Towards one-step enzymatic defucosylation of antibodies

抗体的一步酶促去岩藻糖基化

• 批准号: 10176408
• 负责人: ERIC JOHN SUNDBERG
• 金额: $ 23.4万
• 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/10176408
• 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.

抗体构成了一种正在施用的药物，用于治疗增加范围 人类疾病，包括但不限于自身免疫，感染和癌症。在工程抗体的同时 识别几乎任何抗原在技术上已变得直接，工程抗体会影响 独特的免疫信号或效应子功能，该功能指导体内杀死细胞的杀死在技术上保持 具有挑战性的。此后的属性由FC抗体区域和工程难度进行 抗体FC区域是由于在临床上存在于ASN297附着在ASN297上的保守的N-连接的聚糖所致 相关的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戮机制，取决于我们合理修改化学物质的能力 该ASN297连接的聚糖的结构。该聚糖最重要的分子特征是Fucose糖单元 通过α（1,6）连接到ASN-Proximal n-肌氨基氨基胺（GlcNAC），没有哪个 授予FC域具有增加与激活FcγRFcγR3A的结合亲和力增加，从而大大导致 抗体介导的体内细胞杀死增加。基于我们对Alfc的初步研究，α（1,6） - 从IgG抗体上从ASN297连接的聚糖中除去岩藻糖的岩藻糖苷，但仅在所有分支之后 除去了与其链接的GLCNAC之外的糖单元已被去除，我们建议开发α（1,6） - 核苷酶 可以快速，可靠和完全清除任何抗体上的岩藻糖糖单元的变体，而不管 ASN297连接的聚糖的分支结构。这种酶促工具可以由免疫学使用 社区评估整个抗体目录的体内抗体介导的杀戮机制 目前可用并将在将来开发。在此提案中，我们将解决两个具体目标：（i） 定义α（1-6） - 核苷酶的抗体去糖基化的分子基础； （ii）设计α（1,6） - 核苷酶 活跃于具有完全分支聚糖的抗体上的变体。朝着这些完整性发展 独立的特定目标将大大提高我们对聚糖改良酶的理解。 在Alfc和相关α（1-6） - 核苷酶的背景下利用这些知识将增强我们的能力 自定义抗体，提供免疫学家可以更好地理解抗体介导的工具 体内蜂窝杀死，并进一步释放其巨大的治疗效用并扩大其积极影响 关于人类健康。

项目成果

Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation.

• DOI: 10.1038/s41467-020-20044-z
• 发表时间: 2020-12-04
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Klontz EH;Li C;Kihn K;Fields JK;Beckett D;Snyder GA;Wintrode PL;Deredge D;Wang LX;Sundberg EJ
• 通讯作者: Sundberg EJ