Fang Sheng, Wu Jiankun, Liu Yida, Wang Peiwen, Yuan Guiqiang, Gao Jiajia, Zhang Wenxin, Zhang Junwen, Liu Fusheng
Brain Tumor Research Center, Beijing Neurosurgical Institute, Capital Medical University, Beijing, 100070, China.
Department of Neurosurgery, Beijing Tiantan Hospital Affiliated to Capital Medical University, Beijing, 100070, China.
J Exp Clin Cancer Res. 2025 Jun 5;44(1):173. doi: 10.1186/s13046-025-03431-6.
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in glioblastoma (GBM) due to tumor antigen heterogeneity and the immunosuppressive microenvironment. To address these barriers, we developed a novel combinatorial approach: engineering CAR-T cells with third-generation oncolytic adenoviruses (OAd) to enable targeted viral delivery and sustained immune activation. Unlike conventional OAd administration, this strategy leverages CAR-T cells as tumor-tropic vectors for localized oncolysis and cytokine modulation. METHODS: CD70-specific CAR-T cells were transduced with two third-generation OAds (E1B19K/E3-deleted, replication-selective): OAd-GFP (control) or OAd-IL15 (TS-2021), generating CAR-T and CAR-T. Viral replication kinetics and CAR-T expansion were assessed in vitro. OAd delivery efficiency was quantified by co-culturing CAR-T cells with GBM cells. Flow cytometry was used to analyze IL15-mediated effects on stem-like markers (CCR7, CD45RA) and exhaustion markers (PD-1, TIM-3, and LAG-3) after repeated antigen stimulation. Antitumor activity was evaluated in vitro using cytotoxicity assays and in NCG mice bearing orthotopic GBM xenografts. Mechanistic studies were conducted using RNA-seq and Western blotting. RESULTS: In this study, we found that genetically engineered OAd-GFP can specifically replicate within CAR-T cells and be precisely delivered to GBM through an antigen-specific mechanism. Prolonged antigen stimulation induced T-cell exhaustion, limiting the efficacy of CAR-T therapy. TS-2021-infected CAR-T cells exhibited enhanced expansion and persistence in vitro, with reduced expression of exhaustion markers under sustained antigen stimulation. IL15 autocrine signaling activated JAK-STAT and MAPK-ERK pathways. This process repaired the DNA damage induced by OAd in CAR-T cells and maintained their expansion and persistence. By combining OAd-mediated oncolysis with IL15-driven CAR-T persistence, CAR-T cells demonstrated potent antitumor efficacy against GBM both in vitro and in vivo. CONCLUSIONS: By integrating IL15-armed OAd into CAR-T cells, we demonstrate a synergistic strategy that simultaneously enhances viral oncolysis, sustains T-cell persistence, and counteracts GBM immunosuppression. This approach addresses both antigenic heterogeneity and microenvironment-driven resistance, providing a translatable paradigm for solid tumor immunotherapy.
背景：嵌合抗原受体（CAR）T细胞疗法在胶质母细胞瘤（GBM）中的疗效有限，原因是肿瘤抗原异质性和免疫抑制微环境。为了克服这些障碍，我们开发了一种新的联合方法：用第三代溶瘤腺病毒（OAd）改造CAR-T细胞，以实现靶向病毒递送和持续的免疫激活。与传统的OAd给药不同，该策略利用CAR-T细胞作为肿瘤趋向性载体进行局部溶瘤和细胞因子调节。 方法：用两种第三代OAd（E1B19K/E3缺失、复制选择性）转导CD70特异性CAR-T细胞：OAd-GFP（对照）或OAd-IL15（TS-2021），产生CAR-T和CAR-T。在体外评估病毒复制动力学和CAR-T扩增。通过将CAR-T细胞与GBM细胞共培养来量化OAd递送效率。在反复抗原刺激后，使用流式细胞术分析IL15介导的对干细胞样标志物（CCR7、CD45RA）和耗竭标志物（PD-1、TIM-3和LAG-3）的影响。使用细胞毒性试验在体外和在携带原位GBM异种移植瘤的NCG小鼠中评估抗肿瘤活性。使用RNA测序和蛋白质印迹进行机制研究。 结果：在本研究中，我们发现基因工程改造的OAd-GFP可以在CAR-T细胞内特异性复制，并通过抗原特异性机制精确递送至GBM。长时间的抗原刺激诱导T细胞耗竭，限制了CAR-T疗法的疗效。TS-2021感染的CAR-T细胞在体外表现出增强的扩增和持久性，在持续抗原刺激下耗竭标志物的表达降低。IL15自分泌信号激活JAK-STAT和MAPK-ERK途径。这一过程修复了OAd在CAR-T细胞中诱导的DNA损伤，并维持了它们的扩增和持久性。通过将OAd介导的溶瘤与IL15驱动的CAR-T持久性相结合，CAR-T细胞在体外和体内均表现出对GBM的强大抗肿瘤疗效。 结论：通过将携带IL15的OAd整合到CAR-T细胞中，我们证明了一种协同策略，该策略同时增强病毒溶瘤、维持T细胞持久性并对抗GBM免疫抑制。这种方法解决了抗原异质性和微环境驱动的耐药性问题，为实体瘤免疫治疗提供了一种可转化的范例。
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