Engineering antibody effector functions by Glycan Remodeling Yeast Display

抗体构成了正在被施用用于治疗日益增加的免疫缺陷综合征的日益增长的一类药物。 一系列人类疾病，包括但不限于自身免疫、感染和癌症。虽然工程 识别几乎任何抗原的抗体在技术上已经变得简单， 诱导不同的免疫信号或效应子功能，指导体内细胞的杀伤， 挑战性后一种性质是由抗体的Fc区实现的，并且工程化的困难在于， 在临床上，抗体Fc区的这种差异是由于存在与Asn 297连接的保守的N-连接聚糖。 相关IgG抗体。下一代的免疫抗体，以及我们识别 更好地理解抗体介导的杀伤机制，取决于我们设计IgG Fc结构域的能力 以改变的亲和力和特异性与Fc γ受体（Fcγ R）结合，包括活化和抑制 受体和补体以定制抗体介导的效应子功能。Fc的主要障碍 工程的一个问题是目前没有执行定向进化的方法（即，组合 诱变和选择），同时维持和/或控制聚糖 它们与Fcγ R和补体相互作用所需的化学。我们结合了两种成熟的技术 - 糖蛋白的化学酶促合成（即，糖基化修饰酶和化学品的使用 聚糖合成）和传统酵母展示定向进化-创造一种新的工程方法 糖基化Fc结构域，我们称之为聚糖重塑酵母展示或GRYD。在GRYD中，IgG Fc文库 结构域蛋白展示在酵母细胞表面，在那里它们被高甘露糖聚糖修饰 酵母自然产生的。然后我们使用化学酶合成来重塑Asn 297连接的聚糖， 而仍然在酵母细胞表面上，转化为复合型聚糖，代表人抗体上的那些。 最后，使用荧光标记的FcγR四聚体，我们选择表达Fc结构域变体的酵母细胞， 通过荧光激活细胞分选（FACS）检测到更高的FcγR结合。通过引入化学酶促合成 为了重塑酵母细胞表面上的Fc聚糖，我们不仅产生了一个适当糖基化的 Fc结构域变体，从中选择所需的性质，但我们保持了Fc结构域之间的联系， 基因和它们在同一个细胞中编码的蛋白质-定向进化的关键要求。抗体 使用GRYD技术创建的免疫系统可以构成一套新的工具， 用于操作和评估整个目录的体内抗体介导的杀伤机制， 抗体，无论是目前可用的，并在未来开发。免疫学抗体具有 与此同时，使用GRYD进行工程改造，可以形成全新一代基于抗体的 毒品在拟议的原理验证研究中，我们将优化和实施GRYD技术，以亲和- 成熟无岩藻糖基化（Aim 1）和岩藻糖基化（Aim 2）IgG 1 Fc与活化受体FcγRIIIA结合。

Antibodies constitute a growing class of drugs that are being administered for the treatment of an increasing range of human diseases, including but not limited to autoimmunity, infection and cancer. While engineering antibodies to recognize virtually any antigen has become technologically straightforward, engineering antibodies to induce distinct immune signals, or effector functions, which direct the killing of cells in vivo, remains technically challenging. This latter property is carried out by the Fc region of antibodies and the difficulty in engineering antibody Fc regions is due to the presence of a conserved N-linked glycan attached to Asn297 in clinically- relevant IgG antibodies. The next generation of immunotherapeutic antibodies, as well as our abilities to identify and better understand antibody-mediated killing mechanisms, depends on our ability to engineer IgG Fc domains to bind with altered affinities and specificities to Fc γ receptors (FcγRs), including both activating and inhibitory receptors, and complement in order to customize antibody-mediated effector functions. The major barrier to Fc engineering is that there are currently no methods by which to perform directed evolution (i.e., combinatorial mutagenesis and selection) of glycoproteins, such as Fc domains, while maintaining and/or controlling the glycan chemistry required for their interactions with FcγRs and complement. We combined two established technologies – chemoenzymatic synthesis of glycoproteins (i.e., the use of glycosylation-modifying enzymes and chemical synthesis of glycans) and traditional yeast display directed evolution – to create a novel method for engineering glycosylated Fc domains that we call Glycan Remodeling Yeast Display, or GRYD. In GRYD, a library of IgG Fc domain proteins is displayed on the yeast cell surface, where they are decorated with the high mannose glycans that yeast naturally produce. We then use chemoenzymatic synthesis to remodel the Asn297-linked glycans, while still on the yeast cell surface, to complex type glycans, representative of those on human antibodies. Finally, using a fluorescently-labeled FcγR tetramer, we select yeast cells expressing Fc domain variants with higher FcγR binding by fluorescence-activated cell sorting (FACS). By introducing a chemoenzymatic synthesis step to remodel the Fc glycans on the yeast cell surface, we not only produce a library of properly glycosylated Fc domain variants from which to select for desirable properties, but we maintain the link between the Fc domain genes and the proteins that they encode in the same cell – the key requirement of directed evolution. Antibodies created using the GRYD technology could constitute a novel set of tools that the immunological community can use to manipulate and evaluate the in vivo antibody-mediated killing mechanisms of the entire catalog of antibodies, both currently available and to be developed in the future. Immunotherapeutic antibodies that have been engineered using GRYD, meanwhile, could form the basis of an entirely new generation of antibody-based drugs. In the proposed proof-of-principle studies we will optimize and implement the GRYD technology to affinity- mature afucosylated (Aim 1) and fucosylated (Aim 2) IgG1 Fc to the activating receptor FcγRIIIA.