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

Huan Ma, Weihong Zeng, Xiangzhi Meng, Xiaoxue Huang, Yunru Yang, Dan Zhao, Peigen Zhou, Xiaofang Wang, Changcheng Zhao, Yong Sun, Peihui Wang, Huichao Ou, Xiaowen Hu, Yan Xiang, Tengchuan Jin
doi: https://doi.org/10.1101/2021.02.02.429311
Huan Ma
1Department of pulmonary and critical care medicine, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Weihong Zeng
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Xiangzhi Meng
3Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Xiaoxue Huang
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Yunru Yang
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Dan Zhao
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Peigen Zhou
4Department of Statistics, University of Wisconsin-Madison, Madison, WI 53706, USA
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Xiaofang Wang
5The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
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Changcheng Zhao
6Department of Infectious Diseases, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
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Yong Sun
7Anhui Provincial Center for Disease Control and Prevention, Hefei, Anhui 230001, China
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Peihui Wang
8Key Laboratory for Experimental Teratology of Ministry of Education and Advanced Medical Research Institute, Cheeloo College of Medicine, Shandong University, 250012, Jinan, China
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Huichao Ou
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
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Xiaowen Hu
1Department of pulmonary and critical care medicine, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
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Yan Xiang
3Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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  • For correspondence: jint@ustc.edu.cn xiangy@uthscsa.edu
Tengchuan Jin
1Department of pulmonary and critical care medicine, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, China
2Hefei National Laboratory for Physical Sciences at Microscale, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
9CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Science, Shanghai 200031, China
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  • For correspondence: jint@ustc.edu.cn xiangy@uthscsa.edu
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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.

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 (13-15 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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted February 03, 2021.
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Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain
Huan Ma, Weihong Zeng, Xiangzhi Meng, Xiaoxue Huang, Yunru Yang, Dan Zhao, Peigen Zhou, Xiaofang Wang, Changcheng Zhao, Yong Sun, Peihui Wang, Huichao Ou, Xiaowen Hu, Yan Xiang, Tengchuan Jin
bioRxiv 2021.02.02.429311; doi: https://doi.org/10.1101/2021.02.02.429311
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Potent in vitro Neutralization of SARS-CoV-2 by Hetero-bivalent Alpaca Nanobodies Targeting the Spike Receptor-Binding Domain
Huan Ma, Weihong Zeng, Xiangzhi Meng, Xiaoxue Huang, Yunru Yang, Dan Zhao, Peigen Zhou, Xiaofang Wang, Changcheng Zhao, Yong Sun, Peihui Wang, Huichao Ou, Xiaowen Hu, Yan Xiang, Tengchuan Jin
bioRxiv 2021.02.02.429311; doi: https://doi.org/10.1101/2021.02.02.429311

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