Liu Yanhong, Zhang Min, Shen Xiaoyan, Xia Chengxiang, Hu Fangxiao, Huang Dehao, Weng Qitong, Zhang Qi, Liu Lijuan, Zhu Yanping, Wang Lei, Hao Jie, Zhang Mengyun, Wang Tongjie, Wang Jinyong
Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China.
Cell Prolif. 2024 Dec;57(12):e13727. doi: 10.1111/cpr.13727. Epub 2024 Aug 13.
CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48-72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product.
嵌合抗原受体自然杀伤细胞（CAR-NK）疗法无需进行人类白细胞抗原（HLA）配型，且副作用极小。然而，将CAR导入人组织来源的自然杀伤细胞的传统方法存在异质性、转导效率低和生产成本高的问题。在此，我们提供了一种可靠的方法，可从人类胚胎干细胞（hESC）中大规模生成并冻存间皮素（MSLN）CAR-NK细胞，作为替代细胞来源。我们首先构建了表达MSLN CAR的hESC，以降低CAR工程成本，随后通过高效的类器官诱导系统将这些干细胞分化为MSLN CAR-NK细胞。MSLN CAR-NK细胞表现出自然杀伤细胞激活受体、抑制受体和效应分子的典型表达模式。在存在肿瘤细胞的情况下，与诱导的自然杀伤细胞相比，MSLN CAR-NK细胞显示出干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）分泌增加，以及CD107a表达水平升高。我们使用临床级冷冻培养基（CS10）在液氮中冻存MSLN CAR-NK细胞超过6个月，以模拟即用型CAR-NK细胞产品。解冻后的MSLN CAR-NK细胞在培养48 - 72小时后立即恢复，并有效消除了卵巢肿瘤细胞，包括来自患者的人原发性卵巢肿瘤细胞。解冻后的MSLN CAR-NK细胞在体内有效抑制了卵巢肿瘤的发展，并延长了荷瘤小鼠的生存期。我们的研究为hESC来源的MSLN CAR-NK细胞作为一种有前景的即用型细胞产品的临床转化提供了见解。
