单抗Fc上N-糖末端羟乙酰神经氨酸的免疫原性研究

α-gal and Neu5Gc on biopharmaceuticals expressed in mouse hybridoma cells have potentail immunogenicity to humans. It was well documented that α-gal and Neu5Gc on the Fab portion of monoclonal antibody (mAb) could induce severe anaphylaxis or reduce the half life of the mAb, while no immunogenicity was detected for α-gal on the Fc portion of the mAb, but no study on the immunogenicity of Neu5Gc on the Fc portion was reported. It is essential to clarify the immunogenicity of Neu5Gc on the Fc portion since most mAbs only have glycans on this portion. Based on the qualitative and quantitative analysis of glycan mapping of mAbs expressed from mouse hybridoma cells, we found that 0.1%-20% N-glycan is comprised of Neu5Gc, but our preliminary study revealed that the binding capacity of denatured mAb to anti-Neu5Gc antibody was much stronger than that of undenatured mAb, which suggested that only a small raio of Neu5Gc on the Fc portion of the mAb was exposed on the surface of the mAb. Our project will focus on the in-depth study on the immnogeneicity of Neu5Gc on the Fc portion of mAbs, reveal the distribution pattern of Neu5Gc both with and without immunogenicity on the mAb, and preliminarily unfold the mechanism of their different capacity of inducing imunogeneicity. Our study will lay a solid theoretic foundation for the R&D and QC of mAbs for biopharmaceutical company and for the registering surveillance of mAbs for national regulatory authorities.

鼠杂交瘤细胞所表达的α-1,3-半乳糖（α-gal）和羟乙酰神经氨酸（Neu5Gc）对人体来说为异源性单糖，已有文献证明位于单抗Fab上N-糖末端的α-gal和Neu5Gc具有免疫原性，而位于Fc上N-糖末端的α-gal 则无免疫原性，但是位于Fc上N-糖末端的Neu5Gc的免疫原性则无相关报道，值得注意的是，绝大对数单抗只在Fc上有N-糖。本研究组对多种鼠杂交瘤细胞所表达单抗N-糖糖谱进行了定性定量分析，发现含Neu5Gc的N-糖占整个糖谱的0.1%-20%，但初步研究结果表明变性后单抗与抗Neu5Gc抗体的结合能力远高于未变性单抗，提示Fc上N糖末端的Neu5Gc只有少部分暴露于单抗表面，本课题将阐明具有和不具有免疫原性Neu5Gc在单抗上的分布特点并初步分析其免疫原性差异的机制，为生物制药企业对单抗制品的研发和质控以及药监机构对单抗制品的监管提供理论基础。

西妥昔单抗Fab上所含有两种非人单糖，即α-1,3半乳糖（NEJM，2008）或羟乙酰神经氨酸（Nat Biotechnol.，2010），具有免疫原性，而当α-1,3半乳糖位于单抗Fc上时并不具有免疫原性，但是羟乙酰神经氨酸位于单抗Fc上时是否具有免疫原性则未有报道。由于绝大部分单抗只有在Fc上具有N糖，另外用小鼠骨髓瘤细胞（占上市单抗所用表达体系的40%以上，主要包括SP2/0或NS0细胞）所表达的单抗其所含的相对较高含量的唾液酸主要以羟乙酰神经氨酸的形式存在，另外CHO细胞所表达的单抗也还有痕量的羟乙酰神经氨酸，所以研究单抗Fc上羟乙酰神经氨酸是否具有免疫原性对患者的用药安全具有十分重要的意义。. 该研究证明只有在单抗Fc上含有两个或两个以上唾液酸的情况下，其所包含的羟乙酰神经氨酸才可能具有免疫原性；但是由于即使单抗的N糖含有较高含量的唾液酸如10%，一个单抗含有两个唾液酸的可能性也只有1%，所以其免疫原性是相对较低的。. 本研究于国际上首次解决了治疗用单抗Fc上非人唾液酸即羟乙酰神经氨酸在何种情况下具有免疫原性的问题，对单克隆抗体的生产及质控具有重大的指导意义。