Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG–Fab regions, allowing neighboring IgG–Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1–Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1–Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1–Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.

人类 IgG 包含一个进化上保守的N297 位 Fc 区中的 -连接聚糖。该聚糖对于 Fc 介导的功能至关重要，包括其对经典补体级联反应的诱导。这是在通过 IgG-Fab 区域识别目标后诱导的，允许相邻的 IgG-Fc 尾部通过 Fc:Fc 相互作用结合，最终导致六聚体形成。这种六聚化似乎对于 IgG 能够与第一个补体成分 C1q 的球状头部有效相互作用以及随后的补体激活至关重要。在这项研究中，我们表明掺入 IgG1-Fc 的半乳糖增强了人红细胞和 Raji 细胞中的 C1q 结合、C4、C3 沉积和补体依赖性细胞毒性。IgG1-Fc 唾液酸化略微增强了 C1q 的结合，但对下游补体激活几乎没有影响。使用降低或增加 IgG1 六聚化能力的各种突变，我们表明 IgG1-Fc 半乳糖基化对 C1q 与 IgG1 的结合没有内在影响，但增强了 IgG1 六聚化潜力，从而增强了补体激活。这些数据表明，通过相对简单的糖工程，可以在不引入免疫原性突变的情况下放大抗体的治疗潜力。