Synthesis and Function of Antibody Fc Domain Glycoforms

描述（由申请人提供）：免疫球蛋白G （IgG）型单克隆抗体（mab）是一类重要的治疗糖蛋白。令人信服的证据表明，Fc结构域保守的n-糖基化位点（Asn-297）上的聚糖的精细结构负责单克隆抗体的不同效应功能，包括抗体依赖性细胞毒性（ADCC），补体依赖性细胞毒性（CDC）和凋亡活化。此外，一种特殊的唾液化Fc糖型被确定为静脉注射免疫球蛋白（IVIG）的抗炎活性。然而，了解IgG-Fc糖基化的功能作用的进展受到Fc结构域聚糖巨大的结构异质性的阻碍。此外，控制单克隆抗体的糖基化表达到所需的均匀糖型仍然是一项具有挑战性的任务。在这个应用中，我们建议探索一种化学酶的方法来建立一个均匀糖基化的IgG-Fc和选择的单克隆抗体的糖型库。通过Fc受体结合研究，我们旨在了解不同聚糖结构如何微调IgG和IgG-Fc蛋白的效应功能。我们已经进行了重要的初步研究，表明使用基于内糖苷酶的转糖基化方法构建明确的、均匀的人IgG-Fc糖型是可行的。在这一成功的基础上，我们建议实现三个具体目标。目的1是探索一种化学酶法构建IgG-Fc的各种纯糖型。目的2是评估不同Fc结构域糖型在Fc受体结合中的构效关系，并评估选择性糖工程单克隆抗体的ADCC活性。目的3是合成新的Fc结构域糖型，以评价IgG-Fc糖基化在抗炎活性中的作用。从拟议的研究中获得的知识将最终促进单克隆抗体和IgG-Fc蛋白的新型糖型的发展，作为有效的治疗方法。

DESCRIPTION (provided by applicant): Monoclonal antibodies (MAbs) of the immunoglobulin G (IgG) type are an important class of therapeutic glycoproteins. Compelling evidence has indicated that the fine structures of the glycans at the conserved N-glycosylation site (Asn-297) of the Fc domain are responsible for the distinct effector functions of MAbs, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and activation of apoptosis. In addition, a special sialylated Fc glycoform was identified to be responsible for the antiinflammatory activity of intravenous immunoglobulin (IVIG). However, progress in understanding the functional roles of IgG-Fc glycosylation is hampered by the tremendous structural heterogeneity of Fc domain glycans. In addition, controlling glycosylation of MAbs in expression to a desired homogeneous glycoform is still a challenging task. In this application, we propose to explore a chemoenzymatic method to make a library of homogeneously glycosylated IgG-Fc and selected glycoforms of MAbs. Through performing Fc receptor binding studies, we aim to understand how different glycan structures can fine tune the effector functions of IgG and IgG-Fc protein. We have performed important preliminary studies indicating that it is feasible to use the endoglycosidase-based transglycosylation approach to construct defined, homogeneous glycoforms of human IgG-Fc. Building on this success, we propose to pursue three specific aims. Aim 1 is to explore a chemoenzymatic method for the construction of various pure glycoforms of IgG-Fc. Aim 2 is to evaluate the structure-activity realtionships of different Fc domain glycoforms in Fc receptor binding, and to evaluate ADCC activity of selectively glycoengineered monoclonal antibodies. Aim 3 is to synthesize novel Fc domain glycoforms for evaluating the roles of IgG-Fc glycosylation in anti-inflammatory activity. The knowledge gained from the proposed research will eventually facilitate the development of novel glycoforms of MAbs and IgG-Fc proteins as effective therapeutics.