Team:Shanghai HS/Proof Of Concept

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Summary

 

The fast-evolving of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-binding monoclonal antibody, antibody X (3E8), blocked the S1-subunits and pseudo-typed virus constructs from multiple SARS-CoV-2 mutant variants: B.1.1.7, B.1.351, B.1.617 and P.1 . Cryo-EM and “alanine walk” studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of antibody X heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of antibody X, we provided a potentially potent and “broad-spectrum” management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.

 

Protein level testing

Antibody X (3E8) blocks the bindings of S1-subunits or RBD from multiple coronaviruses to ACE2

 

We investigated the abilities of antibody X to block the ACE2 binding of S1-subunits or RBD from B.1.1.7, B.1.351, B.1.617, P.1. These S1-subunits or RBD can all bind to His-tagged human ACE2 molecules (Fig. 1A-D), and the EC50 values to His-tagged recombinant human ACE2 molecule were 0.8, 6.9, 51.3, 14.9nM, respectively (Fig. 1E).

 

 

 

 

Fig. 1. Binding of 3E8 to His-tagged human ACE2 as measured by ELISA. (A-D) Bindings of recombinant S1-subunits or RBD (in C only) from multiple coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein as measured by ELISA. (H) The EC50 values of recombinant S1-subunit bindings to human ACE2.

 

Furthermore, Incubation with antibody X effectively blocked all S1-subunits or RBD binding to ACE2 (Fig. 2A-D) and the IC50 values were 10.0, 3.7, 10.5, 9.3 nM, respectively (Fig. 2E). Thus, antibody X can broadly block the binding of S1-subunits or RBD from multiple coronaviruses, including the fast-spreading SARS-CoV-2 variants, to human ACE2 molecules.

 

 

 

 

Fig. 2. 3E8 blocked the bindings of recombinant S1 or RBD from multiple coronaviruses and the mutant variants of SARS-CoV-2 to His-tagged recombinant human ACE2 protein. (A-D) Bindings of recombinant S1 or RBD (in C only) from different coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein were blocked by 3E8. (E) The IC50 values of 3E8 in blocking S1 or RBD binding to human ACE2 protein.

 

 

Pseudo-virus level testing

Antibody X abolishes the infectivity of multiple pseudo-typed coronaviruses

 

We next constructed pseudo-typed coronaviruses with full-length S-proteins from B.1.1.7, B.1.351, B.1.617. All pseudo-viruses could infect HEK293F cells that ectopically express human ACE2. Incubation with antibody X fully abolished the infectivity of all pseudo-viruses, with IC50 values at 0.07, 0.3, 0.08nM, respectively (Fig. 3D). In comparison, B38, a SARS-CoV-2 RBD-targeting antibody currently under clinical development, could only suppress the infectivity of B.1.1.7 and B.1.617, but not B.1.351. The suppression of X was not only broader but also remarkably more efficacious and potent, as the IC50 values of X were hundreds of folds improved when compared to that of B38 (Fig. 3D).

 

 

 

 

Fig. 3. 3E8 blocked the infection of ACE2-expressing cells by multiple pseudo-typed coronavirus constructs. ACE2-Fc and B38 were used as positive controls, and the human IgG4 isotype was the negative control. (A-C) 3E8 blocked the infection of ACE2-overexpressing HEK293 cells by different pseudo-typed coronavirus constructed with Env-defective HIV-1 and full-length S-proteins from B.1.1.7, B.1.351, B.1.617. (D) The IC50 values of 3E8 in blocking pseudo-typed coronaviruses.

 

Discussion

 

To block the entry of coronavirus, either virus- or ACE2-directed strategies can be taken. Targeting viruses directly is theoretically safer but vulnerable to viral evolution. By targeting ACE2 with an RBD-blocking antibody, we achieved broader and more effective suppression against ACE2-dependent coronaviruses without causing severe side effects. Furthermore, we revealed a broad-spectrum anti-coronavirus epitope on ACE2.

 

The mechanism by which X is more potent and efficacious than RBD-targeting antibody B38 is not yet fully understood. Limited by the sample size, it is premature to conclude that targeting ACE2 is superior to targeting viral RBD in potency or efficacy. B38 is one of the early anti-SARS-CoV-2 antibodies isolated from COVID-19 patients and due to its urgent nature, it was not well engineered with respect to affinity and developability. More head-to-head studies with more ACE2- and RBD-targeting molecules are necessary before drawing any conclusion.

 

ACE2-Fc (or called ACE2-Ig) fusion protein molecules may act as a “decoy” to interfere with coronaviruses from binding to endogenous ACE2 molecules (Fig. 2 and Fig. 3). Although ACE2-Fc molecules are broad-spectrum in theory, their binding affinity (to RBDs), specificity and developability are usually lower than antibodies. ACE2-Fc was included in our studies as control and mediocre efficacy was observed in vitro. Thus, ACE2-neutralizing antibody appears to be a more favorable approach than ACE2-Fc fusion protein.

 

“Cocktails” or combination therapies have been currently explored in treating COVID-19 [1]. A combination of 3E8 with antibodies recognizing different epitopes (e.g., RBD, NTD and/or glycan) on the viral surface seems a viable option and could be explored in clinic for better efficacy.

 

Overall, we presented evidence that 3E8 is a promising therapeutic candidate for coronavirus pandemic and believe that it represents a significant conceptual advance in fighting COVID-19, which keeps evolving, and may open the door for more ACE2-targeting drug discovery and development.

 

Reference

 

[1] Baum, A. et al. Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies. Science 369, 1014-1018, doi:10.1126/science.abd 0831 (2020).

 

 

 

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