Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.本研究对体外比较免疫原性试验（In Vitro Comparative Immunogenicity Assay, IVCIA）作为预测生物治疗制剂相关属性潜在相对免疫原性的工具开展了评估。研究采集了至多50名健康未致敏人类供者的外周血单个核细胞（Peripheral blood mononuclear cells），对其T细胞增殖情况、分泌白细胞介素2（IL-2）或干扰素γ（IFN-γ）的细胞数量，以及一组分泌型细胞因子的浓度进行了长达8天的动态监测。结果显示，该试验针对10种单克隆抗体（monoclonal antibodies）的响应结果与临床免疫原性表现高度一致，提示该试验可应用于抗体类生物治疗制剂相关属性的免疫原性风险评估。不过，该试验的响应值本质上反映的是T细胞功能活性，其与临床免疫原性的匹配程度受多种其他因素影响。该试验对序列变异体具有良好的敏感性，能够区分同一生物治疗制剂的单点突变体。经搅拌诱导发生高聚集的9株单克隆抗体，相较于搅拌应激处理前的原始单克隆抗体，可在试验中引发更强的细胞响应，但其响应模式与原始单克隆抗体的相对T细胞响应并不匹配。与之相反，经半乳糖、葡萄糖及甘露糖等不同糖类进行糖基化修饰的单克隆抗体，相较于糖基化处理前的原始单克隆抗体，其试验响应几乎未出现显著升高。该试验还成功用于评估同一生产商及不同生产商（生物类似药，biosimilars）来源的多批次同一款单克隆抗体之间的相似性。本研究提出了一种将IVCIA试验应用于生物制药全生命周期开发过程中免疫原性风险评估的可行策略。