Ma Huan, Zeng Weihong, Meng Xiangzhi, Huang Xiaoxue, Yang Yunru, Zhao Dan, Zhou Peigen, Wang Xiaofang, Zhao Changcheng, Sun Yong, Wang Peihui, Ou Huichao, Hu Xiaowen, Xiang Yan, Jin Tengchuan
Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
J Virol. 2021 Apr 26;95(10). doi: 10.1128/JVI.02438-20. Epub 2021 Mar 3.
Cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, while RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity K ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (K ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with non-overlapping epitopes resulted in hetero-bivalent Nbs, namely aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (K of 59.2 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL (∼0.043 nM) and 3.18 ng/mL (∼0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnostic reagents for COVID-19.To date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics, including SARS-CoV-2 targeting antibodies, remains critical. Due to their small size (13-15 kDa), high solubility, and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multivalent formats than the conventional antibody. Here, we report a series of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）进入细胞需要病毒刺突蛋白的受体结合域（RBD）与细胞血管紧张素转换酶2（ACE2）之间的结合。因此，RBD已成为疫苗开发的主要靶点，而RBD特异性抗体则被用作治疗药物。在此，我们报告了从免疫羊驼中开发和鉴定的SARS-CoV-2 RBD特异性重链可变区（VH）/纳米抗体（Nb）。使用噬菌体展示鉴定了七种具有高稳定性的RBD特异性Nb。它们以2.6至113 nM的亲和力K与SARS-CoV-2 RBD结合，其中六种可以阻断RBD与ACE2的相互作用。将Nb与IgG1 Fc融合产生同二聚体，其RBD结合亲和力大大提高（K范围为72.7 pM至4.5 nM），并且具有纳摩尔级的RBD-ACE2阻断能力。此外，将两个具有不重叠表位的Nb融合产生异二价Nb，即aRBD-2-5和aRBD-2-7，其具有明显更高的RBD结合亲和力（K分别为59.2 pM和- 0.25 nM）以及大大增强的SARS-CoV-2中和效力。aRBD-2-5和aRBD-2-7的50%中和剂量（ND）分别为1.22 ng/mL（约0.043 nM）和3.18 ng/mL（约0.111 nM）。这些高亲和力的SARS-CoV-2阻断Nb可进一步开发为治疗药物以及COVID-19的诊断试剂。迄今为止，SARS-CoV-2已在全球范围内造成了巨大的人员生命损失和经济产出损失。尽管一些国家已批准了几种COVID-19疫苗，但开发包括靶向SARS-CoV-2的抗体在内的有效治疗药物仍然至关重要。由于其尺寸小（13 - 15 kDa）、高溶解性和稳定性，纳米抗体特别适合肺部给药，并且比传统抗体更易于工程化为多价形式。在此报告了一系列从免疫羊驼中分离出的新型抗SARS-CoV-2纳米抗体以及两种工程化的异二价纳米抗体。这些强效中和纳米抗体显示出作为抗COVID-19潜在治疗药物的前景。
