Abstract
The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1–Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1–Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1–Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1–Nb2 (designated as Nb1–Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1–Nb2-Fc keeps a firm affinity (KD < 1.0 × 10−12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.
Highlights
The antigenic landscape of the SARS-CoV-2 receptor-binding domain (RBD) can be divided into seven binding communities, including the receptor-binding motif (RBM), the outer face of the RBD, and the inner face of the RBD.[21]
Neutralizing antibodies binding to RBM provide the most potent activity, while neutralizing antibodies associated with the outer face of the RBD demonstrate excellent neutralization breadth.[21]
Several therapeutic antibody cocktails have been approved for postexposure treatment to reduce severe illness.[15,16]
Summary
The current emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes global pandemic and the coronavirus disease (COVID-19)-related deaths had exceeded 5.6 million in January 2022.1,2 The continuing circulation and evolution of SARS-. Through multimers from diverse functional Nb building blocks.[27] Evidence fusion connection, the bivalent Nb1–Nb2 demonstrated high suggests that Nbs can exhibit super-strong activity and a broad affinity (less than or near 0.001 nM) to all RBDs (Fig. 2) These binding spectrum, through combining different Nbs into a new polyvalent molecule.[28] Nbs are becoming a powerful findings reveal that the bivalent format of Nb1 and Nb2 enhances the strength and breadth of its affinity to RBDs. In this study, we obtain several SARS-CoV-2 RBD targeting Nbs Epitope mapping using naturally occurring Spike mutants with either high-affinity or broad neutralization spectrum using a previously developed synthetic nanobody discovery platform.[10] We identified a biparatopic Nb as the best in class broadly. For the Nb1 monomer, varying degrees of resistance were observed in a large proportion of mutant
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