS 小鼠模型中评估了体内肿瘤杀伤能力。CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3 z) 对 RMS 细胞的杀伤力最强，IFN-γ 和颗粒酶的释放量最高B 体外. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) 显示出最特异的杀伤效果。 CD276-CAR Ts 在体内成功根除 RD 和 Rh4 衍生的 RMS 肿瘤，分别在 3/5 和 5/5 小鼠中实现完全缓解。然而，在 CD276low JR 肿瘤中，只有 1/5 的小鼠实现了完全缓解。 FGFR4 CAR T 反而延缓了 Rh4 肿瘤的生长。双 CAR T 促进 5/5 小鼠的 Rh4 肿瘤清除。CD276 和 CD276/FGFR4 导向的 CAR T 在体外显示出有效的 RMS 细胞杀伤作用，并在体内根除 CD276high RMS 肿瘤。 CD276low 肿瘤逃脱了治疗，凸显了抗原密度和有效性之间的相关性。 FGFR4-CAR Ts 在体外表现出特异性杀伤作用，但在体内只能延迟 RMS 生长。我们的结果表明，CD276-CAR 与其他 CAR 的联合表达不会降低其益处。在 RMS 中引入 CD276-CAR T 免疫疗法似乎是可行且有前途的，尽管 CAR 构建体设计和靶标组合必须进一步改进以根除靶标表达低的肿瘤。© 2023。作者。

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo.Four different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models.CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice.CD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.© 2023. The Author(s).