Team:Shanghai HS/Results
Results
Overview
The fast-evolving of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Our results highlighted again the importance of epitope outside or on the verge of RBD/ACE2 interface and would facilitate future endeavors searching for broad-spectrum anti-coronavirus approaches. Overall, we presented evidence that 3E8 is a promising therapeutic candidate for the 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.
1. 3E8 Binds Human ACE2 With Moderate Affinity
Fig. 1. Monoclonal antibody 3E8 and recombinant S1-subunits or RBD from different coronaviruses (and SARS-CoV-2 mutant variants) bound to His-tagged recombinant human ACE2 protein: (A) Binding of 3E8 to His-tagged recombinant human ACE2 protein as measured by ELISA; (B) Binding of 3E8 to His-tagged human ACE2 as measured by BLI; (C-G) Bindings of recombinant S1-subunits or RBD (in F 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.
We measured the binding affinity of 3E8 to His-tagged human ACE2 protein with ELISA and biolayer interferometry (BLI). The EC50 value was 15.3 nM in ELISA (Fig. 1A) and the dissociation constant (KD) was 30.5 nM in BLI (Fig. 1B). It is also bound to HEK293F cells ectopically overexpressing human ACE2 and to Vero E6 cells endogenously expressing human ACE2, as demonstrated by flow cytometry (Fig. S1E).
2. 3E8 Blocks The Bindings Of S1-Subunits Or RBD From Multiple Coronaviruses To ACE2
We investigated the abilities of 3E8 to block the ACE2 binding of S1-subunits or RBD from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, P.1, SARS-CoV and HCoV-NL63. These S1-subunits or RBD can all bind to His-tagged human ACE2 molecules (Fig. 1C-G), and the EC50 values to His-tagged recombinant human ACE2 molecule were 11.8, 2.6, 0.8, 6.9, 51.3, 14.9, 1.1 and 24.2 nM, respectively (Fig. 1H).
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-E) Bindings of recombinant S1 or RBD (in D only) from different coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein were blocked by 3E8. (F) The IC50 values of 3E8 in blocking S1 or RBD binding to human ACE2 protein.
Incubation with 3E8 effectively blocked all S1-subunits or RBD binding to ACE2 (Fig. 2A-E) and the IC50 values were 7.1, 13.8, 10.0, 3.7, 10.5, 9.3, 13.7 and 5.0 nM, respectively (Fig. 2F). Thus, 3E8 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.
3. 3E8 Abolishes The Infectivity Of Multiple Pseudo-Typed Coronaviruses
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 human IgG4 isotype was negative control. (A-G) 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 SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63. (H) The IC50 values of 3E8 in blocking pseudo-typed coronaviruses.
We next constructed pseudo-typed coronaviruses with full-length S-proteins from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, SARS-CoV and HCoV-NL63 (Fig. 3A-G). All pseudoviruses could infect HEK293F cells that ectopically express human ACE2, while SARS-CoV-2-D614G showed significantly enhanced infectivity when compared to the original SARS-CoV-2 (Fig. S2). Incubation with 3E8 fully abolished the infectivity of all pseudoviruses, with IC50 values at 0.1, 0.1, 0.07, 0.3, 0.08, 0.2 and 1.1 nM, respectively (Fig. 3H).
In comparison, B38, a SARS-CoV-2 RBD-targeting antibody currently under clinical development 36, could only suppress the infectivity of SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7 and B.1.617, but not B.1.351, SARS-CoV or HCoV-NL63. The suppression of 3E8 was not only broader but also remarkably more efficacious and potent, as the IC50 values of 3E8 were hundreds of folds improved when compared to that of B38 (Fig. 3H). The ACE2-Fc fusion protein, a virus RBD-targeting molecule consisting of the extracellular domain of human ACE2 and the Fc region of human IgG1, showed broad but mediocre blocking ability on pseudoviruses. Our investigation indicated that 3E8 is potentially a powerful and broad-spectrum blocker on coronaviruses that are dependent on ACE2.
Future Approach
At present, there is no antibody that can completely fight the new coronavirus. Both b38 and ace2fc can only target one virus strain. However, as the number of infected people shows a rebound trend, mutated virus strains continue to mutate. Viruses use the spike protein to bind to the human body's ace2 receptor to cause the human body to infect the new crown, but traditional viral antibodies all interfere with the virus's spike protein, or first bind to the virus's spike protein. Because traditional antibodies can only target some virus strains, because the spike protein of each virus strain is different, each antibody can only target the spike protein of a specific virus. So our team focuses on the ace2 receptor. Our antibody restricts ace2 and prevents ace2 from binding to the new coronavirus, so as to avoid human infection. Our antibody fundamentally refuses the binding of ace2 receptor to the virus, so no matter what kind of virus strain, our antibody is effective.
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