Synthesis and Function of Antibody Fc Domain Glycoforms

说明(申请人提供)：免疫球蛋白G(Ig G)型的单抗是一类重要的治疗性糖蛋白。有证据表明，Fc结构域保守的N-糖基化位点(ASN-297)上的糖链的精细结构是单抗具有独特效应功能的原因，包括抗体依赖的细胞毒性(ADCC)、补体依赖的细胞毒(CDC)和激活细胞凋亡。此外，一种特殊的唾液酸化Fc糖体被确定为静脉注射免疫球蛋白(IVIG)的抗炎活性的原因。然而，Fc结构域的巨大异质性阻碍了对Ig G-Fc糖基化功能作用的理解。此外，控制表达的单抗的糖基化为所需的均一糖形式仍然是一项具有挑战性的任务。在这一应用中，我们建议探索一种化学酶方法来建立均一糖基化的Ig G-Fc和选定的单抗糖型的文库。通过进行Fc受体结合研究，我们旨在了解不同的糖链结构如何微调Ig G和Ig G-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来构建确定的、均一的人免疫球蛋白Fc糖形式是可行的。在这一成功的基础上，我们建议追求三个具体目标。目的1探索一种化学酶法制备各种纯糖形式的Ig G-Fc。目的2评价不同Fc结构域糖形式与Fc受体结合的构效关系，并评价选择性糖工程单抗的ADCC活性。目的3合成新的Fc结构域糖形式，用于评价Ig G-Fc糖基化在抗炎中的作用。从拟议的研究中获得的知识最终将有助于开发新的单抗和Ig G-Fc蛋白糖型作为有效的治疗药物。 公共卫生相关性：免疫球蛋白抗体是一类重要的治疗性糖蛋白。Fc结构域的糖基化对抗体的效应功能包括ADCC和抗炎活性是必不可少的。这项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，这可能会导致发现具有强大治疗效果的新的抗体糖体。

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.