Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

Jianbo Dong,Betty Huang,Pannaga Parthasarathy,Bo Wang,Yue Liu,Meredith Wright,Zhejun Jia,Sachith Gallolu Kankanamalage,Allison Titong

doi:10.1038/s41598-020-74761-y

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.

Highlights

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic
We used two llama VHH libraries to screen for VHHs that bind to the SARS-CoV-2 S1 protein in-vitro[19]
Out of the S1 protein binders, 19 VHHs blocked the interaction between SARS-CoV-2 S1 receptor binding domain (RBD) and ACE2 receptor, with 12 S/ACE2 blockers identified from the naïve library and 7 identified from the humanized library

Summary

Introduction

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. SARS-CoV-2 is a coronavirus that causes the human disease COVID-19, which is contagious and can rapidly spread to cause mild to severe infection, including death [CDC (https://www.cdc.gov/coronavirus/types.html)1] The spread of this newly emergent virus has reached a pandemic level with a significant public impact on the world, leading to more than 25 million infections and more than a 0.85 million deaths worldwide [World Health Organization (WHO) (https://www.who.int/emergencies/diseases/novel-coronavirus-2019)]. The first commonly used method is to clone the antibody V genes from the B cells of surviving COVID-19 patients who have mounted a natural immune response against SARS-CoV-210,11,13 This strategy has yielded a number of neutralizing monoclonal antibodies; it is important to note that the patients’ antibody repertoire condition and the timing of blood sample collection play a critical role in its success. The approach of using camelid antibody VHHs is advantageous because the VHH regions are easy to produce, are stable, and are smaller sized, which increases the possibility to target unique epitopes that are not accessible to conventional VH/VL a ntibodies[17,18]

Methods

Results

Conclusion