Potent Neutralization of SARS-CoV-2 by Hetero-Bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain

Cellular entry of SARS-CoV-2 requires the binding between the receptor-binding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, whereas RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity K-D ranging from 2.6 to 113 nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (K-D ranging from 72.7 pM to 45 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with nonoverlapping epitopes resulted in hetero-bivalent Nbs, namely, aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (K-D of 592 pM and 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization doses of aRBD-2-5 and aRBD-2-7 were 1.22 ng/ml (similar to 0.043 nM) and 3.18 ng/ml (similar to 0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics, as well as diagnostic reagents for COVID-19. IMPORTANCE To date, SARS-CoV-2 has caused tremendous loss of human life and economic output worldwide. Although a few COVID-19 vaccines have been approved in several countries, the development of effective therapeutics, including SARS-CoV-2 targeting antibodies, remains critical. Due to their small size (13 to 15 kDa), high solubility, and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multivalent formats than the conventional antibody. Here, we report a series of new anti-SARS-CoV-2 Nbs isolated from immunized alpaca and two engineered hetero-bivalent Nbs. These potent neutralizing Nbs showed promise as potential therapeutics against COVID-19.