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

Abstract

Neutralizing antibodies (nAbs) play an important role against SARS-CoV-2 infections. Previously, we have reported one potent receptor binding domain (RBD)-binding nAb Ab08 against the SARS-CoV-2 prototype and a panel of variants, but Ab08 showed much less efficacy against the variants harboring the L452R mutation. To overcome the antibody escape caused by the L452R mutation, we generated several structure-based Ab08 derivatives. One derivative, Ab08-K99E, displayed the mostly enhanced neutralizing potency against the Delta pseudovirus bearing the L452R mutation compared to the Ab08 and other derivatives. Ab08-K99E also showed improved neutralizing effects against the prototype, Omicron BA.1, and Omicron BA.4/5 pseudoviruses. In addition, compared to the original Ab08, Ab08-K99E exhibited high binding properties and affinities to the RBDs of the prototype, Delta, and Omicron BA.4/5 variants. Altogether, our findings report an optimized nAb, Ab08-K99E, against SARS-CoV-2 variants and demonstrate structure-based optimization as an effective way for antibody development against pathogens.

Keywords: COVID-19; L452R mutation; neutralizing antibody; structure-based antibody optimization.

Figure 1

Structure-based design of Ab08 derivatives. (A) Overview ((i), cartoon) and expanded view ((ii), electropotential surface) of the Ab08-RBD structure. Various parts, including the side chain of Leu453 (green sphere), are appropriately shown and labeled. (B–F) Expanded view of the L452R-binding site (cartoon and ribbon, left) and electropotential surface of Ab08 (right) of Ab08. In electropotential representations, blue and red indicates positively and negatively charged surfaces, respectively.

Figure 2

Neutralization activity of Ab08 and Ab08-K99E. (A–D) Neutralization assays of Ab08 and Ab08-K99E using pseudoviruses from SARS-CoV-2 prototype (A), Delta (B), and Omicron variants, as indicated (C,D). Data are plotted as mean ± SEM of four replicate wells. Numbers in brackets are IC₅₀ values in μg/mL. p values were analyzed with an unpaired t-test and indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Binding activities and kinetics of Ab08 and Ab08-K99E. (A–C) Reactivities of Ab08 and Ab08-K99E to the SARS-CoV-2 RBDs measured via ELISA. Data are mean ± SEM of triplicate wells. Numbers in brackets are EC₅₀ values in μg/mL. Comparisons between Ab08 and Ab08-K99E were performed using an unpaired t-test. p values were indicated as follows: ** p < 0.01; *** p < 0.001; **** p < 0.0001. hIgG synthesized by AtaGenix served as IgG1 isotype control (hIgG ctr). (D–F) Binding affinities of Ab08 and Ab08-K99E to SARS-CoV-2 prototype and variant RBDs using surface plasmon resonance (SPR). Gray dashed lines indicate experimental data and solid colored lines indicate fitted curves. One representative experiment out of three is shown.