There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.

人们普遍对产生针对 SARS-CoV-2 和相关病毒的有效中和抗体、纳米抗体和其他亲和蛋白的简便方法感兴趣，以应对当前和未来的流行病。虽然从动物和人类中分离抗体是行之有效的方法，但这些方法仅限于免疫系统产生的抗体的亲和力、特异性和功能活性。在这里，我们报告了一种非常简单的定向进化方法，用于生成对 SARS-CoV-2 具有高亲和力和中和活性的纳米抗体。我们证明了低亲和力先导纳米体之间的互补决定区域交换，这是我们无意中发现的，但发现很容易系统地实施，导致成熟的纳米体具有异常大的亲和力增加。重要的，成熟的纳米抗体可有效中和 SARS-CoV-2 假病毒和活病毒，并具有类似药物的生物物理特性。我们期望我们的方法会改进体外纳米抗体的发现和加速产生针对多种冠状病毒的有效中和纳米抗体。