Here, we show a cocktail of two mAbs (CoV2-06+CoV2-14) that target the RBD and cooperate with each other to prevent escape mutations. The two mAbs bind to non-overlapping epitopes of the RBD and independently block RBD and ACE2 interaction. The cocktail prevents SARS-CoV-2 escape mutations through a mechanism of imposing stronger mutational constraints on the RBD than individual mAbs. Individual mAbs and the cocktail confer protections against SARS-CoV-2 infection in mice. Overall, our comprehensive study provides important molecular insights for the development of antibody cocktail therapies for COVID-19.

We used the authentic live SARS-CoV-2 to evaluate neutralization escape. We passaged the SARS-CoV-2-mNG virus in the presence of CoV2-06, CoV2-14, CoV2-06+CoV2-14 for three rounds (Fig. 5a). We could recover virus in the presence of individual CoV2-06 or CoV2-14 mAbs but not in the presence of the cocktail mAbs (Fig. 5a). We then sequenced the S region of the viruses recovered from the four replicative selections to identify escape mutations. Under CoV2-06 selection, three independently selected viruses had a K444R mutation and one selected virus had a K444S mutation. Under CoV2-14 selection, three independently selected viruses had an E484A mutation and one selected virus had an F486S mutation (Fig. 5b). Outside the RBD, additional mutations in the N-terminal domain (NTD) of S were also observed in some selected viruses. For example, H66R or H66R+R190K were observed under CoV2-06 selection, and N74K was observed under CoV2-06 or CoV2-14 selection. We sought to confirm whether the mutations in the RBD but not the NTD are responsible for resistance. We focused on the most frequent K444R and E484A mutations and constructed two recombinant SARS-CoV-2 viruses with point mutation K444R or E484A. The two mutant viruses were then analyzed for neutralization by individual CoV2-06, CoV2-14 mAbs, and the CoV2-06+CoV2-14 cocktail. The K444R mutant virus could escape the neutralization by CoV2-06 but not CoV2-14; the E484A mutant virus could escape the neutralization by CoV2-14 but not neutralization by CoV2-06 (Fig. 5b). The mAb cocktail maintained neutralization against both the K444R and E484A mutant viruses (Fig. 5b). These results demonstrated that the mAb cocktail of CoV2-06 and CoV2-14 is effective in preventing SARS-CoV-2 escape mutations in vitro.

While some RBD mutations are well tolerated, other mutations are deleterious for RBD function and therefore constrained in SARS-CoV-222. We reasoned that simultaneous mutations on the two distinct binding sites of CoV2-06 and CoV2-14, which are required for virus to escape neutralization by the cocktail, would be more constrained than mutations on the binding sites of individual mAbs. To test this hypothesis, we generated eight sCoV2-RBD mutant proteins, four with individual mutations of single binding sites (K444R, K444S, E484A, and F486S) and four with simultaneous mutations of both binding sites (K444R+E484A, K444R+F486S, K444S+E484A, and K444S+F486S) (Supplementary Fig. 5a). These single-site or double-site RBD mutants were analyzed for their affinity to ACE2 (Fig. 6a and Supplementary Fig. 5b–j), protein expression (Fig. 6b), and folding stability (Fig. 6c). Single-site mutations of K444R, K444S, E484A, and F486S reduced the sCoV2RBD/ACE2 binding affinities to 56%, 61%, 79%, and 6% of the wild-type (WT), respectively. In comparison, double-site mutations of K444R+E484A, K444R+F486A, K444S+E484A, and K444S+F486S further reduced the sCoV2-RBD/ACE2 binding affinities to 23%, 9%, 19%, and 3% of the WT, respectively (Fig. 6a and Supplementary Fig. 5b–j). Similarly, while single-site mutations altered the RBD expression to 69–110.1% of the WT, double-site mutations reduced the expression to 25.5–84.2% of the WT (Fig. 6b). The size exclusion chromatography (SEC) analysis showed protein aggregates of 0.88–11.42% and 4.99–14.74% for RBD with single-site mutations and double-site mutations, as compared to only 0.21% of aggregates for WT RBD (Fig. 6c). These data indicate that double mutations at both the CoV2-06 and CoV2-14 epitope sites attenuated the receptor-binding affinity and stability of the RBD more than that of the single-site mutations, suggesting that such double-site mutations would have deleterious effects on viral fitness.

n summary, we report the molecular determinants and mechanism for a mAb cocktail that prevents SARS-CoV-2 viral escape mutations. We also identified an epitope combination potentially suitable for the design of other cocktail mAbs, as well as a conserved epitope for selecting cross-reactive neutralizing mAbs. Our study is informative for the evaluation of the clinical-stage cocktail mAbs, benefits further selection of other cocktail antibodies against SARS-CoV-2, and aids the assessment of vaccines. Finally, the mAbs we isolated hold promise for further development as antibody therapies for COVID-19.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817669/

They found 2 monoclonal antibodies that work in the face of escape mutations.

The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies

https://www.sciencedirect.com/science/article/pii/S0092867421007030

It protects AGAINST mutations escape.

Found 2 that if escape happens, it still protects..

So they move it towards PEP.

*mutational

So if it protects against mutational escape, now they will try to make it a vaccine to develop these 2 antibodies.

With this combo of Ab's given…

The people who take it…. what are the chances of mutations within those individuals occurring and can they be transferred to others? Or does the cocktail neutralize the mutants? Have they taken the half life of the mAB's into account against this theory?

What about the worldwide vaccinated subjects….? Why did we approve that? If mutations can be tolerated in the mab cocktail group is there a chance you are furthering the vaccinated's demise?

Do we need to give the cocktail to people who are not at high risk….. modify your high risk category….. BMI 25 is not that high risk…..

Regeneron's cocktail was sold to the fed government.

Reg made some money.

Now the cocktail is being given to us for free.

If it prevents mutant from forming you are golden.

IV over injection has benefits, instant immunity right into the blood.

The data described herein strongly support the notion that cocktail therapy may provide a powerful way to minimize mutational escape by SARS-CoV-2; in particular, our studies point to the potential value of antibody cocktails in which two antibodies were chosen so as to bind to distinct and non-overlapping regions of the viral target (in this case, the RBD of the spike protein), and thus require the unlikely occurrence of simultaneous mutations at two distinct genetic sites for viral escape. A clinical candidate selection criterion for broad potency that includes functional assessment against naturally circulating sequence variants, as well as inclusion of multiple antibodies with non-overlapping epitopes, may provide enhanced protection against loss of efficacy. Future in vivo animal and human clinical studies need to pay close attention to possible emergence of escape mutants and potential subsequent loss of drug efficacy.