Clinical success of chimeric antigen receptor (CAR) T cell immunotherapy requires the engineering of autologous T cells, which limits the broader implementation of CAR cell therapy. The development of allogeneic and universal cell products will significantly broaden their application and reduce costs. Allogeneic natural killer (NK) cells can be used for universal CAR immunotherapy. Here, we develop an alternative approach for the rapid expansion of primary NK and CAR-NK cells with superior expansion capability and in vivo cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, “off-the-shelf” NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.

嵌合抗原受体（CAR）T细胞免疫疗法的临床成功需要自体T细胞的工程改造，这限制了CAR细胞疗法的更广泛实施。同种异体和通用细胞产品的开发将大大拓宽其应用范围并降低成本。同种异体自然杀伤（NK）细胞可用于通用CAR免疫疗法。在这里，我们开发了一种替代方法，用于以优异的扩增能力和体内快速扩增原代NK和CAR-NK细胞来自各种来源（包括外周血，脐带血和肿瘤组织）的细胞毒性。我们应用具有膜结合白介素21（mIL-21）的人类B淋巴母细胞系721.221（以下称221）为基础的人工饲养细胞系统来繁殖NK和CAR-NK细胞。用221-mIL-21饲养细胞扩增的NK细胞的扩增能力，纯度和细胞毒性优于常规K562-mIL-21饲养细胞。RNA测序（RNA-seq）数据显示，221-mIL-21饲养细胞扩增的NK细胞显示出分化程度较低，未耗尽，有限的自相残杀，记忆样表型，与丰富的代谢途径相关，这解释了其潜在机制。从而，