Synthesis and Function of Antibody Fc Domain Glycoforms

描述（由申请方提供）：免疫球蛋白G（IgG）型单克隆抗体（MAb）是一类重要的治疗性糖蛋白。令人信服的证据表明，Fc结构域保守N-糖基化位点（Asn-297）处聚糖的精细结构负责MAb的独特效应子功能，包括抗体依赖性细胞毒性（ADCC）、补体依赖性细胞毒性（CDC）和细胞凋亡激活。此外，一种特殊的唾液酸化的Fc糖型被鉴定为负责静脉内免疫球蛋白（IVIG）的免疫活性。然而，在理解IgG-Fc糖基化的功能作用方面的进展受到Fc结构域聚糖的巨大结构异质性的阻碍。此外，控制MAb在表达中的糖基化为期望的同质糖型仍然是一项具有挑战性的任务。在本申请中，我们提出探索化学酶促方法来制备均一糖基化IgG-Fc和选择的单克隆抗体糖型的文库。通过进行Fc受体结合研究，我们旨在了解不同的聚糖结构如何微调IgG和IgG-Fc蛋白的效应子功能。我们已经进行了重要的初步研究，表明使用基于内切糖基化酶的转糖基化方法来构建人IgG-Fc的确定的、均一的糖型是可行的。在这一成功的基础上，我们建议实现三个具体目标。目的1：探索一种化学酶法构建各种纯IgG Fc糖型的方法。目的二是评价不同Fc结构域糖型在Fc受体结合中的构效关系，并评价选择性糖基化单克隆抗体的ADCC活性。目的3：合成新型的Fc结构域糖型，以评价IgG-Fc糖基化在抗炎活性中的作用。从所提出的研究中获得的知识将最终促进MAb和IgG-Fc蛋白的新型糖型作为有效治疗剂的开发。 公共卫生相关性：IgG抗体是一类重要的治疗性糖蛋白。Fc结构域糖基化对于抗体的效应子功能（包括ADCC和抗炎活性）是必不可少的。该研究旨在通过糖基化工程和Fc受体结合研究来破译Fc糖基化的功能作用，这可能导致发现具有有效治疗功效的新型抗体糖型。

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. PUBLIC HEALTH RELEVANCE: IgG antibodies are an important class of therapeutic glycoproteins. The Fc domain glycosylation is essential for antibody's effector functions including ADCC and anti-inflammatory activity. The proposed research aims to decipher the functional roles of Fc glycosylation through glycosylation engineering and Fc receptor binding studies, which may lead to the discovery of novel antibody glycoforms with potent therapeutic efficacy.