Rationalizing glycoengineering strategies for immunotherapeutic antibodies

为了逃避宿主免疫，许多细菌分泌免疫调节酶。链球菌 化脓性链球菌是最常见的人类病原体之一，分泌独特的内切糖苷酶，包括EndoS， 其以高度特异性的方式从人IgG抗体中去除复合型聚糖， EndoS 2，可额外去除IgG连接的高甘露糖聚糖。这使得抗体不能 通过补体或Fc γ受体（FcγRs）引发宿主效应子功能， 生存优势。因为抗体是许多人类免疫反应的核心参与者， 桥梁的先天和适应性武器的免疫，分析和操纵的酶活性， EndoS和EndoS 2影响着生物医学的各个领域。特别地，修饰抗体聚糖结构 可对其与Fcγ R结合的能力和随后的免疫系统反应产生显著影响 他们诱导。下一代的治疗性抗体已经在构建中， 聚糖化学来调整它们的免疫反应并增加它们的临床效力。EndoS和EndoS 2， 由于抗体特异性糖苷酶及其衍生的糖苷酶是未来免疫治疗中的关键酶， 抗体工程我们认为，如果不同的内切糖苷酶的分子机制 特异性识别和水解不同糖蛋白底物被更好地理解为EndoS和 EndoS 2变体可以被合理地工程化以产生一类新的抗体修饰酶， 具有独特的聚糖特异性，以修饰表现出增强的临床特性的抗体。在这 我们将提出三个具体目标：（1）通过以下方法确定聚糖特异性的分子基础： （2）确定碳水化合物结合模块-非酶蛋白结构域的作用 具有聚糖结合特性-内切糖苷酶特异性和活性;和（3）阐明 基于内切糖苷酶的蛋白特异性。在实现这些相辅相成但又相互独立的 具体的目标将大大推进我们对聚糖修饰酶的理解。利用这一 EndoS和EndoS 2背景下的知识将增强我们定制抗体的能力， 释放其巨大的治疗效用并扩大其对人类健康的积极影响。

In order to evade host immunity, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes unique endoglycosidases, including EndoS, which removes complex-type glycans in a highly specific manner from human IgG antibodies, and its homolog EndoS2, which can additionally remove IgG-linked high-mannose glycans. This renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors (FcγRs), providing the bacteria with a survival advantage. Because antibodies are central players in many human immune responses and bridge the innate and adaptive arms of immunity, the analysis and manipulation of the enzymatic activities of EndoS and EndoS2 impact diverse fields in biomedicine. In particular, modifying antibody glycan structures can have significant impacts on their abilities to bind to FcγRs and the subsequent immune system reactions that they induce. The next generation of therapeutic antibodies is already being constructed with modified glycan chemistries to tailor their immune reactions and to increase their clinical potency. EndoS and EndoS2, as antibody-specific glycosidases, and glycosynthases derived thereof, are key enzymes in the future of antibody engineering. We propose that if the molecular mechanisms by which diverse endoglycosidases specifically recognize and hydrolyze distinct glycoprotein substrates are better understood that EndoS and EndoS2 variants can be rationally engineered to create a new class of antibody-modifying enzymes endowed with unique glycan specificities in order to modify antibodies that exhibit enhanced clinical properties. In this proposal, we will address three Specific Aims: (1) to determine the molecular basis of glycan specificity by endoglycosidases; (2) to define the role of carbohydrate binding modules – non-enzymatic protein domains with glycan binding properties – in endoglycosidase specificity and activity; and (3) to elucidate the molecular basis of protein specificity by endoglycosidases. Progress towards these complementary, yet independent, Specific Aims will significantly advance our understanding of glycan-modifying enzymes. Leveraging this knowledge in the context of EndoS and EndoS2 will enhance our ability to customize antibodies, further unleashing their vast therapeutic utility and expanding their positive impact on human health.