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

总结 一种新型冠状病毒（CoV），SARS-CoV-2，正在引起全球大流行，每天的病例数不断增加 和死亡SARS-CoV-2与引起2002-2003年SARS疫情的SARS-CoV密切相关， 与MERS-CoV的关系不太密切，后者导致感染患者的高死亡率。没有治疗 已经批准了药物或疫苗来控制这些CoV在人类中的感染， 立即努力制定有效的对策。CoV刺突（S）蛋白是CoV的重要靶标。 治疗学它们通过其S1亚基与宿主受体结合，引导病毒进入宿主细胞， 通过它们的S2亚基融合病毒和宿主细胞膜。S1亚基含有受体结合的 域（RBD）。RBD和S蛋白的S2区均能诱导产生中和抗体。RBD是主要的 靶向诱导有效的中和抗体，但它在不同的CoV之间存在分歧，而S2区的 S蛋白在不同的CoV中更为保守。纳米抗体（Nbs）是来源于纳米抗体的单结构域抗体。 来自骆驼科动物的抗体。它们正在成为新的治疗剂，其中许多已经被发现。 在临床试验中获得批准或测试，以预防和治疗其他人类疾病。Nbs拥有许多独特的 作为治疗剂的优点：它们具有高的物理和化学稳定性， 渗透能力（上级药代动力学），易于表达，产量高， 储存和运输的坚固性。此外，Nbs可以潜在地识别表位（例如，隐藏的或非隐藏的）。 部分隐藏的表位），这些表位是常规抗体不能接近的。在本提案中，我们将设计 并利用噬菌体开发Nbs作为抗SARS-CoV-2和其他致病性人类CoV的治疗剂 展示和结构生物学作为指导工具。我们建议发展高效能的非典型肺炎疫苗- CoV-2阻止当前的COVID-19大流行，以及针对未来CoV的广谱中和Nbs 感染.我们已经建立了Nb文库，并鉴定了几种靶向以下RBD的中和Nbs： SARS-CoV和MERS-CoV，为拟议的研究提供了坚实的基础。在以前的工作中，我们 广泛表征了MERS-CoV和SARS-CoV S蛋白的结构和功能， 最近表征了SARS-CoV-2的RBD，并解决了其与病毒受体复合的结构， 建立了快速筛选和鉴定SARS-CoV-2S蛋白Nbs的方法。具体目标是 建议是：1）开发高效靶向SARS-CoV-2 S（RBD/S2）蛋白的Nbs; 针对CoV感染的谱Nbs，2）表征这些CoV S靶向Nbs，和3）体内评价 这些Nbs对SARS-CoV-2和其他CoV感染的有效性。总的来说，这项提案将发展 针对SARS-CoV-2 S蛋白的高效Nbs，旨在阻止当前的COVID-19大流行。它将 还开发针对未来CoV感染的广谱Nbs。这一建议对以下方面具有重要意义： 抗击致病性冠状病毒并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.