Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

Xin Zeng; Lingfang Li; Jing Lin; Xinlei Li; Bin Liu; Yang Kong; Shunze Zeng; Jianhua Du; Huahong Xiao; Tao Zhang; Shelin Zhang; Jianghai Liu

RBD-ACE2 interaction initiated viral infection of both SARS-CoV and SARS-CoV-2. Their RBDs share high sequence identities (73%) and structure homology, so the well-established SARS-CoV antibodies were firstly assumed short-cut therapeutic candidates for SARS-CoV-2. However, the real scenario is much more problematic. Several independent peer-reviewed studies as well as preprinted ones have proved that all structurally known SARS-CoV specific antibodies, including S230, 80R, m396 and F26G19, have no cross-reactivity of SARS-CoV-2 [4, 5, 9] . These antibodies all compete with ACE2 to bind SARS-CoV RBD, but their epitopes only have limited overlaps of the full ACE2-RBD interface, which could be the reason of lacking cross-reactivity. CR3022 is a special case with 86% conserved key residues in the epitope between SARS-CoV-2 and SARS-CoV. Its cross-reactivity was remarkable, but one site loss of N-glycan results in 1~2 magnitude reduction of binding affinity to SARS-CoV-2 RBD [9] . As in human life, RBD-specific monoclonal antibodies derived from COVID-19 recovered individuals indicated similar patterns of no cross-reactivities with either SAR-CoV or MERS-CoV [10] . In general, structural and functional analysis suggests that targeting SARS-CoV-2 RBD could be a direct and promising therapeutic strategy, while focusing on previous SARS-CoV antibodies is not very ideal or efficient. No SARS-CoV-2 RBD-specific monoclonal antibody has been reported from human antibody libraries (up to April 17 th , 2020). In the meantime, SARS-CoV-2 spreads unexpectedly fast around the world, and a new study just shifted its basic reproductive number (R0) from 2.2 to 5.7 [11] . A rapid and effective method of obtaining the SARS-CoV-2 neutralizing antibodies is much required. Naï ve antibody libraries derived from natural immune systems have their capacity limits, while synthetic libraries with higher diversity have more opportunities to isolate binders especially for novel infectious antigens. Compared to a naï ve antibody library of 10 8~1 0 9 diversity, a synthetic library with additional artificial randomization on CDRs can reach diversity as high as 10 12~1 0 13 . When the recombinant RBD and ACE2 proteins were ready, it took 3 weeks to isolate, produce and verify the antibodies in this study. Using the standard biopanning method, we enriched RBDspecific phages from our synthetic Lib AB1 but not from our naï ve antibody libraries (data not shown). Unfortunately, the top1 lead rRBD-16 from the standard biopanning of Lib AB1 couldn't block the RBD-ACE2 interaction (Fig. 4) , although it did bind to RBD with an EC50 of 5.3nM (Fig. 3) . The clinical potential and applications of an antibody often depends on its binding epitopes of the target protein. A high-affinity antibody against the target protein can be screened from a phage display antibody library using the standard biopanning process, but its binding epitopes are identified by some extra steps, such as epitope mapping and competitive ELISA. We therefore developed a new competitive biopanning strategy to efficiently isolate isotype-specific antibodies from libraries. As expected, the top1 lead rRBD-15 successfully bind to RBD in compete with ACE2 both in solution and in pseudovirus, and its binding affinity is quite high in 1~10nM differing from measuring methods. In conclusion, our strategic discovery of human monoclonal antibodies against SARS-CoV-2 RBD may fill the blanks of antibody-related pharmaceu