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 receptorbinding 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 KD ranging from 2.6 to 113nM, 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 (KD ranging from 72.7 pM to 4.5nM) 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 (KD of 59.2 pM and 0.25nM) 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 (∼0.043 nM) and 3.18 ng/ml (∼0.111nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics, as well as diagnostic reagents for COVID-19.