Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold-promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

中文翻译：

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通过高效、超热稳定性和耐突变的纳米抗体中和 SARS-CoV-2

单克隆抗 SARS-CoV-2 免疫球蛋白代表了 COVID-19 的一种治疗选择。然而，它们在哺乳动物细胞中的生产无法满足全球需求。单域 (VHH) 抗体（也称为纳米抗体）提供了一种适用于微生物生产的替代方案。我们使用针对 SARS-CoV-2 刺突蛋白受体结合域 (RBD) 的羊驼免疫文库，分离出 45 种感染阻断 VHH 抗体。其中包括可以承受 95°C 的纳米抗体。最有效的 VHH 抗体在 17–50 pM 浓度（每升 0.2–0.7 µg）下中和 SARS-CoV-2，结合 Spike 的打开和关闭状态，并在 X 射线和冷冻中显示出紧密的 RBD 相互作用。电磁结构。最好的 VHH 三聚体即使在 40 ng/L 时也能中和。我们构建了纳米抗体串联并鉴定了能够耐受在 Alpha、Beta、Gamma、Epsilon、Iota 和 Delta/Kappa 谱系中发现的 K417N/T、E484K、N501Y 和 L452R 免疫逃逸突变的纳米抗体单体。我们还展示了在低皮摩尔 VHH 浓度下对 Beta 菌株的中和作用。我们进一步发现了 VHH 抗体，它们在大肠杆菌胞质溶胶，其折叠通常会失败。这种“促进折叠”的纳米抗体可以简化疫苗的生产及其对病毒逃逸突变的适应。