Mesothelin CAR‐engineered NK cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals

Abstract

AbstractCAR‐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.