Mazigi Ohan, Langley David B, Henry Jake Y, Burnett Deborah L, Sobti Meghna, Walker Gregory J, Rouet Romain, Balachandran Harikrishnan, Lenthall Helen, Jackson Jennifer, Ubiparipovic Stephanie, Schofield Peter, Brown Simon H J, Schulz Sebastian R, Hoffmann Markus, Pöhlmann Stefan, Post Jeffrey, Martinello Marianne, Ahlenstiel Golo, Kelleher Anthony, Rawlinson William D, Turville Stuart G, Bull Rowena A, Stewart Alastair G, Jäck Hans-Martin, Goodnow Christopher C, Christ Daniel
Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
University of New South Wales Sydney, St. Vincent's Clinical School, Faculty of Medicine, Sydney, NSW 2010, Australia.
Proc Natl Acad Sci U S A. 2025 Jan 7;122(1):e2417544121. doi: 10.1073/pnas.2417544121. Epub 2025 Jan 2.
The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails and of first-generation broadly neutralizing antibodies such as S309 (Sotrovimab). In contrast, antibodies targeting cryptic conformational epitopes of the receptor binding domain (RBD) have demonstrated broad activity against emerging variants, but exert only moderate neutralizing activity, which has so far hindered clinical development. Here, we utilize in vitro display technology to identify and affinity-mature antibodies targeting the cryptic class 6 epitope, accessible only in the "up" conformation of the SARS-CoV-2 spike trimer. Increasing antibody affinity into the low picomolar range endowed potent neutralization of VOCs and protection of hACE2 mice from viral challenge. Cryoelectron microscopy and crystal structures of two affinity-matured antibodies (4C12-B12 and 4G1-C2) in complex with RBD highlighted binding modes and epitopes distal from mutational hotspots commonly overserved in VOCs, providing direct structural insights into the observed mutational resistance. Moreover, we further demonstrate that antibodies targeting the class 6 epitope, rather than being an artifact of in vitro selection, are common in the IgG1 memory B cell repertoire of convalescent patients and can be induced in human antibody V-gene transgenic mice through immunization. Our results highlight the importance of very high (picomolar) affinity in the development of neutralizing antibodies and vaccines and suggest an affinity threshold in the provision of broad and long-lasting immunity against SARS-CoV-2.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）关注变异株（VOCs）的出现，极大地削弱了先前美国食品药品监督管理局（FDA）批准的单克隆抗体（mAbs）的中和活性，包括抗体鸡尾酒以及第一代广泛中和抗体（如S309，即索托维单抗）的中和活性。相比之下，靶向受体结合域（RBD）隐蔽构象表位的抗体已显示出对新兴变异株具有广泛活性，但仅具有中等程度的中和活性，这迄今为止阻碍了其临床开发。在此，我们利用体外展示技术来鉴定和亲和力成熟靶向隐蔽6类表位的抗体，该表位仅在SARS-CoV-2刺突三聚体的“向上”构象中可及。将抗体亲和力提高到低皮摩尔范围赋予了对VOCs的有效中和作用，并保护人血管紧张素转换酶2（hACE2）小鼠免受病毒攻击。两种亲和力成熟抗体（4C12-B12和4G1-C2）与RBD复合物的冷冻电镜和晶体结构突出了远离VOCs中常见突变热点的结合模式和表位，为观察到的突变抗性提供了直接的结构见解。此外，我们进一步证明，靶向6类表位的抗体并非体外选择的假象，在康复患者的IgG1记忆B细胞库中很常见，并且可以通过免疫在人抗体V基因转基因小鼠中诱导产生。我们的结果突出了非常高（皮摩尔）亲和力在中和抗体和疫苗开发中的重要性，并表明在提供针对SARS-CoV-2的广泛和持久免疫力方面存在一个亲和力阈值。
