Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity and efficacy. One of the most important variations concerns the N-glycosylation pattern, which directly affects an anti-tumor mechanism called antibody-dependent cell-meditated cytotoxicity (ADCC). Thus, careful engineering of mAbs is expected to enhance both protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Fc region on the interaction with the FcγRIIIa/CD16a receptor in native and label-free conditions. The single mAb molecule comprises variants with minimal and maximal galactosylation, as well as α2,3 and α2,6-sialic acid isomers. Here, we apply native electrospray ionization mass spectrometry to determine the solution-phase antibody-receptor equilibria and by using temperature-controlled nanoelectrospray, a thermal stability of the complex is examined. Based on these, we prove that the galactosylation of a fucosylated Fc region increases the binding to CD16a 1.5-fold when compared with the non-galactosylated variant. The α2,6-sialylation has no significant effect on the binding, whereas the α2,3-sialylation decreases it 1.72-fold. In line with expectation, the galactoslylated and α2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured in the temperature gradient from 20 to 50°C. The similar binding pattern is observed based on surface plasmon resonance analysis and immunofluorescence staining using natural killer cells. The results of our study provide new insight into N-glycosylation-based interaction of the mAb:CD16a complex.

中文翻译：

---

糖工程化抗体与 CD16a 的相互作用分析：一种天然质谱方法。


生物制造的单克隆抗体 (mAb) 质量的微小变化可能会影响其生物活性和功效。最重要的变异之一涉及 N-糖基化模式，它直接影响称为抗体依赖性细胞介导的细胞毒性 (ADCC) 的抗肿瘤机制。因此，精心设计的 mAb 有望增强蛋白质-受体结合和 ADCC。本研究的具体目的是评估 Fc 区内末端碳水化合物对天然和无标记条件下与 FcγRIIIa/CD16a 受体相互作用的影响。单个 mAb 分子包含具有最小和最大半乳糖基化的变体，以及 α2,3 和 α2,6-唾液酸异构体。在这里，我们应用天然电喷雾电离质谱法来确定溶液相抗体-受体平衡，并通过使用温控纳电喷雾来检查复合物的热稳定性。基于这些，我们证明，与非半乳糖基化变体相比，岩藻糖基化 Fc 区的半乳糖基化使与 CD16a 的结合增加了 1.5 倍。 α2,6-唾液酸化对结合没有显着影响，而α2,3-唾液酸化则使其结合降低1.72倍。与预期一致，在 20 至 50°C 的温度梯度下测量时，半乳糖基化和 α2,6-唾液酸化 mAb:CD16a 复合物表现出更高的热稳定性。基于表面等离子共振分析和使用自然杀伤细胞的免疫荧光染色观察到类似的结合模式。我们的研究结果为 mAb:CD16a 复合物基于 N-糖基化的相互作用提供了新的见解。