A novel nanobody with good druggability to prevent and treat MERS-CoV infection

摘要 由中东呼吸综合征冠状病毒（MERS-coronavirus，MERS- CoV）已对全球公共卫生构成威胁，呼吁开发创新和有效的 干预策略。我们以前的研究已经证明，受体结合域（RBD）中， MERS-CoV刺突（S）蛋白，其介导病毒与受体二肽基肽酶4（DPP 4）结合， 含有关键中和结构域（CND），作为疫苗开发的重要靶标 和中和抗体。许多具有强效抗RBD作用的靶向中和性单克隆抗体（mAb）， 已经报道了中和活性和/或针对MERS-CoV感染的保护。尽管他们 上级安全性和强特异性，这些常规抗体具有大尺寸（例如，IgG为160 kDa）和 复杂的结构。这些特性导致不稳定性并限制了快速、大规模的生产。 与常规抗体不同，骆驼科动物仅重链抗体（VHH）的可变结构域，或 纳米抗体（Nb），具有小分子尺寸（~15 kDa）、强热稳定性、高产量和其它优点。 良好的可药用性，使其特别适合于作为抗病毒药物的快速和大规模生产， 价格合理，以满足疫情期间的医疗需求。我们以前已经开发了几种有效的 RBD特异性小鼠和人中和mAb，并鉴定了三种RBD靶向中和Nbs。基于 根据我们的经验和初步数据，我们假设一种新的、稳定的、有效的RBD特异性 可以开发中和Nb以对抗MERS。本提案的具体目标是：1）产生 具有延长的半衰期的人源化MERS-CoV RBD特异性中和Nbs; 2）表征MERS-CoV RBD特异性人源化Nbs的体外和体内功效;以及3）评估RBD特异性人源化Nbs的体外和体内功效。 针对MERS-CoV感染的Nbs。这项研究的长期目标是开发一种新的 用于预防和治疗MERS-CoV感染的具有良好可药性的纳米抗体。类似的方法 可用于开发针对其它I类包膜病毒的预防和治疗剂， 流行病/大流行病的可能性。

Abstract The continuous epidemic of Middle East respiratory syndrome (MERS) caused by MERS-coronavirus (MERS- CoV) has posed a threat to global public health, calling for the development of innovative and effective intervention strategies. Our previous studies have demonstrated that the receptor-binding domain (RBD) in the MERS-CoV spike (S) protein, which mediates virus binding to the receptor dipeptidyl peptidase 4 (DPP4), contains a critical neutralizing domain (CND), serving as an important target for the development of vaccines and neutralizing antibodies. A number of RBD-targeting neutralizing monoclonal antibodies (mAbs) with potent neutralizing activity and/or protection against MERS-CoV infection have been reported. In spite of their superior safety and strong specificity, these conventional antibodies have large size (e.g., 160 kDa for IgG) and complicated structures. These characteristics result in instability and limit rapid, large-scale production. Different from conventional antibodies, variable domains of camelid heavy chain-only antibody (VHH), or nanobody (Nb), has small molecular size (~15 kDa), strong thermal stability, high production yield and other good druggabilities, making it particularly suitable for rapid and large-scale production as an antiviral drug with affordable price to meet healthcare needs during an outbreak. We have previously developed several effective RBD-specific mouse and human neutralizing mAbs and identified three RBD-targeting neutralizing Nbs. Based on our experience and preliminary data, we hypothesize that a novel, stable, and effective RBD-specific neutralizing Nb can be developed to combat MERS. The specific aims of this proposal are: 1) to generate humanized MERS-CoV RBD-specific neutralizing Nbs with extended half-lives; 2) to characterize MERS-CoV RBD-specific humanized Nbs; and 3) to evaluate the in vitro and in vivo efficacy of RBD-specific humanized Nbs against MERS-CoV infection. The long-term goal of the proposed study is to develop a novel nanobody with good druggability for the prevention and treatment of MERS-CoV infection. Similar approaches can be applied to develop prophylactic and therapeutic agents against other class I enveloped viruses with epidemic/pandemic potential.