α-L-岩藻糖苷酶的Loop环改造用于抗体药物去岩藻糖研究

The pharmacy market has witnessed the fast growing of therapeutic antibodies in the past decades, which have been widely used for the treatment of cancers and various diseases nowadays. The removal of α1,6-fucose on N-glycan of Fc fragment (referred to as “core fucose”) increases the cell-mediated cytotoxicity of antibodies dozens of times, making the defucosylation of antibodies a research hotspot. However, all known α-L-fucosidases are unable to remove core fucose of antibodies directly, thus greatly hinders the development of afucosylated therapeutic antibodies. α-L-Fucosidases AlfC, BfFucH, Eo0918 and FucO showed great potential for glyco-engineering of antibodies. Thus, with the aim to eliminate the space hindrance between α-L-fucosidases and antibody substrate, we plan to engineer surrounding loops of the catalytic cave which might related to the space hindrance: (i) Comparative study of the substrate specificity of four α-L-fucosidases towards N-glycopetides and antibodies with different N-glycoform; (ii) substitution of six surrounding loops individually by the shortest sequence of the corresponding loop among four α-L-fucosidases; (iii) engineering of loops of α-L-fucosidase which possesses the most broad substrate specificity by substituting loops that affect substrate reorganization in combination, and deletion, replacing or adding amino acid, to expand the space around the catalytic cave further. Finally we will test defucosylation ability of the best mutant α-L-fucosidases towards therapeutic antibodies. Those researches provide deeper view of the structure and function of glycosidase, especially on the role of loops on the substrate specificity of α-L-fucosidases. It will also improve the development of afucoslyated therapeutic antibodies.

抗体药物是目前药物市场增长最快的生物药，广泛用于癌症等多种疾病的治疗。去除Fc片段N-糖链核心岩藻糖能几十倍提高抗体依赖细胞毒作用，成为新一代抗体药物研发热点。但现有α-L-岩藻糖苷酶均不能直接去除该糖基，严重制约了去岩藻糖化糖工程抗体发展。AlfC、BfFucH、Eo0918和FucO为目前最具抗体糖基化改造潜力的α-岩藻糖苷酶，拟针对酶与抗体底物空间阻碍，对反应腔口与空间位阻相关loop环分步进行改造：1)四种酶对N-糖肽和不同N-糖型抗体水解底物特异性的比较；2)六个loop环单独置换为相应最短序列，研究其对抗体底物结合影响；3)最优α-岩藻糖苷酶中影响底物结合loop环的组合置换及氨基酸删减、替换及添加，进一步拓宽酶反应空间，并检测去抗体药物核心岩藻糖基的活性。该研究将深入解析糖苷酶结构与功能的关系，尤其是loop环对底物特异性影响；同时将推进新一代去岩藻糖化糖工程抗体研发及应用。

抗体Fc片段N-糖链核心岩藻糖基的有效去除对于抗体药物的糖基化改造和非岩藻糖基化抗体的研发与应用具有重要的意义，但现有α-L-岩藻糖苷酶对该核心岩藻糖基均表现出较低的水解效率或无活性。为了探究α-L-岩藻糖苷酶结构与活性的关系，并为抗体糖基化改造寻找更有效的工具酶，本项目从多种α-L-岩藻糖苷的异源表达和活性检测、α-L-岩藻糖苷工具酶AlfC和BfFucH的Loop环改造、以及融合蛋白的构建三个方面开展了研究。(1) α-L-岩藻糖苷的表达显示细菌来源的Eo0918和FucoO及黑曲霉来源的AnFuc-1均表达困难，并且水解活性较弱；而AlfC和BfFucH表现出较高的表达效率和水解活性。(2) Loop环置换中，将AlfC和BfFucH催化腔口的两个Loop环分别置换为Eo0918相应较短的Loop环，结果显示多数Loop环单置换和双置换突变酶均表达效率下降或水解活性降低，只有BfFucH的双置换有较好的表达，但水解活性损失较大。(3) 内切β-N-乙酰葡糖苷酶EndoS和EndoS2的非催化域推测参与到两酶对抗体底物的识别中，为此将AlfC与EndoS和EndoS2非催化域融合，构建了不同linker连接的6个融合蛋白。活性检测显示6个融合蛋白均对去除外侧糖链的抗体底物表现出显著高于原始AlfC的去岩藻糖基活性，水解动力学研究表明融合蛋白AlfC-EndoS-2和AlfC-EndoS2-2均具高出AlfC原始酶3.7倍以上的kcat。在化学酶法抗体糖基化改造中，AlfC-EndoS2-2与EndoS共同作用可以将原需多小时甚至数天的去糖基化反应缩短至10分钟，极大的提高了改造效率。这些研究结果拓展了α-L-岩藻糖苷酶结构和功能关系的认识，并为抗体药物的N-糖基化改造提供了更有效的工具酶。