背景:可以通过用引发抗体依赖性细胞介导的细胞毒性(ADCC)的治疗性抗体进行特异性靶向或通过基因工程以表达嵌合抗原受体(CAR)来增强自然杀伤(NK)细胞的抗肿瘤活性。尽管抗体或CAR靶向,一些肿瘤仍然对NK细胞攻击具有抗性。虽然ICAM-1/LFA-1相互作用对NK细胞的天然细胞毒性的重要性是已知的,其对ErbB2(HER2)特异性抗体曲妥珠单抗诱导的ADCC和ErbB2-CAR介导的NK细胞对乳腺癌细胞的细胞毒性的影响尚未研究.
方法:在这里,我们将表达高亲和力Fc受体FcγRIIIa的NK-92细胞与曲妥珠单抗或ErbB2-CAR工程化NK-92细胞(NK-92/5.28。z)以及与曲妥珠单抗组合或用ErbB2-CAR修饰的原代人NK细胞,并测试了针对ICAM-1表达不同或阻断NK细胞上LFA-1的癌细胞的细胞毒性。此外,我们特别刺激Fc受体,CAR和/或LFA-1研究它们在免疫突触处的串扰以及它们在抗体靶向或CAR靶向NK细胞中对脱粒和细胞内信号传导的贡献。
结果:在曲妥珠单抗介导的抗ErbB2阳性乳腺癌细胞的ADCC过程中,阻断LFA-1或不存在ICAM-1显著降低了细胞杀伤和细胞因子释放,但在针对CAR的NK细胞中并非如此。用5-氮杂-2'-脱氧胞苷预处理诱导ADCC中ICAM-1上调并逆转NK细胞抗性。曲妥珠单抗单独不能充分激活NK细胞,需要额外的LFA-1共刺激,而CAR-NK细胞中ErbB2-CAR的激活诱导了独立于LFA-1的有效脱粒。全内反射荧光单分子成像显示,CAR-NK细胞与排除ICAM-1的肿瘤细胞形成不规则的免疫突触,而曲妥珠单抗形成典型的外周超分子激活簇(pSMAC)结构。机械上,缺乏ICAM-1不影响ADCC期间的细胞-细胞粘附,而是导致通过Pyk2和ERK1/2的信号传导减少,这本质上是由CAR介导的靶向提供的。此外,而抑制性NK细胞检查点分子NKG2A的刺激显着减少FcγRIIIa/LFA-1介导的脱颗粒,CAR的重定向仅受到轻微影响。
结论:乳腺癌细胞上ICAM-1的下调是曲妥珠单抗触发的ADCC的关键逃逸机制。相比之下,CAR-NK细胞能够克服由ICAM-1减少引起的癌细胞耐药性,强调CAR-NK细胞在癌症免疫治疗中的潜力。
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 FcγRIIIa 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 FcγRIIIa/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.