A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

Abstract

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

Fast and accurate genome-wide predictions and structural modeling of protein-protein interactions using Galaxy

“…High-scoring models were also generated for dipeptidyl peptidase-4 (DPP4, see Fig. 5 ), confirming the computational modeling results presented by Li et al 2020. DPP4 is a cell surface glycoprotein receptor involved in T-cell activation and assumed to play a role in cell adhesion, migration and tube formation (Durinx, 2000).…”

“…Additionally several suitable multimeric template frameworks were identified. The corresponding PDB entries are 7C8V (Li, 2020), 6XC2 (Yuan, 2020), 6XC4 (Yuan, 2020), 7BZ5 (Wu, 2020) and 7C01 (Shi, 2020). All of these structures, except 7C8V, were crystalized with a potent neutralizing antibody of SARS-CoV2.…”

Potent Molecular Feature-based Neutralizing Monoclonal Antibodies as Promising Therapeutics Against SARS-CoV-2 Infection

“…The methods adopted to develop mAbs against SARS-CoV-2 range from empirical to recently developed innovative methods ( BalcioGlu et al, 2020 ). Empirical methods used to produce anti-SARS-CoV-2 antibodies include ribosome, phage, yeast, and retained display technologies ( Beasley et al, 2020 ; Li T. et al, 2020 ; Wu et al, 2020a ), allowing to produce more than hundreds of antibodies. Phage and yeast display-based mAbs development technologies have subsequently been improved for fast isolation of antibodies ( Wu et al, 2020b ; Zost et al, 2020b ).…”

“…Besides, techniques such as Machine Learning technologies ( Magar et al, 2020 ), in the context of the fight against SARS-CoV-2, made it possible to produce numerous mAbs. In addition, single-cell sequencing, CDR3 H structure similarity, high-efficiency screening, mutation-based maturation and engineering, and synthetic approaches ( Custódio et al, 2020 ; Han et al, 2020 ; Li T. et al, 2020 ; Miersch et al, 2020 ; Rouet et al, 2020 ; Schoof et al, 2020 ) or approaches from genetically humanized mice led to the production of mAbs with well-proven potency. Furthermore, the use of proteomics, an innovative approach in sorting neutralizing immunoglobulins in hilled COVID-19 patients allowed Voss et al to develop outstanding neutralizing antibodies with high ability to protect efficiently against experimental SARS-CoV-2 infection ( Voss et al, 2020 ).…”

“…Specifically, 20 of these reported SARS-CoV-2 RBD-specific nhNmAbs including ACE2-Ig, mACE2-Ig ( Lei et al, 2020 ), MR3, MR4, MR17, SR4 ( Li T. et al, 2020 ), rRBD-15 ( Zeng et al, 2020a ; Zeng et al, 2020b ), IgG1 ab1 ( Li W. et al, 2020 ), 15031, 15032, 15033, 15034 ( Miersch et al, 2020 ), RU169–178, RU167–230, RU171–155 ( Beasley et al, 2020 ), Nb6 ( Schoof et al, 2020 ), and Nb11, Nb12, Nb15, Nb19 ( Schoof et al, 2020 ), were demonstrated to target RBD-epitopes overlapping with ACE2 binding site directly. Besides, recently, Ma et al ( Ma et al, 2021 ) developed six efficient alpaca RBD-specific nanobodies, including aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, and aRBD-54, and more interestingly, engineered two hetero-bivalent nanobodies named aRBD-2-5 (from aRBD-2 and aRBD-5) and aRBD-2-7 (from aRBD-2 and aRBD-7) with higher potent neutralizing ability against WT SARS-CoV-2 ( Ma et al, 2021 ).…”