Comparative Assessment of the Binding and Neutralisation Activity of Bispecific Antibodies Against SARS-CoV-2 Variants

Abstract

Background: Neutralising antibodies against SARS-CoV-2 are a vital component in the fight against COVID-19 pandemic, having the potential of both therapeutic and prophylactic applications. Bispecific antibodies (BsAbs) against SARS-CoV-2 are particularly promising, given their ability to bind simultaneously to two distinct sites of the receptor-binding domain (RBD) of the viral spike protein. Such antibodies are complex molecules associated with multi-faceted mechanisms of action that require appropriate bioassays to ensure product quality and manufacturing consistency.

Methods: We developed procedures for biolayer interferometry (BLI) and a cell-based virus neutralisation assay, the focus reduction neutralisation test (FRNT). Using both assays, we tested a panel of five BsAbs against different spike variants (Ancestral, Delta and Omicron) to evaluate the use of these analytical methods in assessing binding and neutralisation activities of anti-SARS-CoV-2 therapeutics.

Results: We found comparable trends between BLI-derived binding affinity and FRNT-based virus neutralisation activity. Antibodies that displayed high binding affinity against a variant were often followed by potent neutralisation at lower concentrations, whereas those with low binding affinity also demonstrated reduced neutralisation activity.

Conclusion: The results support the utility of BLI and FRNT assays in measuring variant-specific binding and virus neutralisation activity of anti-SARS-CoV-2 antibodies.

Keywords: COVID-19; SARS-CoV-2; bioassay; bispecific antibody; product quality.

Figure 1

Binding kinetics of bispecific antibodies against Spike RBDs. (A–C) Kinetics experiments were performed using BLI with bispecific antibodies against SARS-CoV-2 RBDs. Representative sensorgrams of binding of BsAbs with (A) Ancestral, (B) Delta and (C) Omicron variants are shown. Due to the lack of binding signals, a sensorgram of CoV-X2 antibodies against Omicron is not displayed. Each experiment was run in duplicate.

Figure 2

Binding kinetics of bispecific antibodies against Spike trimers. (A–C) Kinetics experiments were performed using BLI with bispecific antibodies against SARS-CoV-2 trimer proteins. Representative sensorgrams of binding of BsAbs with (A) Ancestral, (B) Delta and (C) Omicron variants are shown. Due to the lack of binding signals, a sensorgram of CoV-X2 antibodies against Omicron is not displayed. Experiments were run in duplicate.

Figure 3

Schematic of fluorescent focus-reduction-neutralisation-test 50% using H1299-hACE2 cells. (A) Layout of a 96-well plate with H1299-hACE2 cells infected with known concentrations of SARS-CoV-2 isolates. Wells are treated with antibodies that are diluted 2-fold along with control antibodies and no-antibody negative controls. (B) Visualisation of infected cells by using fluorescent antibodies following overnight incubation under treatment conditions. (C) The Cytation7 machine can be used to count foci. (D) Resulting foci counts can be used to generate 50% end-point neutralisation titers.

Figure 4

Neutralisation of infectious Ancestral and Delta variant SARS-CoV-2 isolates using reduction of fluorescent foci in H1299-hACE2 cells. A panel of bispecific antibodies was tested by FRNT50 against Ancestral (A) and Delta (B) SARS-CoV-2 with n = 4 biological replicates per antibody. FRNT50, IC50 (4PL nonlinear regression) and R² values are listed below the relevant panel.