Novel nanobodies to prevent and treat SARS-CoV-2 and other pathogenic human coronaviruses

摘要 一种新的冠状病毒，SARS-CoV-2，正在引起全球大流行，每天有越来越多的病例 和死亡。SARS-CoV-2与导致2002-2003年SARS疫情的SARS-CoV密切相关，以及 与MERS-CoV关系不那么密切，MERS-CoV导致感染患者的高死亡率。没有治疗效果 已经批准了药物或疫苗来控制这些冠状病毒在人类中的感染，呼吁 立即努力制定有效的对策。冠状病毒尖峰蛋白(S)是病毒的重要靶标 治疗学。它们通过S1亚基与宿主受体结合，引导病毒进入宿主细胞 通过S2亚基融合病毒和宿主细胞膜。S1亚基含有与受体结合的 域(RBD)。S蛋白的RbD区和S2区均可诱导产生中和抗体。RBD是主要的 靶向诱导有效的中和抗体，但在不同的冠状病毒之间存在差异，而 S蛋白在不同冠状病毒间比较保守。纳米抗体(NBS)是一种单域抗体 来自骆驼抗体。它们正在成为新的治疗剂，其中许多已经被 在临床试验中被批准或测试以预防和治疗其他人类疾病。国家广播公司拥有许多独特的 作为治疗剂的优点：它们具有很高的物理和化学稳定性，组织性能优良 渗透能力(卓越的药代动力学)，易于表达，产量高，以及 储存和运输的坚固性。此外，NBS可以潜在地识别表位(例如，隐藏的或 部分隐藏的表位)，这些表位是常规抗体无法获得的。在这份提案中，我们将设计 并利用噬菌体开发NBS作为治疗SARS-CoV-2和其他致病性人类CoV的试剂 展示和结构生物学作为指导工具。我们建议发展高效的国家防疫系统，对抗SARS- CoV-2，以阻止当前的新冠肺炎大流行，并广谱中和针对未来CoV的NBS 感染。我们已经建立了Nb文库，并确定了几个针对RBDS的中和NBS SARS冠状病毒和MERS冠状病毒，为拟议的研究提供了坚实的基础。在我们之前的工作中，我们 广泛表征了MERS冠状病毒和SARS冠状病毒S蛋白的结构和功能，我们有 最近对SARS-CoV-2的RBD进行了表征，并在与病毒受体的复合体中解决了其结构，为 基于S蛋白的SARS-CoV-2NBS快速筛查鉴定方法。这样做的具体目的是 建议：1)发展针对SARS-CoV-2 S蛋白的高效免疫球蛋白；发展广谱的免疫球蛋白； 抗冠状病毒感染的NBS谱，2)鉴定这些冠状病毒S靶向NBS，3)体内评价 这些NBS对SARS-CoV-2和其他CoV感染的疗效。总体而言，这项提议将得到发展 针对SARS-CoV-2 S蛋白的高效国家广播公司，旨在阻止当前的新冠肺炎大流行。会的 还开发针对未来冠状病毒感染的广谱国家广播公司。这项建议对以下方面有重要影响 抗击致病冠状病毒，消除其对人类健康的威胁。

Summary A novel coronavirus (CoV), SARS-CoV-2, is causing global pandemics with growing numbers of daily cases and deaths. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2002-2003 SARS epidemic, and less closely related to MERS-CoV, which causes a high mortality rate in infected patients. No therapeutic agents or vaccines have been approved to control the infections of these CoVs in humans, calling for immediate efforts to develop effective countermeasures. The CoV spike (S) proteins are important targets for therapeutics. They guide virus entry into host cells by binding to a host receptor through their S1 subunits and fusing the viral and host cell membranes through their S2 subunits. The S1 subunits contain a receptor-binding domain (RBD). Both RBD and S2 region of S protein can elicit neutralizing antibodies. The RBD is the major target to induce potent neutralizing antibodies, but it diverges among different CoVs, whereas the S2 region of S protein is more conserved among different CoVs. Nanobodies (Nbs) are single-domain antibodies derived from camelid antibodies. They are emerging as novel therapeutic agents, numbers of which have been approved or tested in clinical trials to prevent and treat other human diseases. Nbs possess many unique advantages as therapeutic agents: they have high physical and chemical stabilities, excellent tissue penetration capability (superior pharmacokinetics), easy expression with great production yields, and robustness for storage and transportation. Moreover, Nbs can potentially recognize epitopes (e.g. hidden or partially hidden epitopes) that are not accessible to conventional antibodies. In this proposal, we will design and develop Nbs as therapeutic agents against SARS-CoV-2 and other pathogenic human CoVs using phage display and structural biology as guiding tools. We propose to develop highly efficacious Nbs against SARS- CoV-2 to stop the current COVID-19 pandemic and also broad-spectrum neutralizing Nbs targeting future CoV infections. We have established Nb libraries and identified several neutralizing Nbs targeting the RBDs of SARS-CoV and MERS-CoV, providing a solid foundation for the proposed studies. In our previous work, we extensively characterized the structures and functions of MERS-CoV and SARS-CoV S proteins, and we have recently characterized the RBD of SARS-CoV-2 and solved its structure in complex with viral receptor, paving the way for rapid screening and identification of SARS-CoV-2 S protein-based Nbs. The specific aims of this proposal are to: 1) develop highly efficacious Nbs targeting SARS-CoV-2 S (RBD/S2) protein; develop broad- spectrum Nbs against CoV infections, 2) characterize these CoV S-targeting Nbs, and 3) evaluate in vivo efficacy of these Nbs against SARS-CoV-2 and other CoV infections. Overall, this proposal will develop highly efficacious Nbs targeting SARS-CoV-2 S protein, aiming to stop the current COVID-19 pandemic. It will also develop broad-spectrum Nbs targeting future CoV infections. This proposal has important implications for combating pathogenic coronaviruses and neutralizing their threat to human health.