Evaluation of strategies to modify Anti-SARS-CoV-2 monoclonal antibodies for optimal functionality as therapeutics

Abstract

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease, the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study, a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus, preventing it from infecting cells in an in vitro cell-based assay, including multiple viral variants that are currently circulating in the human population. In addition, we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease.

Fig 1. Viral neutralization studies.

The results of the viral variant testing. Heat map represents activity as measured by the average ratio of the mutation IC50/wild type (D614G). Ratios are provided in the Figures. The average is calculated from multiple experiments so specific mutation IC50 and WT IC50 are not provided. IC50 (dark grey represents <0.3; light grey represents 0.3–5.0; yellow represents 5.0–10.0; orange represents 10.0–50.0; red represents >50.0). (A) Neutralization results against VOCs; (B) Neutralization results against a panel of mutant variants of the non-RBD, (C) Neutralization results against a panel of mutant variants of the RBD.

Fig 2. Binding of the monoclonal antibody variants to Fc receptors.

The bar graphs present the overall binding of the monoclonal antibody variants to the indicated Fc receptor represented as the area under the curve calculated from the dilution curves presented in S1 Fig. Data are presented as the mean AUC ± standard deviation from two independent experiments. (A) FCGR2A 131H; (B) FCGR2A 131R; (C) FCGR2B; (D) FCGR3A 158F; (E) FCGR3A 158V; (F) FCGR3B; (G) FCRN pH6; (H) FCRN pH 7.4. The Ebola virus GP–specific antibody KZ52 was used as the irrelevant antibody. Significance was calculated using a one-way ANOVA with post hoc Holm-Šídák’s multiple comparisons test. *: p ≤ 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 3. Extra-neutralizing functional activity of the monoclonal antibody variants.

The data are presented both as bar graphs representing the overall activity of the monoclonal antibody variants in the indicated functional assays represented as the area under the curve calculated from the individual dilution curves presented in S2 Fig. Data are presented as the mean AUC ± standard deviation from two independent experiments. For assays using primary cells, cells isolated from two independent donors were used. (A) Antibody-dependent cellular phagocytosis; (B) antibody-dependent neutrophil phagocytosis; (C) antibody-dependent cellular cytotoxicity; (D–F) antibody-dependent NK cell activation; (G) antibody-dependent complement deposition; (H) antibody-dependent mucin (MUC5A/C) binding; (I) antibody-dependent mucin (MUC5B) binding. The Ebola virus GP–specific antibody KZ52 was used as the irrelevant antibody. Significance was calculated using a one-way ANOVA with post hoc Holm-Šídák’s multiple comparisons test. *: p ≤ 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.