DataSheet_1_Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron.pdf

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.

可逃逸免疫中和作用的新型冠状病毒（SARS-CoV-2）变异株的出现，使得针对新冠大流行开发的疫苗与抗体研发面临严峻挑战。因此，构建针对多株或特定SARS-CoV-2变异株的治疗策略具有重要价值。新型冠状病毒的包膜刺突（S）糖蛋白是中和抗体（Abs）的关键靶标。我们从经S蛋白受体结合域（RBD）免疫的单峰驼体内筛选得到一组共9株纳米抗体（Nbs），并将其工程化改造为人源化重链抗体（hcAbs）。
纳米抗体及其衍生的人源化重链抗体可与S蛋白及其RBD以亚纳摩尔或皮摩尔级亲和力结合；通过S蛋白结合交叉竞争实验，可将其聚类为两个不同组别。绝大多数人源化重链抗体可阻断RBD与其人类血管紧张素转换酶2（hACE2）受体的结合，抑制携带S蛋白的假型病毒的细胞侵入过程，并在细胞培养体系中中和新型冠状病毒的感染。
4株强效中和性人源化重链抗体可在hACE2转基因小鼠体内阻止致死性新型冠状病毒感染的进展，证实了其治疗潜力。冷冻电子显微镜分析明确了纳米抗体在受体结合基序（RBM）内外的结合表位，并揭示了其阻断病毒结合细胞侵入受体的不同分子机制。纳米抗体的结合模式与其对新型冠状病毒RBD变异株的识别特性高度一致；单特异性与双特异性人源化重链抗体可高效结合除奥密克戎之外的所有关切变异株，这一结果凸显了该最新变异株的免疫逃逸能力。