CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, China.
PLoS Pathog. 2021 Mar 3;17(3):e1009328. doi: 10.1371/journal.ppat.1009328. eCollection 2021 Mar.
A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
SARS-CoV-2 感染的一个关键步骤是其 Spike 受体结合域（S RBD）与宿主受体 ACE2 的附着。已经有大量研究致力于开发中和抗体，包括来自骆驼科的单链纳米抗体，以靶向受体结合基序（RBM）并阻断 ACE2-RBD 结合。当抗体的效果仅为中等时，对于此类研究，需要采用简单有效的策略来提高效力。在这里，我们鉴定并表征了一种高亲和力的合成纳米抗体（sybody，SR31）作为融合伙伴，以提高 RBM 抗体的效力。晶体学研究表明，SR31 结合到 RBD 的一个保守的、位于 RBM 远端的“油腻”位点。尽管 SR31 在界面上使 RBD 变形，但它不会使 RBM 构象发生扰动，因此本身不具有中和活性。然而，将 SR31 融合到两种中和活性中等的 sybody 上，可极大地提高它们与 RBD 的亲和力和对 SARS-CoV-2 假病毒的中和活性。我们的工作提供了一种工具蛋白和一种提高纳米抗体效力的有效策略。
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