The AAPS Journal ( IF 5.0 ) Pub Date : 2022-07-01 , DOI: 10.1208/s12248-022-00729-7
Michael A Partridge 1 , Jihua Chen 1 , Elif Kabuloglu Karayusuf 1 , Thanoja Sirimanne 1 , Colin Stefan 1 , Ching Ha Lai 1 , Meghna Gathani 1 , Lisa DeStefano 1 , Michal Rozanski 1 , Sean McAfee 1 , Manoj Rajadhyaksha 1, 2 , Matthew D Andisik 1 , Albert Torri 1 , Giane Sumner 1
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Twenty percent of baseline patient samples exhibited a pre-existing response in a bridging anti-drug antibody (ADA) assay for a human IgG4 monoclonal antibody (mAb) therapeutic. In some cases, assay signals were more than 100-fold higher than background, potentially confounding detection of true treatment-emergent ADA responses. The pre-existing reactivity was mapped by competitive inhibition experiments using recombinant proteins or chimeric human mAbs with IgG4 heavy chain regions swapped for IgG1 sequences. These experiments demonstrated that the majority of the samples had reactivity to an epitope containing leucine 445 in the CH3 domain of human IgG4. The pre-existing reactivity in baseline patient samples was mitigated by replacing the ADA assay capture reagent with a version of the drug containing a wild type IgG1 proline substitution at residue 445 without impacting detection of drug-specific, treatment-emergent ADA. Finally, purification on Protein G or anti-human IgG (H + L) columns indicated the pre-existing response was likely due to immunoglobulins in patient samples.
Graphical abstract
中文翻译:
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对 IgG4 Fc 表位的预先存在的反应性:桥接抗药物抗体测定中干扰的表征和缓解
20% 的基线患者样本在人 IgG4 单克隆抗体 (mAb) 治疗的桥接抗药物抗体 (ADA) 测定中表现出预先存在的反应。在某些情况下,检测信号比背景高 100 倍以上,可能会混淆对真正治疗出现的 ADA 反应的检测。预先存在的反应性通过使用重组蛋白或嵌合人 mAb 的竞争性抑制实验来绘制,其中 IgG4 重链区域交换为 IgG1 序列。这些实验表明,大多数样品对人 IgG4 CH3 结构域中含有亮氨酸 445 的表位具有反应性。通过用在残基 445 处含有野生型 IgG1 脯氨酸取代的药物版本替换 ADA 测定捕获试剂,减轻了基线患者样本中预先存在的反应性,而不影响对药物特异性、治疗出现的 ADA 的检测。最后,在蛋白 G 或抗人 IgG (H + L) 柱上的纯化表明预先存在的反应可能是由于患者样本中的免疫球蛋白所致。