Engineering antibody effector functions by Glycan Remodeling Yeast Display

抗体构成了越来越多的药物类别，用于治疗越来越多的疾病 一系列人类疾病，包括但不限于自身免疫、感染和癌症。在工程的同时 识别几乎任何抗原的抗体已变得技术简单，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，在技术上仍然存在 具有挑战性。后一种特性是由抗体的 Fc 区和工程难度来实现的 抗体 Fc 区是由于临床上存在与 Asn297 相连的保守 N 连接聚糖 相关 IgG 抗体。下一代免疫治疗抗体，以及我们识别的能力 并更好地了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 (FcγR) 结合，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。 Fc的主要障碍 工程的缺点是目前没有方法可以执行定向进化（即组合 糖蛋白（例如 Fc 结构域）的诱变和选择，同时维持和/或控制聚糖 它们与 FcγR 和补体相互作用所需的化学反应。我们结合了两种成熟的技术 – 糖蛋白的化学酶合成（即使用糖基化修饰酶和化学合成 聚糖的合成）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，我们称为聚糖重塑酵母展示 (Glycan Remodeling Yeast Display)，或 GRYD。在 GRYD 中，IgG Fc 文库 结构域蛋白展示在酵母细胞表面，并用高甘露糖聚糖装饰 酵母自然产生的。然后我们使用化学酶合成来重塑 Asn297 连接的聚糖， 仍在酵母细胞表面上，形成复合型聚糖，代表人类抗体上的聚糖。 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) 获得更高的 FcγR 结合。通过引入化学酶合成 一步重塑酵母细胞表面的 Fc 聚糖，我们不仅产生了正确糖基化的文库 Fc 结构域变体，从中选择所需的特性，但我们保持 Fc 结构域之间的链接 基因和它们在同一细胞中编码的蛋白质——定向进化的关键要求。抗体 使用 GRYD 技术创建的可以构成免疫学界可以使用的一套新颖的工具 用于操纵和评估整个目录的体内抗体介导的杀伤机制 目前可用的和未来将开发的抗体。免疫治疗抗体具有 同时，使用 GRYD 进行设计可以构成全新一代基于抗体的基础 药物。在拟议的原理验证研究中，我们将优化和实施 GRYD 技术以亲和力 成熟的无岩藻糖基化 (Aim 1) 和岩藻糖基化 (Aim 2) IgG1 Fc 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.