Synthesis and Function of Antibody Fc Domain Glycoforms

项目摘要/摘要 抗体是一类重要的糖蛋白。令人信服的实验数据表明 在IgG型抗体中高度保守的Fc多糖的精细结构 对抗体的生物学功能产生深远影响，在治疗性抗体的情况下， 临床疗效，如抗体依赖的细胞毒性(ADCC)，补体- 依赖细胞毒性(CDC)、激活细胞凋亡和抗炎活性。更多 最近，据报道，在IgE-Fc结构域上有一个单一的寡注型糖链。 负责与肥大细胞上的IgE受体的高亲和力结合，是启动 过敏反应。然而，了解抗体的功能作用的进展 Fc糖基化的巨大结构异质性阻碍了糖基化。 基因工程的方法已经取得了一些成功，但抗体 通过遗传方法可以产生的糖型是有限的。目前还没有一般的 一种可将糖基化控制到预定均一形式的方法 重组抗体的生产。在这个应用中，我们建议探索一种化学酶 我们最近开发了一种制备各种均一的Ig G和Ig E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糖链 结构调节免疫球蛋白和免疫球蛋白E的效应器功能 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。目标1 通过研究进一步探索抗体糖工程的化学酶方法 酶结构，寻找新的酶和突变体，并评估新的多糖 底物。目的2是建立一种通用的位点特异性结合方法，用于构建 均一抗体-药物结合物。功能标签将被引入Fc多糖网站- 具体地说，毒素将被化学选择性地连接到Fc多糖上，以提供 同质ADC。目的3是对IgE Fc进行位点特异性糖基化重塑 探讨糖基化在变态反应中的作用。对提议的成功追求 研究可能导致产生具有新生物学特性的新的免疫球蛋白和免疫球蛋白E糖体 属性。化学酶法有可能成为现场检测的通用平台技术。 特异性抗体-药物偶联和标记。免疫球蛋白Fc糖基化的研究 成功，将填补我们在与过敏反应和糖免疫学相关的知识空白 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.