The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility

Abstract

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.

Keywords: SARS-CoV-2; antibody discovery; neutralizing antibodies; phage display; phage panning.

Fig. 1

Kinetic and competition results with corresponding epitope binning of anti-SARS-CoV-2 antibodies. (A) Kinetic measurements were performed using biotinylated S1 protein and the panel of anti-SARS-CoV-2 antibodies was competed against reference antibodies CR3022 and Ab 12 and recombinant ACE2 in a BLI-based competition assay. Competition and percent blockade is determined by comparison to an unblocked sensor. Competition is indicated by a black block, and percent blockade is shown on a sliding scale, with red blocks displaying near complete inhibition and green blocks showing minimal inhibition. (B) Competition matrices and SARS-CoV cross reactivity were used to create antibody sub-bins, each with a different competition pattern or binding properties. The majority of antibodies bound to the RBD, with one-third targeting the CR3022 epitope and two-third targeting the ACE2 interface. N.d., not determined

Fig. 2

Neutralization activity of anti-SARS-CoV-2 scFv-Fcs. (A) Selected antibodies were tested against authentic SARS-CoV-2 virus in full neutralization curves. (B) Anti-SARS-CoV-2 scFv-Fcs demonstrate a wide range of neutralization activity, with Abs 12 and 27 on the potent end and Abs 28 and 23 on the weak/non-neutralizing end. Bin 3 antibodies typically have the most potent activity and Bins 1-2 display considerably weaker activity.

Fig. 3

Characterization of converted IgGs. (A) Kinetic measurements via BLI of selected antibodies show that conversion from scFv-Fc to IgG does not have a significant effect on the KD values for these antibodies when binding the RBD. (B) scFv-Fcs and IgGs were tested in parallel SARS-CoV-2 neutralization assays (PRNT). Abs 12 and 38 showed minimal loss in neutralization efficacy, however Abs 14, 27, 29 and 2-7 displayed substantial loss. Of the three most potent scFv-Fcs, Abs 12 and 38 maintain their ability to neutralize as IgGs, whereas the IC50 for Abs 14 and 27 shifts to the right by 40- and 950-fold respectively. In this assay, Ab 29 and 2-7 IgGs appear to lose all neutralization ability; however, due to the high IC50 for the scFv-Fcs, this may be due to the curve shifting further to the right than our assay tests.

Fig. 4

FACS binding curves comparing selected IgG and scFv-Fc pairs. (A) FACS binding curves with 293T-Spike cells show a pronounced decrease in binding for Abs 14, 27 and 2-7, whereas Ab 12 shows an increase in binding.

Fig. 5

Spike shedding induced by anti-SARS-CoV-2 antibody binding. (A) Selected scFv-Fcs were tested in a spike shedding experiment using 293T cells stably expressing the SARS-CoV-2 spike. Percent change is relative to the median fluorescence signal for each sample at the 5 min time point, and antibody traces are colored based on their epitope sub-bins. Bin 2 antibodies Ab 2-7 and Ab 28 do not lead to shedding of the spike as seen by the increase in fluorescent signal over time. Antibodies in Bin 3E (Abs 17, 19, 35) trigger minimal to low shedding (green traces), whereas Abs in Bin 3C (Abs 14, 18, 27, 38) all lead to significant levels of spike shedding. Ab 12 (Bin 3A) leads to the most significant amount of shedding, whereas sub bin member Ab 7 induces significantly less shedding. (B) Selected scFv-Fcs were tested against their respective IgGs. Abs 12 and 38 display similar levels of spike shedding as an IgG and scFv-Fc, correlating to a minimal change in IC50 in in vitro virus neutralization assays. Abs 14, 27 and 29 show pronounced decreases in the amount of spike shedding in the IgG compared with the original scFv-Fc, providing a rational to their drastic loss of neutralization efficacy seen with in vitro neutralization assays.

Fig. 6

Spike shedding assay comparing mono- versus bivalent binding. scFv-Fc, Fab and IgG formats of Ab 12 were tested in a spike shedding experiment. Only the bivalent scFv-Fc and IgG lead to spike shedding, whereas the monovalent Fab signal remains constant throughout the experiment.