Wang Yao, Zheng Xiujuan, Wang Zhiqian, Xiao Ziyun, Lin Yunqing, Zhang Fan, Liu Yanhong, Liu Pengcheng, Weng Qitong, Zhang Leqiang, Xia Chengxiang, Huang Dehao, Liu Lijuan, Zhu Yanping, Zhang Qi, Qi Hanmeng, Chen Yi, Shen Yiyuan, Zhang Chenyuan, Xu Jiacheng, Zhao Yaoqin, Wu Jiaxin, Wang Tongjie, Zhang Mengyun, Li Minming, Qian Wenbin, Liang Aibin, Du Xin, Yang Wenyu, Hu Tianyuan, Chen Qi, Zhu Xiaofan, Hu Fangxiao, Wang Jinyong
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
J Immunother Cancer. 2025 Sep 8;13(9):e011887. doi: 10.1136/jitc-2025-011887.
Patients with acute myeloid leukemia (AML) are often older, which brings challenges of endurance and persistent efficacy of autologous chimeric antigen receptor (CAR)-T cell therapies. Allogenic CAR-natural killer (NK) cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. CD33 CAR-NK cells have been investigated for AML therapy. However, the fratricide-mediated lysis of CD33-expressing NK cells by CD33 CAR-NK cells limits the expansion and efficacy of CD33 CAR-NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is highly expressed in a fraction of patients with AML, making it a promising target for AML therapy.
We designed a novel CD33-MSLN Loop CAR (Loop CAR) and evaluated its antitumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established an hPSC-derived cell line via knockout of the gene (CD33) and engineered Loop CAR. We generated CD33-Loop CAR-iNK cells using an organoid induction approach. The efficacy of CD33-Loop CAR-iNK cells against tumor cells expressing CD33 and MSLN was investigated both in vitro and in AML xenograft mice.
Loop CAR-NK cells exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells compared with CD33 CAR-NK and MSLN CAR-NK cells. Moreover, Loop CAR-NK cells showed upregulated signaling pathways related to NK cell activation and cytotoxic function. The loss of CD33 in iNK cells effectively avoided fratricide, improved expansion ability, and significantly enhanced CD33 and MSLN-mediated specific cytotoxicity of Loop CAR-iNK cells. Moreover, the CD33-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mouse models and significantly prolonged mouse survival.
Loop CAR empowered both UCB-NK cells and hPSC-iNK cells with superior cytotoxicity against CD33MSLN tumor cells. Genetic disruption of CD33 avoided fratricide and improved efficacy of Loop CAR-iNK cells against AML. This innovative strategy possesses unique advantages and translational potential for treating AML.
急性髓系白血病（AML）患者往往年龄较大，这给自体嵌合抗原受体（CAR）-T细胞疗法的耐受性和持续疗效带来了挑战。同种异体CAR-自然杀伤（NK）细胞疗法可能具有更低的毒性，并增强对AML的抗白血病潜力。CD33 CAR-NK细胞已被研究用于AML治疗。然而，CD33 CAR-NK细胞对表达CD33的NK细胞的自相残杀介导的裂解限制了CD33 CAR-NK细胞的扩增和疗效。间皮素（MSLN）是一种肿瘤分化抗原，在一部分AML患者中高表达，使其成为AML治疗的一个有前景的靶点。
我们设计了一种新型的CD33-MSLN环型CAR（环型CAR），并在人脐带血来源的NK（UCB-NK）细胞和人多能干细胞来源的NK（hPSC-iNK）细胞中评估了其抗肿瘤疗效。为了进一步避免NK细胞内源性CD33表达引起的自相残杀，我们通过敲除基因（CD33）并构建环型CAR建立了一种hPSC来源的细胞系。我们使用类器官诱导方法生成了CD33-环型CAR-iNK细胞。在体外和AML异种移植小鼠中研究了CD33-环型CAR-iNK细胞对表达CD33和MSLN的肿瘤细胞的疗效。
与CD33 CAR-NK和MSLN CAR-NK细胞相比，环型CAR-NK细胞对双抗原阳性肿瘤细胞系和原发性AML细胞表现出更强的细胞毒性。此外，环型CAR-NK细胞显示出与NK细胞激活和细胞毒性功能相关的信号通路上调。iNK细胞中CD33的缺失有效地避免了自相残杀，提高了扩增能力，并显著增强了环型CAR-iNK细胞的CD33和MSLN介导的特异性细胞毒性。此外，CD33-环型CAR-iNK细胞在AML异种移植小鼠模型中表现出卓越的肿瘤杀伤活性，并显著延长了小鼠存活时间。
环型CAR赋予UCB-NK细胞和hPSC-iNK细胞对CD33/MSLN肿瘤细胞的卓越细胞毒性。CD33的基因破坏避免了自相残杀，并提高了环型CAR-iNK细胞对AML的疗效。这种创新策略在治疗AML方面具有独特优势和转化潜力。
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