Towards one-step enzymatic defucosylation of antibodies

抗体构成了一类不断增长的药物，用于治疗越来越多的疾病。 人类疾病，包括但不限于自身免疫、感染和癌症。当工程抗体 识别几乎任何抗原在技术上已经变得简单，工程抗体诱导 指导体内细胞杀伤的独特免疫信号或效应器功能仍然是技术上的， 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 并了解抗体介导的杀伤机制，取决于我们合理修饰化学物质的能力 这种Asn 297连接的聚糖的结构。这种聚糖最重要的分子特征是岩藻糖单位 通过α（1，6）键连接至Asn近端N-乙酰葡糖胺（GlcNac），如果没有 赋予Fc结构域与活化FcγR，Fcγ R3 A的结合亲和力增加， 增加抗体介导的体内细胞杀伤。在我们对AlfC的初步研究的基础上，发现了一个α（1，6）- 岩藻糖苷酶，其从IgG抗体上的Asn 297连接的聚糖中去除岩藻糖，但仅在所有的分支 除了与之连接的GlcNac之外的糖单元已经被去除，我们建议开发α（1，6）-岩藻糖苷酶 这些变体可以快速、可靠和完全地去除任何抗体上的岩藻糖单元，而不管抗体的结构如何。 Asn 297连接聚糖的分支结构。这种酶工具可以被免疫学 社区来评估整个抗体目录的体内抗体介导的杀伤机制， 目前可用，并将在未来开发。在这项建议中，我们将针对两个具体目标：（i） 确定α（1-6）-岩藻糖苷酶使抗体去岩藻糖基化的分子基础;和（ii）设计α（1，6）-岩藻糖苷酶 对带有完全分支聚糖的抗体有活性的变体。在实现这些互补方面取得的进展， 独立的，具体的目的将大大提高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，提供免疫学家可以更好地了解抗体介导的免疫反应的工具。 体内细胞杀伤，以及进一步释放其巨大的治疗效用和扩大其积极影响 对人类健康的影响。

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.