Martins-Ribeiro Ana, Kizhedath Arathi, Ahmed Shaheda Sameena, Glassey Jarka, Ishaq Abbas, Freer Matthew, Dickinson Anne Mary
Alcyomics Ltd., The Biosphere, Draymans Way, Newcastle Helix, Newcastle Upon Tyne NE4 5BX, UK.
Translational and Clinical Research Institute Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK.
Toxics. 2024 Apr 30;12(5):332. doi: 10.3390/toxics12050332.
Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. : Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II-III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.
单克隆抗体（mAb）是重要的治疗药物。然而，在单克隆抗体制备过程中，聚集可能性的增加已成为一个关键的质量参数。此外，在临床环境中给患者施用单克隆抗体治疗药物时，单克隆抗体聚集也可能带来潜在的健康风险。虽然患者群体中免疫毒性的程度尚不确定，但报告显示它可通过细胞活化和细胞因子释放引发免疫反应。在本研究中，一种旨在预测包括皮肤致敏在内的不良免疫事件的自体体外皮肤试验被用作评估单克隆抗体聚集引起的免疫毒性的新方法。通过热应激方案诱导单克隆抗体聚集，随后通过分析超速离心和透射电子显微镜对蛋白质含量进行表征，结果显示总蛋白质含量的聚集水平为4%。然后，在皮肤外植体试验中测试聚集物引起的免疫毒性和潜在的皮肤致敏性。聚集的赫赛汀和利妥昔单抗引起了皮肤致敏，组织病理学损伤（II-III级阳性反应）以及热休克蛋白70（HSP70）的阳性染色表明了这一点。未观察到T细胞增殖的变化。细胞因子分析显示，在最极端的聚集条件下（pH3时65°C），白细胞介素-10显著增加，并且干扰素-γ总体上有增加的趋势，尤其是对利妥昔单抗的反应。皮肤外植体试验表明，聚集的单克隆抗体表现出不良免疫反应，如组织病理学II-III级的皮肤致敏。因此，该试验可能是评估单克隆抗体聚集引起的免疫毒性和皮肤致敏性的一种新工具。
