肿瘤细胞治疗研究
英文原题：CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.
乳腺癌细胞上ICAM-1的下调是逃逸trastuzumab触发的ADCC的关键机制。相反，CAR-NK细胞能够克服由ICAM-1减少引起的癌细胞耐药，凸显了CAR-NK细胞在肿瘤免疫治疗中的潜力。
自然杀伤（NK）细胞的抗肿瘤活性可通过治疗性抗体特异性靶向来增强，这些抗体触发抗体依赖性细胞介导的细胞毒性（ADCC），或通过基因工程使其表达嵌合抗原受体（CARs）来增强。尽管有抗体或CAR靶向，一些肿瘤仍对NK细胞攻击具有抗性。虽然ICAM-1/LFA-1相互作用对NK细胞自然细胞毒性的重要性已知，但其对ErbB2（HER2）特异性抗体trastuzumab诱导的ADCC以及ErbB2-CAR介导的NK细胞对乳腺癌细胞的细胞毒性的影响尚未被研究。
在此，我们使用了表达高亲和力 Fc 受体 Fc RIIIa 的 NK-92 细胞联合 trastuzumab，或使用经 ErbB2-CAR 改造的 NK-92 细胞（NK-92/5.28.z），以及联合 trastuzumab 或经 ErbB2-CAR 修饰的原代人 NK 细胞，并检测了针对 ICAM-1 表达不同的癌细胞的细胞毒性，或检测了另外阻断 NK 细胞上的 LFA-1 后的细胞毒性。此外，我们特异性刺激 Fc 受体、CAR 和/或 LFA-1，以研究它们在免疫突触处的串扰及其对抗体靶向或 CAR 靶向 NK 细胞脱颗粒和细胞内信号传导的贡献。
阻断 LFA-1 或缺失 ICAM-1 可显著降低 trastuzumab 介导的针对 ErbB2 阳性乳腺癌细胞的 ADCC 期间的细胞杀伤和细胞因子释放，但在 CAR 靶向的 NK 细胞中并非如此。用 5-aza-2'-deoxycytidine 预处理可诱导 ICAM-1 上调，并逆转 ADCC 中的 NK 细胞抗性。单独使用 trastuzumab 不足以激活 NK 细胞，并需要额外的 LFA-1 共刺激；而在 CAR-NK 细胞中激活 ErbB2-CAR 可诱导不依赖 LFA-1 的高效脱颗粒。全内反射荧光单分子成像显示，CAR-NK 细胞与肿瘤细胞形成排除 ICAM-1 的不规则免疫突触，而 trastuzumab 形成典型的外周超分子激活簇 (pSMAC) 结构。从机制上看，ICAM-1 缺失并不影响 ADCC 期间的细胞间黏附，而是导致经 Pyk2 和 ERK1/2 的信号传导减少，而该信号传导是由 CAR 介导的靶向所内在提供的。此外，虽然刺激抑制性 NK 细胞检查点分子 NKG2A 显著降低了 Fc RIIIa/LFA-1 介导的脱颗粒，但通过 CAR 的再靶向仅受到轻微影响。
BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor Fc RIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced Fc RIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.
MEMBER ACCOUNT
登录成功会直接打开下一页。
