Towards one-step enzymatic defucosylation of antibodies

抗体的一步酶促去岩藻糖基化

• 批准号: 10041315
• 负责人: ERIC JOHN SUNDBERG
• 金额: $ 19.5万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041315
• 关键词: Address; Affinity; Antibodies; Antigens; Autoimmunity; Binding; Catalogs; Cells; Chemical Structure; Cleaved cell; Clinical; Communities; Complement; Custom; Directed Molecular Evolution; Disease; Engineering; Enzymatic Biochemistry; Enzymes; Excision; Family; Fc domain; Fucose; Fucosidase; Funding Opportunities; Future; Glycoside Hydrolases; Goals; Health; Homo; Human; Immune signaling; Immune system; Immunoglobulin Constant Region; Immunoglobulin G; Immunologic Receptors; Immunologics; Immunologist; Immunotherapeutic agent; In Vitro; Infection; Inflammatory; Knowledge; Libraries; Link; Malignant Neoplasms; Mediating; Methods; Molecular; Molecular Conformation; Pharmaceutical Preparations; Pharmacotherapy; Polysaccharides; Property; Reaction; Role; Structure; Therapeutic; United States National Institutes of Health; Variant; antibody engineering; base; cancer therapy; cell killing; clinical efficacy; clinically relevant; design; experimental study; human disease; in vivo; macromolecule; next generation; novel; receptor; recruit; response; sugar; tool; tool development; virtual

Antibodies constitute a growing class of drugs that are being administered for 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 technologically 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 understand antibody-mediated killing mechanisms, depends on our ability to rationally modify the chemical structure of this Asn297-linked glycan. The most important molecular feature of this glycan is a fucose sugar unit connected through an α(1,6) linkage to the Asn-proximal N-actylglucosamine (GlcNac), the absence of which imparts Fc domains with increased binding affinity to an activating FcγR, FcγR3A, resulting in substantially increased antibody-mediated in vivo cellular killing. Based on our preliminary studies of AlfC, an α(1,6)- fucosidase that removes fucose from Asn297-linked glycans on IgG antibodies, but only after all of the branched sugar units beyond the GlcNac to which it is linked have been removed, we propose to develop α(1,6)-fucosidase variants that can rapidly, reliably and entirely remove the fucose sugar unit on any antibody, regardless of the branched structure of the Asn297-linked glycan. Such an enzymatic tool could be used by the immunological community to evaluate the in vivo antibody-mediated killing mechanisms of the entire catalog of antibodies, both currently available and to be developed in the future. In this proposal, we will address two Specific Aims: (i) to define the molecular basis of antibody defucosylation by α(1-6)-fucosidases; and (ii) to design α(1,6)-fucosidase variants active on antibodies bearing fully branched glycans. Progress towards these complementary, yet independent, Specific Aims will significantly advance our understanding of glycan-modifying enzymes. Leveraging this knowledge in the context of AlfC and related α(1-6)-fucosidases will enhance our ability to customize antibodies, providing the tools with which immunologists can better understand antibody-mediated in vivo cellular killing, as well as further unleashing their vast therapeutic utility and expanding their positive impact on human health.

抗体构成了越来越多的药物类别，用于治疗范围越来越广 人类疾病，包括但不限于自身免疫、感染和癌症。在设计抗体时 识别几乎任何抗原在技术上已经变得简单，设计抗体来诱导 指导杀死体内细胞的不同免疫信号或效应器功能在技术上仍然存在 具有挑战性的。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 并了解抗体介导的杀伤机制，取决于我们合理修改化学物质的能力 该 Asn297 连接聚糖的结构。这种聚糖最重要的分子特征是岩藻糖单元 通过 α(1,6) 连接与 Asn 近端 N-乙酰葡萄糖胺 (GlcNac) 相连，缺少该键 赋予 Fc 结构域对活化 FcγR、FcγR3A 的结合亲和力增加，从而基本上 增加抗体介导的体内细胞杀伤。根据我们对 AlfC 的初步研究，α(1,6)- 岩藻糖苷酶可从 IgG 抗体上的 Asn297 连接的聚糖中去除岩藻糖，但仅在所有支链之后 GlcNac 之外的糖单元已被去除，我们建议开发 α(1,6)-岩藻糖苷酶 可以快速、可靠和完全去除任何抗体上的岩藻糖单元的变体，无论其 Asn297 连接聚糖的支链结构。这种酶促工具可以被免疫学使用 社区评估整个抗体目录的体内抗体介导的杀伤机制，两者 目前可用并在未来开发。在本提案中，我们将解决两个具体目标：(i) 定义 α(1-6)-岩藻糖苷酶对抗体去岩藻糖基化的分子基础； (ii) 设计 α(1,6)-岩藻糖苷酶 对带有全支链聚糖的抗体有活性的变体。在这些互补性方面取得进展，但 独立的、具体的目标将显着增进我们对聚糖修饰酶的理解。 在 AlfC 和相关 α(1-6)-岩藻糖苷酶的背景下利用这些知识将增强我们的能力 定制抗体，为免疫学家提供了更好地了解抗体介导的工具 体内细胞杀伤，以及进一步释放其巨大的治疗效用并扩大其积极影响 对人类健康。