Rapid engineering of novel SARS-CoV-2 therapeutic antibodies

In a recent study published on bioRxiv* preprint server, researchers assessed the influence of rapid engineering of therapeutic antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to broaden the neutralization spectrum.

Background

Antibody therapies serve as the standard of care for immunocompromised people prone to adverse infection with coronavirus disease 2019 (COVID-19). However, antibody therapies approved for COVID-19 treatment showed waning efficacy soon after new strains emerged. Given the threat of reinfection, broadly active monoclonal antibodies (MABs) are urgently needed for the prevention and/or treatment of those at high risk.

Existing methodologies for designing therapeutic neutralizing mAbs depend on the isolation of antibodies from vaccinated or infected individuals or vaccinated humanized mice. However, this method is not well suited to rapidly evolving targets like SARS-CoV-2, for which the main discovery phase must be repeated each time a new variant emerges.

About the study

In the current study, the researchers developed a method that adapts the extent of antibody neutralization in real time as SARS-CoV-2 variants evolve.

Shortly after the onset of the COVID-19 pandemic, the team developed a therapeutic candidate mAb using the rabbit discovery approach, which combined rabbit vaccination with Fab-phage display. The team immunized rabbits with the receptor binding domain of the SARS-CoV-2 strain Wuhan-Hu-1 (RBD) and selected phage libraries on the Wuhan-Hu-1 spike protein trimer. Using the Fab neutralization assay, an efficient C-A11 clone was identified. Next, complementarity determining regions (CDRs) were grafted onto a human framework to generate hN2Y, a humanized clone, which showed robust neutralization of Wuhan-Hu-1.

The team used the Step-Enhanced Maturation (STEM) platform to increase neutralization efficiency of hN2Y against newly emerging SARS-CoV-2 variants. This STEM platform sampled the greatest diversity of sequences among all CDRs, such as mutations that work cooperatively and include developability criteria during selection.

Unique CDR mutant hN2Y libraries were generated. These distinct CDR libraries were initially screened on the Wuhan-Hu-1 spike trimer to eliminate non-binding and separate a large number of unique clones. To broaden the scope of neutralization, the combined light chain library was chosen for the SARS-CoV-2 Beta variant, while the combined heavy chain library was chosen independently for the Alpha, Gamma, Beta, and Epsilon variants .

Neutralization titers associated with monoclonal antibodies or serum samples were determined using the surrogate virus neutralization assay (sVNT), which measured the blockade of the recombinant spike trimer against the recombinant protein of the enzyme conversion of angiotensin-2 (ACE-2) by enzyme immunoassay (ELISA). In this experiment, the team immobilized the recombinant human Fcγ-tagged ACE2 receptor and pre-incubated immunoglobulin (Ig)-G with the tip trimer before adding the blocked wells coated with ACE2. The IgGs tested included the original neutralizing rabbit mAb C-A11, LxC1-G10 with mutations in the light chain CDRs, the parent humanized clone hN2Y, and 6R8/6R9 clones with alterations in the heavy and light chain CDRs.

Results

The results of the study showed that LxC1-G10, a remarkable candidate from the combined light chain library, demonstrated excellent neutralization of SARS-CoV-2 Omicron BA.1 and BA.2 variants. The library was chosen based on variants BA.2.75.2, BA.5, XBB and BQ.1.1, all of which have an F486 mutation, which is suspected to be a key contributor to the loss of LxC1 activity -G10. After eight or nine pannings, a heterogeneous collection of seven highly effective and broadly neutralizing IgGs (6R8/6R9 clones) was found.

Notably, humanization of the rabbit antibody did not significantly reduce efficacy, as hN2Y maintained comparable neutralizing activity for Wuhan-Hu-1 strain and Delta variants like C-A11. However, IgGs showed remarkably reduced neutralization of the original Omicron BA.1 variant.

LxC1-G10-mediated neutralization of early Omicron variants was significantly enhanced at low half-maximal inhibitory concentration (IC50) values ​​in ng/mL compared to BA.1, BA.2, BA2.3.20 and BA2 .75 as well as BN. 1. However, the IgGs lost their effectiveness against the BA.2.75.2 variant, which differed significantly from the others due to the F486S and R346T mutations. Additionally, BQ.1 and BA.5 lacked the R346T mutation but carried the F486V mutation, while LxC1-G10 did not substantially neutralize these variants. This indicated that the F486 mutation was the most important factor contributing to the loss of activity in LxC1-G10.

The affinity of hN2Y for the beta variant was slightly lower than that of the original rabbit clone C-A11, whereas the affinity of LxC1-G10 increased to low pM K binding._D. LxC1-G10 displays a K_D 24 pM affinity for BA.1, which was a five-fold improvement over hN2Y. However, the binding affinity of LxC1-G10 decreased nearly 100-fold from 24 pM for BA.1 to 2.1 nM for BQ.1.1. Additionally, seven 6R8/6R9 IgGs had a K_D 7:00 p.m. or less for BQ.1.1 and less than 70:00 a.m. for XBB.1.5.

Bebtelovimab demonstrated weak binding to BQ.1.1 but no binding to XBB.1.5. Neither tixagevimab nor cilgavimab binds to XBB.1.5 or BQ.1.1. On the other hand, modified IgG exhibited low pM K binding_D for the most recent circulating Omicron variants.

Conclusion

The results of the study highlighted the validation of the innovative STEM platform. The team could successfully regain potency via engineering against SARS-CoV-2 variants. The researchers believed that the current strategy could be used for the future generation of therapeutic mAbs against SARS-CoV-2 or other viruses, allowing the rapid development of broadly neutralizing therapeutic mAbs.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.