Synthesis and Function of Antibody Fc Domain Glycoforms

项目总结/摘要 抗体是一类重要的糖蛋白。令人信服的实验数据表明 IgG型抗体中高度保守的Fc聚糖的精细结构可以具有 对抗体的生物学功能有深远的影响，在治疗性抗体的情况下， 临床疗效，如抗体依赖性细胞毒性（ADCC），补体- 依赖性细胞毒性（CDC）、细胞凋亡激活和抗炎活性。更 最近，据报道，在IgE-Fc结构域处的单个寡甘露糖型聚糖被 负责与肥大细胞上的IgE受体的高亲和力结合，并对启动 过敏反应然而，了解抗体功能作用的进展 Fc糖基化的巨大结构异质性阻碍了糖基化。 糖工程的遗传学方法已经取得了一些成功，但抗体 可以通过遗传方法产生的糖型是有限的。目前，没有将军。 一种可用于控制糖基化为预定的均质形式的方法， 重组抗体生产。在本申请中，我们提出探索一种化学酶促 我们最近开发了一种制备各种均一IgG和IgE糖型的方法。 通过进行Fc受体结合研究，我们旨在了解不同的聚糖 结构调节IgG和IgE的效应子功能，并应用所获得的知识 来开发更好的治疗方法为实现这一目标，提出了三个具体目标。要求1 目的是进一步探索抗体糖基化的化学酶促方法， 酶结构，寻找新的酶和突变体，并评估新的聚糖 印刷受体.目的二是建立一种通用的位点特异性接合方法， 均匀的抗体-药物缀合物。功能标签将被引入Fc聚糖位点- 并且毒素将化学选择性地连接至Fc聚糖以提供 均相ADC。目的3是进行IgE Fc的位点特异性糖基化重塑， 探索糖基化在过敏反应中的作用。一个成功的追求所提出的 这项研究可能导致产生新的IgG和IgE糖型， 特性.化学酶法可能成为一种通用的定点酶解平台技术。 特异性抗体-药物缀合和标记。IgE Fc糖基化的研究，如果 成功，将填补我们在糖免疫学与过敏反应相关的知识空白， 疾病

Project Summary/Abstract Antibodies are an important class of glycoproteins. Compelling experimental data have shown that the fine structures of the highly conserved Fc glycans in IgG type antibodies can have profound effects on the antibody’s biological functions and, in the case of therapeutic antibodies, the clinical efficacy, such as antibody-dependent cellular cytotoxicity (ADCC), complement- dependent cytotoxicity (CDC), activation of apoptosis, and anti-inflammatory activities. More recently, it was reported that a single oligomannose type glycan at the IgE-Fc domain was responsible for high-affinity binding to IgE receptor on mast cells and was critical for initiation of allergic reactions. However, progress in understanding the functional roles of antibody glycosylation is hampered by the tremendous structural heterogeneity of Fc glycosylation. Gnetic approach to glycoengineering has achieved some successes but the antibody glycoforms that can be generated by genetic approach are limited. Currently there is no general method avaiable to control glycosylation to a pre-defined homogeneous form during recombinant antibody production. In this application, we propose to explore a chemoenzymatic method that we recently developed to make various homogeneous IgG and IgE glycoforms. Through performing Fc receptor binding studies, we aim to understand how different glycan structures modulate the effector functions of IgG and IgE and to apply the knowledge obtained for developng better therapeutics. Three specific aims are proposed to achieve the goal. Aim 1 is to further explore the chemoenzymatic method for antibody glycoengineering by investigating enzyme structures, searching for new enzymes and mutants, and evaluating new glycan substrates. Aim 2 is to establish a general site-specific conjugation method for constructing homogeneous antibody-drug conjugates. Functional tags will be introduced to Fc glycan site- specifically and a toxin will be chemoselectively ligated to the Fc glycan to provide homogeneous ADCs. Aim 3 is to perform site-specific glycosylation remodeling of IgE Fc for probing the glycosylation functions in allergic reactions. A successful pursuit of the proposed research could lead to the creation of new IgG and IgE glycoforms with novel biological properties. The chemoenzymatic method may become a general platform technology for site- specific antibody-drug conjugation and labeling. The study on IgE Fc glycosylation, if successful, will fill a gap of our knowledge in glyco-immunology related to allergic reactions and diseases.