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

Ma, H.; Zeng, W.; Meng, X.; Huang, X.; Yang, Y.; Zhao, D.; Zhou, P.; Wang, X.; Zhao, C.; Sun, Y.; Wang, P.; Ou, H.; Hu, X.; Xiang, Y.; Jin, T.

doi:10.1101/2021.02.02.429311

bioRxiv · 10.1101/2021.02.02.429311

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

Ma, H.·Zeng, W.·Meng, X.·Huang, X.·Yang, Y.·Zhao, D.·Zhou, P.·Wang, X.·Zhao, C.·Sun, Y.·Wang, P.·Ou, H.·Hu, X.·Xiang, Y.·Jin, T.

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Abstract

Cell entry by 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, while 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 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 (KD ranging from 72.7 pM to 4.5 nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, fusion of two Nbs with non-overlapping 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 0.25 nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization dose (ND50) of aRBD-2-5 and aRBD-2-7 was 1.22 ng/mL ([~]0.043 nM) and 3.18 ng/mL ([~]0.111 nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics as well as diagnosis reagents for COVID-19. ImportanceTo 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 (13-15 kDa), highly solubility and stability, Nbs are particularly well suited for pulmonary delivery and more amenable to engineer into multi-valent formats, compared to the conventional antibody. Here, we report a serial 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.

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Ma, H., Zeng, W., Meng, X., Huang, X., Yang, Y., Zhao, D., Zhou, P., Wang, X., Zhao, C., Sun, Y., Wang, P., Ou, H., Hu, X., Xiang, Y., Jin, T.. 2021-02-03. Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain. https://doi.org/10.1101/2021.02.02.429311

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