Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody

Abstract

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.

High-throughput ML-guided design of diverse single-domain antibodies against SARS-CoV-2

“…5). In agreement with previous reports, we found that VHH-72 bound >5x weaker to SARS-CoV-2 than to SARS-CoV-1 15,26 . Overall, 13 VHH sequences (Seqs 1-12 and VHH-72), showed high protein expression (>10 mg/L) in E. coli.…”

“…Overall 5,361/7,716 (69.5%) ML-designed sequences with up to 15 substitutions showing signi cant (p <= 0.05 Mann-Whitney U-test, Bonferroni correction) improvement over VHH-72 against SARS-CoV-2 WT, compared to 338/1496 (22.6%) baseline sequences with up to nine substitutions (Figure 2D, E, F). We note that previously reported VHH-72 variants with improved binding a nity that have up to 8 substitutions 15,25,26 . Overall these results provide evidence that the ML models extrapolate accurately to identify functional sequence variants with multiple mutations.…”

Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern

“…Pseudovirus neutralization assay was performed in 384-well plate format adapted from previously described protocol and modification 27 , 28 . Briefly, 4-fold serial dilutions of the serum samples were incubated with diluted virus at a 2:1 ratio for 1 h at 37 °C before addition to HEK293-ACE2/TMPRSS2 cells obtained from BEI Resources repository of ATCC and the NIH (NR-55293).…”

Structure-Based Optimization of One Neutralizing Antibody against SARS-CoV-2 Variants Bearing the L452R Mutation

“…The increased binding capacity between the mutant antibodies and the RBD is mainly due to the increase in the electrostatic interactions and the free energy of solvation of the polar species. Additionally, using ADAPT (Assisted Design for Antibody and Protein Therapeutics), a platform that combines structure-based computational predictions with experimental testing, the binding affinity can be further optimized [ 21 , 22 ].…”