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How to mix the best anti-SARS-CoV-2 cocktail

Neutralisation resistance generated by Spike (S) protein mutations in Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) poses a challenge to antibody therapies for COVID-19. Antibody cocktails targeting the S protein receptor binding domain (RBD) are one way to avoid viral neutralisation escape, however only certain antibody combinations are currently effective. Here, Dr Zhiqiang Ku shares insight into his team’s development of a unique antibody cocktail with neutralising activity against COVID-19.

Antibodies have emerged as important tools in the global fight against SARS‑CoV-2. Therapeutic antibodies targeting the S protein of SARS-CoV-2 can curb disease progression in individuals with asymptomatic or mild COVID-19. They can also act as a prophylaxis in cases of potential exposure.

The first step of SARS-CoV-2 infection involves docking of the viral S protein at the cellular receptor angiotensin-converting enzyme 2 (ACE2). The hot spots on the S protein that directly interact with ACE2 are concentrated in the RBD, making it the leading target for antibody intervention. Unsurprisingly, SARS‑CoV-2-neutralising antibodies isolated from COVID-19 patients predominantly target the RBD. Understanding the molecular determinants of the interaction between the RBD and neutralising antibodies is therefore vital to antibody therapeutics and vaccine design.

As is the case for all antiviral drugs, antibody therapeutics face the challenge of mutation-based drug resistance. Antibody cocktails, however, represent a potential solution to the problem of neutralisation escape, as shown by in vitro data on the Middle East Respiratory Syndrome (MERS) and SARS coronaviruses. The premise behind this strategy is that while viral pathogens may tolerate one hotspot mutation as a means of evolving drug resistance to monotherapies, they cannot withstand the deleterious effects of multiple hotspot mutations as required by a cocktail regimen. Two dual‑antibody cocktails, REG10987+REG10933 (Regeneron) and LY‑COV555+LY-COV016 (Eli Lilly), have received Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA). Another, COV2-2130+COV2-2196 (AstraZeneca), has entered Phase III clinical trials. These cocktails target different molecular sites on the RBD with mixed results in avoiding escape mutations.

We sought to understand which molecular sites on the RBD are important for designing effective antibody cocktails and importantly, the mechanisms to prevent viral escape.

It is crucial for a mAb cocktail to prevent neutralisation escape with live virus”

In our study, published in the January 2021 issue of Nature Communications, we identified a potent antibody pair that targets independent sites on the RBD. We used mutagenesis and binding studies to elucidate the molecular interactions underpinning their synergistic neutralisation activity. Furthermore, we determined the mechanism by which the antibody pair prevented viral escape with a live SARS-CoV-2 culture assay.

Using a single-chain variable fragment (scFv) phage display antibody library, we isolated 30 RBD‑specific monoclonal antibodies (mAbs), 11 of which demonstrated robust neutralisation of a live SARS‑CoV-2 virus. Next, we performed epitope binning for the 11 mAbs to identify antibody pairs that could bind the RBD simultaneously. We used a sandwich assay on a biolayer interferometry (BLI) instrument to rapidly bin a total of 15×15 sets of mAbs. Two high-potency mAbs, CoV2-06 and CoV2‑14, could bind the RBD simultaneously and showed synergistic SARS-CoV-2 neutralising activity in vitro.

To understand which RBD residues are critical for binding with CoV2-06 and CoV2-14, we performed epitope mapping with a comprehensive SARS-CoV-2 RBD alanine scanning library of 184 mutations on the full-length protein. High‑throughput flow cytometry enabled us to easily measure the binding of mAbs to each mutant clone using small sample volumes. We observed no overlap between the critical RBD residues for either mAb. Moreover, we identified that critical epitope residues for both CoV2-06 and CoV2-14 make direct contact with K31 and K353 residues in ACE2, two known virus-binding hotspots at the RBD-ACE2 interface. Similar epitopes have been observed with the Regeneron cocktail (REG10987+REG10933) and the AstraZeneca cocktail (COV2-2130+COV2-2196).

Antibody development

Figure 1: Selection of anti-SARS-CoV-2 cocktail. High-throughput epitope binning was performed to find non-competing antibody pairs (top). High-throughput mapping was performed using an alanine scanning library and a flow cytometer to find non‑overlapping epitopes (lower). The two antibodies with simultaneous binding and non-overlapping epitopes were combined as an antibody cocktail to prevent SARS-CoV-2 escape mutations (right).

Based on extensive analysis of critical RBD epitopes and our top five mAbs, we learned that non-overlapping epitopes and lack of competition for binding are important selection criteria for choosing antibody combinations for cocktails. We also identified a novel neutralising epitope with mAb CoV2-09; this mAb has potential to form an effective cocktail pair with VH3-53-like antibodies, which are well characterised mAbs with potent neutralising activity against SARS-CoV-2.

It is crucial for a mAb cocktail to prevent neutralisation escape with live virus. Studies with the Regeneron cocktail (REG10987+REG10933) for SARS-CoV-2 evaluated neutralisation escape with a VSV-SARS-CoV-2 recombinant virus; we felt it was important to test our CoV2-06 and CoV2‑14 mAb pair using authentic SARS-CoV-2. After three passages, we recovered virus with either CoV2‑06 or CoV2-14 alone but not with both mAbs, indicating that the cocktail could prevent the development of viral escape mutants.

Sequencing analysis revealed K444R and E484A as the dominant escape mutations associated with CoV2-06 and CoV2-14, respectively. We verified this observation that our CoV2-06 and CoV2-14 cocktail prevents viral escape by showing that the cocktail could maintain neutralisation against engineered SARS-CoV-2 strains with either the K444R or E484A point mutations.

Ongoing research and development on cocktail antibody therapies must evaluate viral escape in vivo”

We reasoned that while the virus can tolerate the individual mutations, a double mutation of the critical K444 and E484 residues would significantly impair RBD function. Indeed, binding studies with recombinant RBD mutant protein and ACE2 revealed that double-site RBD mutations reduced binding affinities to less than 23 percent of wild-type RBD.

Interestingly, K444 and E484 were also reported as critical residues in studies with the Regeneron and AstraZeneca mAb cocktails. Additionally, in an analysis of 70,934 publicly available genome sequences for naturally occurring SARS-CoV-2 strains, we found no strains with double mutations of the critical residues we identified for viral escape. These shared observations point to K444 and E484 being key determinants on the RBD for optimising mAb cocktails against SARS-CoV-2.

Ongoing research and development on cocktail antibody therapies must evaluate viral escape in vivo. We demonstrated protection against SARS‑CoV-2 infection in mice with both individual and cocktail dosing of CoV2-06 and CoV2-14. We did not detect viral escape mutants in either single- or cocktail-treated mice; however, these results require further follow up and cannot be directly extrapolated to conclusions about viral escape mechanisms in humans.

Our study therefore provides mechanistic insights into how mAb cocktails prevent viral escape and informs ongoing efforts to generate successful cocktail mAbs and vaccines against SARS-CoV-2.

About the author

Dr Zhiqiang Ku is a Postdoctoral Fellow at the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, US. He received his PhD at the Institute Pasteur of Shanghai, Chinese Academy of Sciences. Subsequently, Zhiqiang worked as a scientist at the Boehringer-Ingelheim R&D Center in Shanghai. He joined Dr Zhiqiang An’s lab in 2017 to work on antibody discovery. Two antibodies discovered by Zhiqiang are now being developed with biotech partners for the treatment of cancers and COVID-19.