新型糖基工程酵母表达具有不同糖基化修饰的抗体及其功能特性研究

N-glycosylation is crucial for antibodies’ property. It has been shown that the fucosylation of glycans on human IgG1 affects ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity) efficacy. For instance, afucosylated glycans can significantly increase IgG1 binding to FcγRIII and enhance ADCC efficacy over 10-fold. However, N-glycosylation modifications on glycoproteins are uncontrolled to date. The glycan profiles of recombinant proteins produced in mammalian cells and yeast are predominantly heterogeneous. Therefore, the relationships between certain glycans and protein functions cannot be clarified by means of current expression systems. The use of methylotrophic yeast system for foreign protein expression is increasing dramatically. Yeasts are capable of performing many human posttranslational modification reactions, including N-linked glycosylation. However, glycoproteins produced in wild type yeast contain potentially immunogenic high-mannose type N-glycans, limiting the use of yeast expression systems for glycoprotein. This study will be performed to optimize and the recreation of the sequential nature of mammalian cell glycosylation in the ER and Golgi. (ii) The monoclonal antibodies (anti-Her2 mAb or anti-CD20 mAb) were used as examples to study glycoprotein production in glycoengineered Pichia pastoris. In this study, we will measure the levels of recombinant yeast derived antibodies protein in heterotetramer folding, physical stability and binding affinity. The combination of glycoengineered Pichia pastoris expression system with established quality control methods maybe provide an alternative production platform for therapeutic monoclonal antibodies with higher ADCC and CDC.

抗体的糖基化修饰对其功能活性具有重要影响，如岩藻糖的有无会直接影响抗体依赖细胞介导的细胞毒作用（ADCC）和补体依赖的细胞毒作用（CDC），研究证实无岩藻糖化的抗CD20抗体比含有岩藻糖抗体的ADCC提高10倍以上。现有各种糖蛋白表达系统对糖基的修饰过程都是不可控的，因此一般只能通过糖基化位点的突变来研究糖基化"有无"对糖蛋白的影响，而不能详细研究特定结构糖基对糖蛋白的影响。本研究将在前期研究基础上，优化酵母糖基化修饰通路，获得各种具有哺乳动物细胞糖基化修饰能力的新型酵母系统。利用抗CD20或抗Her抗体为模型蛋白，在细胞水平、动物水平检测具有不同糖基化（甘露糖型、杂合型、复杂型糖基）修饰的抗体活性，如ADCC和CDC，建立基于糖基工程酵母的抗体高效表达、规模化发酵和产品制备技术，研究并发现不同的N-糖基结构对抗体蛋白活性的影响，为研制高ADCC或CDC活性的新型抗体提供技术与理论基础。

酵母已成为最为成熟的表达系统之一，现有各种糖蛋白表达系统对糖基的修饰过程都是不可控的，无法详细研究特定结构糖基对糖蛋白的影响。酵母原有的过度甘露糖修饰限制了其在糖蛋白药物领域中的应用。本课题通过对酵母N-糖基修饰系统的研究和改造，在酵母中阻断其原有的高甘露糖型糖基合成途径，并构建了具有类似于哺乳动物细胞的复杂型糖基合成途径，使其糖基化修饰更接近、甚至更优于天然糖基结构，工程酵母菌株分别具有哺乳动物细胞杂合型GlcNAcMan3GlcNAc2、复杂型糖基结构GlcNAc2Man3GlcNAc2和Gal2GlcNAc2Man3GlcNAc2合成能力。基于糖基工程酵母表达系统，分别构建了抗Her2单克隆抗体、抗腺病毒单克隆抗体、抗埃博拉病毒单克隆抗体的高效表达酵母菌株，制备了抗体蛋白，以哺乳动物表达的抗体蛋白为对照，通过细胞和动物实验，发现糖基工程酵母表达的单克隆抗体和哺乳动物细胞表达的抗体在药物代谢动力学和药效学方面表现基本一致，这将为酵母成为抗体药物、基因工程糖蛋白疫苗产业化的新平台提供基础。