大肠埃希菌中功能性糖表位修饰重组抗体及其作用分析

组织穿透性和血浆半衰期是决定组织靶向类抗体治疗效果的两个重要因素。重组小分子抗体相对全抗体组织穿透性有所提高，但是其血浆半衰期非常短，限制了其在临床上的应用，需要修饰后提高其血浆半衰期和组织穿透性后才能应用。基于白细胞通过其细胞表面上的唾液酸路易斯糖表位(sLeX）与血管内皮细胞上的选择素作用介导其高效穿越血管内皮细胞机制，以及唾液酸修饰重组抗体可以显著提高其血浆半衰期的事实，本项目将在大肠埃希菌中生产sLeX修饰的新型重组抗体。本项目以流感嗜血杆菌寡糖合成机制、空肠弯曲菌寡糖转移酶、翻转酶、唾液酸合成酶及其他原核生物来源的唾液酸转移酶、岩藻糖基转移酶等基因，在大肠埃希菌中构建sLeX高效修饰重组抗体代谢途径的菌株；分析sLeX糖表位修饰重组抗体组织穿透性、血浆半衰期等，探索高效低成本生产具有高效组织穿透性和较长血浆半衰期sLeX糖表位修饰的新型重组抗体新途径，推动重组抗体在临床上的应用。

Tissue penetration and plasma half-life are important factors that determine the therapeutic efficacy of tissue-targeted antibodies. Although recombinant antibody fragments, such as Fabs and scFvs, can achieve greater tissue penetration than whole antibody molecules due to their smaller size, their lack of constant regions and of glycosylation motifs limits their plasma half-life, which in turn restricts their clinical application..Selectins are carbohydrate-binding proteins that recognize the sialyl Lewis X structure (sLeX) and mediate the rolling adhesion of cells, such as leukocytes, along vascular surfaces, leading to leukocyte arrest and migratation through the endothelium. Meanwhile, recombinant antibodies modified with terminally sialylated glycans exhibit significantly increased plasma half-lives. Based on these facts, we propose that recombinant antibodies modified with sLeX carbohydrate could combine the advantages of high tissue penetration and increased plasma half-life for improved therapeutic effects..In this project, we will engineer a pathway in Escherichia coli to produce sLeX glycan and N-glycosylated recombinant antibodies. This entails: 1. cloning glycosyltransferases from Haemophilus influenza, as well as NeuBCA, flippase and oligosaccharyltransferase from Campylobacter jejuni, and alpha2,3-sialyltransferase, alpha-(1,3)-fucosyltransferase from different prokaryotes to engineer a pathway in E. coli to efficiently produce recombinant antibodies N-glycosylated with sLeX glycan; 2. investigation of the physicochemical properties of recombinant antibodies modified with the sLeX epitope. We propose in this way to produce in E. coli a novel, reshaped antibody fragment, containing a functional carbohydrate epitope, which combines high tissue penetration and long plasma half-life for improved therapeutic effects.