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Updated: Dec 5, 2025
Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection
Published on: June 5, 2021
Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2
Jianbo Dong1, Betty Huang2, Bo Wang2
1Ab Studio Inc., Hayward, CA, USA. jianbo.dong@antibodystudio.com.
Abstract:
SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.
Insights
New tri-specific nanobodies targeting the SARS-CoV-2 spike protein offer potent neutralization against COVID-19. These engineered antibodies demonstrate enhanced binding and blocking capabilities, paving the way for effective COVID-19 therapeutics.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- The COVID-19 pandemic, caused by SARS-CoV-2, highlights the urgent need for effective antiviral therapies.
- Therapeutic antibodies, particularly nanobodies (VHH), present a promising strategy for neutralizing SARS-CoV-2.
- Previous work established VHH antibodies that bind the SARS-CoV-2 spike protein's S1 subunit and block ACE2 interaction.
Purpose of the Study:
- To engineer multi-specific VHH antibodies fused to human IgG1 Fc domains for enhanced SARS-CoV-2 neutralization.
- To evaluate the potency of tri-specific VHH-Fc antibodies compared to bi-specific or individual VHH-Fc antibodies.
- To assess the developability and stability of engineered VHH-Fc antibodies for therapeutic applications.
Main Methods:
- Computer-aided design was employed to construct multi-specific VHH-Fc antibodies based on epitope predictions.
- The binding affinity to the S1 subunit and the blocking of S1/ACE2 interaction were assessed.
- SARS-CoV-2 pseudovirus neutralization assays were performed.
- Protein stability and developability analyses were conducted.
Main Results:
- Tri-specific VHH-Fc antibodies exhibited significantly more potent S1 binding compared to bi-specific or individual VHH-Fcs.
- Enhanced S1/ACE2 blocking activity was observed with the tri-specific constructs.
- Superior neutralization of SARS-CoV-2 pseudoviruses was demonstrated by the tri-specific VHH-Fcs.
- Engineered VHH-Fcs displayed favorable protein stability and developability characteristics.
Conclusions:
- Multi-specific VHH-Fc antibodies represent a highly effective therapeutic strategy against SARS-CoV-2.
- The engineered tri-specific nanobodies show enhanced potency and favorable developability for COVID-19 treatment.
- This approach facilitates the rapid development of novel therapeutics to combat the COVID-19 pandemic.
