Towards one-step enzymatic defucosylation of antibodies

抗体构成了越来越多的药物，用于治疗范围越来越大的药物 人类疾病，包括但不限于自身免疫、感染和癌症。在设计抗体的同时 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 指导体内细胞杀伤的独特免疫信号或效应器功能在技术上仍然存在 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 而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Asn297连接的糖链的结构。这种多糖的最重要的分子特征是岩藻糖单位。 通过α(1，6)连接到天冬氨酸-近端N-乙酰氨基葡萄糖(GlcNAc)，不存在 将具有增加的结合亲和力的Fc结构域赋予激活的FcγR，FcγR3A，导致基本上 增加抗体介导的体内细胞杀伤。基于我们对AlFC的初步研究，α(1，6)- 岩藻糖苷酶从Asn297连接的多糖上去除岩藻糖的抗体，但只有在所有分支之后 超出其所连接的GlcNAc的糖单元已被移除，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糖单位的变体，而无论 Asn297连接的多糖的分支结构。这样的酶工具可以用于免疫学 社区评估体内抗体介导的整个抗体类别的杀伤机制，两者 目前可用的，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)-岩藻糖苷酶催化抗体脱糖的分子基础；(Ii)设计α(1，6)-岩藻糖苷酶 对含有完全分枝的多糖的抗体起作用的变异体。在这些互补性方面取得的进展，但 独立的、特定的目标将极大地促进我们对糖链修饰酶的理解。 在ALFC和相关的α(1-6)-岩藻糖苷酶的背景下利用这一知识将增强我们的能力 定制抗体，为免疫学家提供工具以更好地了解抗体介导的 活体细胞杀伤，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.