Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)–SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

自然杀伤（NK）细胞具有抗癌的临床潜力；然而，多种限制阻碍了 NK 细胞疗法的成功。在这里，我们使用体内腺相关病毒（AAV）-SB（睡美人）-CRISPR（成簇规则间隔短回文重复序列）筛选在四种实体瘤小鼠模型中对肿瘤浸润 NK (TINK) 细胞进行了无偏见的功能图谱。与此同时，我们对 TINK 细胞的单细胞转录组景观进行了表征，鉴定了以前未探索的 NK 细胞亚群和差异表达的 TINK 基因。作为一种聚合攻击，CALHM2 敲除 (KO) NK 细胞在小鼠原代 NK 细胞和人嵌合抗原受体 (CAR)-NK 细胞中显示出增强的细胞毒性和肿瘤浸润。 CALHM2 mRNA 逆转了 CALHM2-KO 表型。人原代 NK 细胞中的 CALHM2 KO 增强了其细胞毒性、脱颗粒和细胞因子的产生。转录组学分析揭示了基线和刺激条件下 CALHM2-KO 改变的基因和通路。在对未修饰的 CAR-NK 细胞产生耐药性的实体瘤模型中，CALHM2-KO CAR-NK 细胞显示出强大的体内抗肿瘤功效。这些数据确定了自然限制 NK 细胞功能的内源性遗传检查点，并证明了 CALHM2 KO 用于设计增强的基于 NK 细胞的免疫疗法。