SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

在 COVID-19 大流行期间，引起人们关注和关注的 SARS-CoV-2 变体将继续出现。为了绘制影响与 SARS-CoV-2 受体血管紧张素转换酶 2 (ACE2) 结合的刺突蛋白受体结合域 (RBD) 中的突变，我们应用体外进化来使 RBD 亲和力成熟。RBD 的多轮随机诱变文库根据 ACE2 浓度的降低进行分类，从而选择了更高亲和力的 RBD 结合剂。我们发现在我们的高通量筛选中优先选择存在于更具传染性的病毒（S477N、E484K 和 N501Y）中的突变。进化的 RBD 突变体主要包括在 B.1.620、B.1.1.7 (Alpha)、B1.351 (Beta) 和 P.1 (Gamma) 变体的 RBD 中发现的氨基酸取代。而且，GISAID 数据库（2021 年 4 月）中显示的人群中 RBD 突变的发生率与与 ACE2 的结合亲和力增加呈正相关。进一步的体外进化使结合增加了 1,000 倍，并确定了如果它们在循环病毒群体中进化可能更具传染性的突变，例如，Q498R 对 N501Y 具有上位性。我们表明，我们的高亲和力变体 RBD-62 可用作抑制体外 SARS-CoV-2 和变体 Alpha、Beta 和 Gamma 感染的药物。在仓鼠 SARS-CoV-2 挑战模型中，RBD-62 在感染之前或之后给药时显着减少了临床疾病。RBD-62 和 ACE2 的高亲和力复合物的 2.9 Å 低温电子显微镜结构，包括所有快速扩散的突变，