CAR-T 细胞正在被研究作为胶质母细胞瘤的一种新型免疫疗法，但临床成功有限。我们最近将成纤维细胞激活蛋白（FAP）描述为胶质母细胞瘤免疫治疗的理想靶抗原，在肿瘤细胞和肿瘤血管上都有表达。然而，靶向 FAP 的 CAR-T 细胞从未被研究作为胶质母细胞瘤的治疗方法。我们生成了一种新型 FAP 靶向 CAR，具有 CD3 z 和 CD28 信号传导域，并在体外测试了所得 CAR-T 细胞的裂解活性和细胞因子分泌功能（使用实时阻抗、流式细胞术、成像和基于珠子的细胞因子测定）和体内（使用模仿人类胶质母细胞瘤自然异质性的异种移植物）。FAP-CAR-T细胞表现出针对模型细胞系的靶特异性和有效的细胞毒性对抗患者来源的神经胶质瘤神经干细胞，即使只有一个亚群表达 FAP，这表明旁观者杀伤机制。通过共培养测定，我们证实 FAP-CAR-T 细胞介导旁观者杀死抗原阴性肿瘤细胞，但只有在被 FAP 阳性靶细胞激活后才有效。这种旁观者杀伤至少部分是由可溶性因子介导的，并由激活 CAR-T 产品的非转导部分的 IL-2 放大。最后，低剂量静脉注射的 FAP-CAR-T 细胞控制了使用抗原阴性和抗原阳性胶质母细胞瘤细胞混合物产生的皮下肿瘤的生长，没有明显的毒性。我们的研究结果使 FAP 成为临床的主要候选者胶质母细胞瘤的 CAR-T 疗法，并强调了未被充分认识的抗原非特异性机制，这些机制可能对 CAR-T 细胞的抗肿瘤活性做出有意义的贡献。© 2024 作者。临床

CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma.We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function in vitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma).FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells.Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.© 2024 The Author(s). Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.