SARS-CoV-2, the causative agent of COVID-19¹, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein^1,2,3,4,5,6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics^{7,8,9,10,11,12,13,14,15,16,17}. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (K_D = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC₅₀ = 0.42 μg mL⁻¹). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log₁₀. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

SARS-CoV-2 是 COVID-19 ¹的病原体，具有一个受体结合域 (RBD)，用于与宿主细胞 ACE2 蛋白^1,2,3,4,5,6结合。阻断 RBD-ACE2 相互作用的中和抗体是开发靶向治疗的候选药物^{7,8,9,10,11,12,13,14,15,16,17} 。美洲驼衍生的单域抗体（纳米抗体，~15 kDa）由于其尺寸小和稳定性高，在生物利用度、适用性以及生产和储存方面具有优势。在这里，我们报告了使用三个文库通过体外选择快速选择了 99 种针对 RBD 的合成纳米抗体 (sybodies)。最好的sybody，MR3以高亲和力（ K _D = 1.0 nM）与RBD结合，并对SARS-CoV-2假病毒表现出高中和活性（IC ₅₀ = 0.42 μg mL ^-1 ）。结构、生化和生物学特征表明了一种常见的中和机制，其中 RBD-ACE2 相互作用被 sybody 竞争性抑制。通过基于结构的设计、双互补位构建和二价工程已经产生了各种形式的具有改进效力的sybody。两种二价形式的 MR3 可保护仓鼠在活病毒攻击后免受临床症状的影响，单剂量的 MR3 Fc 融合构建体可将病毒 RNA 负载量降低 6 Log ₁₀ 。我们的研究结果为开发针对 COVID-19 的治疗性纳米抗体铺平了道路，并提出了在疫情爆发期间快速开发有针对性的医疗干预措施的策略。