Bioscience COPD/IPF, Research and Early Development, Respiratory and Immunology, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, MD, USA.
R. Dagher and R. Kolbeck contributed equally to this article as lead authors and supervised the work.
Eur Respir J. 2022 Mar 3;59(3). doi: 10.1183/13993003.04306-2020. Print 2022 Mar.
Benralizumab is a humanised, anti-interleukin-5 receptor α monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remains elusive.
Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities by employing relevant primary human autologous cell co-cultures and real-time-lapse imaging combined with flow cytometry.
In the presence of NK cells, benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of Caspase-3/7 and upregulation of cytochrome . In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cellular phagocytosis. Using live cell imaging, we unravelled the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays, we identified a novel role for macrophage-derived tumour necrosis factor (TNF) to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with cytochrome upregulation, mitochondrial membrane depolarisation and increased Caspase-3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of interferon-γ, subsequently driving TNF expression by macrophages.
Our data provide deeper insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab . Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.
本瑞鲁单抗是一种人源化抗白细胞介素-5 受体α单克隆抗体，具有抗嗜酸性粒细胞活性。缺乏岩藻糖（去岩藻糖基化）增加了其与 CD16a 的亲和力，并通过自然杀伤 (NK) 细胞显著增强抗体依赖性细胞介导的细胞毒性。尽管本瑞鲁单抗在严重哮喘和嗜酸性粒细胞增多综合征患者的临床试验中已被证明具有临床疗效，但深入了解其抗嗜酸性粒细胞作用机制仍不清楚。
在这里，我们通过使用相关的原发性人自体细胞共培养物和实时延时成像结合流式细胞术，进一步研究了本瑞鲁单抗抗嗜酸性粒细胞活性的相关机制。
在 NK 细胞存在的情况下，本瑞鲁单抗诱导 Caspase-3/7 的上游诱导和细胞色素上调，从而导致嗜酸性粒细胞凋亡。此外，我们发现了一种以前未被识别的机制，即本瑞鲁单抗可以诱导巨噬细胞吞噬/胞吐嗜酸性粒细胞，这一过程称为抗体依赖性细胞吞噬作用。通过活细胞成像，我们揭示了导致激活的巨噬细胞吞噬嗜酸性粒细胞凋亡和摄取的逐步过程。通过仔细观察细胞共培养试验，我们确定了巨噬细胞衍生的肿瘤坏死因子 (TNF) 的新作用，通过激活嗜酸性粒细胞上的 TNF 受体 1 进一步增强本瑞鲁单抗介导的嗜酸性粒细胞凋亡。TNF 诱导的嗜酸性粒细胞凋亡与细胞色素上调、线粒体膜去极化和 Caspase-3/7 活性增加有关。此外，激活的 NK 细胞被发现通过分泌干扰素-γ 放大这一轴，随后驱动巨噬细胞表达 TNF。
我们的数据提供了对导致本瑞鲁单抗诱导的嗜酸性粒细胞凋亡的事件及时出现的更深入了解，并表明其他机制可能有助于本瑞鲁单抗的强大抗嗜酸性粒细胞活性。重要的是，本瑞鲁单抗的去岩藻糖基化强烈增强了其所有研究机制的效力。
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