Engineering antibody effector functions by Glycan Remodeling Yeast Display

抗体构成了越来越多的药物，这些药物正在被用于治疗日益增长的 一系列人类疾病，包括但不限于自身免疫、感染和癌症。在工程设计中 识别几乎任何抗原的抗体在技术上变得简单，即工程抗体 诱导不同的免疫信号或效应器功能，指导体内细胞的杀伤，从技术上讲仍然是 很有挑战性。后一种特性是通过抗体的Fc区域和工程上的困难来实现的 抗体Fc区是由于在临床上存在与Asn297连接的保守的N-连接的糖链所致。 相关免疫球蛋白抗体。下一代免疫治疗性抗体，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Ig G Fc结构域的能力 结合Fcγ受体(FcγR)的亲和力和特异性的改变，包括激活和抑制 受体和补体，以定制抗体介导的效应器功能。影响FC的主要障碍 工程学是指目前没有执行定向进化(即，组合进化)的方法 突变和选择)糖蛋白，例如Fc结构域，同时维持和/或控制多糖 它们与Fc、γ、Rs和补体相互作用所需的化学物质。我们结合了两项成熟的技术 -化学酶法合成糖蛋白(即使用糖基化修饰酶和化学试剂 糖链合成)和传统酵母展示定向进化--创造一种新的工程方法 糖基化的Fc结构域，我们称之为糖链重塑酵母展示，或GRYD。在GRYD中，Ig G Fc的文库 结构域蛋白显示在酵母细胞表面，在那里它们被高甘露糖聚糖装饰 酵母菌自然产生。然后我们使用化学酶合成来重塑Asn297连接的多糖， 同时还在酵母细胞表面，以复合型多聚糖为代表的那些对人体产生抗体。 最后，使用荧光标记的FcγR四聚体，我们选择了表达Fc结构域变体的酵母细胞 通过荧光激活细胞分选(FAC)获得更高的FcγR结合率。通过引入一种化学酶合成 步骤重塑酵母细胞表面的Fc糖链，我们不仅产生了一个正确糖基化的文库 FC域变体，从中选择所需的属性，但我们维护FC域之间的链接 基因和它们在同一细胞中编码的蛋白质--这是定向进化的关键要求。抗体 使用GRYD技术创建的可能构成一套新的工具，免疫学社区可以 用来操纵和评估体内抗体介导的整个目录的杀伤机制 抗体，既有目前可用的，也有未来开发的。免疫治疗性抗体具有 同时，使用GRYD进行工程设计，可以形成全新一代基于抗体的基础 毒品。在拟议的原则证明研究中，我们将优化和实施GRYD技术，以亲和力- 成熟的果糖基化(目标1)和岩藻糖基化(目标2)的IgG1Fc与激活受体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.