Antibodies mediate effector functions through Fcγ receptor (FcγR) interactions and complement activation, causing cytokine release, degranulation, phagocytosis, and cell death. They are often undesired for development of therapeutic antibodies where only antigen binding or neutralization would be ideal. Effector elimination has been successful with extensive mutagenesis, but these approaches can potentially lead to manufacturability and immunogenicity issues. By switching the native glycosylation site from position 297 to 298, we created alternative antibody glycosylation variants in the receptor interaction interface as a novel strategy to eliminate the effector functions. The engineered glycosylation site at Asn298 was confirmed by SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB code 6X3I). The lead NNAS mutant (S298N/T299A/Y300S) shows no detectable binding to mouse or human FcγRs by surface plasmon resonance analyses. The effector functions of the mutant are completely eliminated when measured in antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. In vivo, the NNAS mutant made on an antibody against a human lymphocyte antigen does not deplete T cells or B cells in transgenic mice, in contrast to wild-type antibody. Structural study confirms the successful glycosylation switch to the engineered Asn298 site. The engineered glycosylation would clash with approaching FcγRs based on reported Fc-FcγR co-crystal structures. In addition, the NNAS mutants of multiple antibodies retain binding to antigens and neonatal Fc receptor, exhibit comparable purification yields and thermal stability, and display normal circulation half-life in mice and non-human primate. Our work provides a novel approach for generating therapeutic antibodies devoid of any ADCC and CDC activities with potentially lower immunogenicity.

抗体通过Fcγ受体（FcγR）相互作用和补体激活介导效应子功能，从而导致细胞因子释放，脱粒，吞噬和细胞死亡。它们通常不期望用于开发治疗性抗体，其中仅抗原结合或中和才是理想的。通过广泛的诱变已经成功消除了效应子，但是这些方法可能潜在地导致可制造性和免疫原性问题。通过将天然糖基化位点从位置297切换到298，我们在受体相互作用界面中创建了替代性抗体糖基化变体，作为消除效应子功能的新策略。通过SDS-PAGE，质谱和X射线晶体学（PDB代码6X3I）确认了Asn298处的工程糖基化位点。NNAS前导突变体（S298N / T299A / Y300S）通过表面等离振子共振分析显示未检测到与小鼠或人FcγR的结合。当在抗体依赖性细胞介导的细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）分析中进行测量时，该突变体的效应子功能被完全消除。与野生型抗体相比，在体内，针对人类淋巴细胞抗原的抗体制成的NNAS突变体不会耗尽转基因小鼠中的T细胞或B细胞。结构研究证实成功糖基化转换到工程Asn298位点。基于报道的Fc-FcγR共晶体结构，工程化的糖基化将与接近的FcγR发生冲突。此外，多种抗体的NNAS突变体保留了与抗原和新生儿Fc受体的结合，具有可比的纯化产率和热稳定性，并在小鼠和非人灵长类动物中显示出正常的循环半衰期。我们的工作提供了一种新颖的方法来产生治疗性抗体，该抗体没有任何ADCC和CDC活性，且免疫原性可能较低。