采用养育的嵌合抗原受体（CAR）工程化自然杀伤细胞（NK）在治疗不同类型的癌症方面显示出潜力。然而，免疫记忆力有限以及获得足够数量的异基因供体细胞是限制其更广泛的临床应用的因素。在这里，我们首先评估了八种不同的CAR构建体，它们使用抗PD-L1纳米抗体和/或通用的荧光素（FITC）单链可变片段（scFv）来增强针对异种实体瘤的NK-92细胞的抗原特异性增殖和抗肿瘤细胞毒性。然后，我们将优化的CAR基因工程到人类多能干细胞（hPSCs）中，并将其分化为功能性的双CAR-NK细胞。PD-L1诱导的记忆样hPSC-NK细胞的抗肿瘤活性通过给予荧光素-叶酸双特异性适配器而得到进一步增强，该适配器在可编程抗FITC CAR与叶酸受体α表达的乳腺肿瘤细胞之间桥接。总之，我们的hPSC CAR-NK工程平台是模块化的，并且可以构成一种现实的策略，用于制造具有免疫记忆样表型的现成CAR-NK细胞，以进行靶向免疫治疗。© 2023作者们。

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.© 2023 The Authors.