The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K_D of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody–RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD–ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4–6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

SARS-CoV-2 病毒比以前的冠状病毒更具传播性，导致的疾病比流感更严重。刺突蛋白的 SARS-CoV-2 受体结合域 (RBD) 与人血管紧张素转换酶 2 (ACE2) 受体结合，作为病毒进入细胞的前奏。使用原始美洲驼单域抗体库和基于 PCR 的成熟，我们生产了两个密切相关的纳米抗体，H11-D4 和 H11-H4，它们结合 RBD ( K _D分别为 39 和 12 nM）并阻断其与 ACE2 的相互作用。单粒子冷冻电镜显示，两种纳米抗体都与尖峰三聚体中的所有三个 RBD 结合。每个纳米体-RBD 复合物的晶体结构揭示了两个纳米体如何识别相同的表位，该表位与 ACE2 结合表面部分重叠，解释了 RBD-ACE2 相互作用的阻断。纳米抗体-Fc 融合体显示出针对 SARS-CoV-2 的中和活性（H11-H4 为 4-6 nM，H11-D4 为 18 nM），并与 SARS-CoV-1/2 抗体 CR3022 相加中和。