Centre for Oncology, Radiopharmaceuticals and Research, Biologics and Radiopharmaceutical Drugs Directorate, Health Canada, Ottawa, ON, Canada.
Front Immunol. 2022 Oct 12;13:972168. doi: 10.3389/fimmu.2022.972168. eCollection 2022.
Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined -glycans, we show that galactosylation at a specific location of -glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex -glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-H-N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.
单克隆抗体（mAbs）是生物治疗的重要类型，因其具有抗癌和抗炎特性，以及能够预防呼吸道感染，因此被用于治疗疾病。其生产涉及翻译后糖基化，这是一个将聚糖连接到蛋白质上的生物合成过程，在 mAb 的生物活性中起着至关重要的作用，包括效应功能和药代动力学。这些聚糖是异质的，具有不同的化学结构，其组成对制造条件敏感，这使得理解特定糖基结构如何影响 mAb 的生物活性具有挑战性。需要阐明特定糖基对 mAb 生物活性的影响，以确定制造过程中某些糖基化谱的变化是否会显著影响产品质量。使用具有化学定义的 -聚糖的酶转糖基化，我们表明，在无岩藻糖基化抗病毒 mAb 中 -聚糖的特定位置上的半乳糖基化负责 FcγRIIIA 结合和抗体依赖性细胞介导的细胞毒性（ADCC）活性。我们报告了一种简便的方法来获得纯化的不对称单半乳糖基化双天线复合 -聚糖，并研究了它们在掺入无岩藻糖基化 mAb 中对生物活性的影响。通过 ELISA、表面等离子体共振和流式细胞术，我们表明，α6 天线而不是α3 天线的半乳糖基化一致增加了 FcγRIIIA 的结合亲和力。我们在呼吸道合胞病毒（RSV）的抗病毒模型中使用适应性 ADCC 报告测定法证实了这一点。我们通过二维 H-N-NMR 进一步将这种结构-功能关系与α6 天线的半乳糖残基与蛋白质骨架的相互作用相关联，结果表明，与缺乏该半乳糖残基的糖型相比，该位置的半乳糖基化表现出化学位移扰动。我们的结果强调了识别和定量特定聚糖异构体的重要性，以确保在批次间和类似物比较中进行充分的质量控制。
