Novel sarbecovirus bispecific neutralizing antibodies with exceptional breadth and potency against currently circulating SARS-CoV-2 variants and sarbecoviruses

Figures

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  Fig. 1. Isolation of sarbecovirus-neutralizing antibody GW01 from COVID-19 patients. a Binding of five mAbs isolated from COVID-19 patients to the RBD proteins of SARS-CoV-2, RaTG13, GD-Pangolin, GX-Pangolin, and SARS-CoV in ELISA. b Binding affinity of GW01 to RBD proteins of SARS-CoV-2, RaTG13, GD-Pangolin, GX-Pangolin, or SARS-CoV was measured by bilayer interferometry experiments. c Neutralization of mAbs against pseudovirus of SARS-CoV-2 and its variants B.1.1.7, B.1.351, B.1.617.1, B.1.617.2, B.1.1.529, and C.37, SARS-CoV, and bat SARSr-CoVs (WIV1 and Rs3367). d Neutralization summary of GW01, S309, and other NAbs against pseudoviruses. e Neutralization of GW01 against authentic SARS-CoV-2 and its variants B.1.1.7, B.1.351, B.1.617.2, and B.1.525. f Neutralization of GW01 against authentic bat SARSr-CoVs (WIV1 and SHC014). g IC50s of GW01 against seven authentic viruses.

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  Fig. 2. GW01 binds to an RBD epitope distinct from that of REGN10989 and overlaps with the ACE2-binding site. a Binding of GW01 to SARS-CoV-2 RBD (left) and SARS-CoV RBD (right) in competition with S309 and REGN10989 as measured by bilayer interferometry experiments. S309 was used as a control. b Binding of ACE2 to SARS-CoV-2 RBD (left) and SARS-CoV RBD (right) in competition with GW01 (red), S309 (blue), REGN10989 (green), control IgG1, and ACE2.

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  Fig. 3. GW01 targets a conserved RBD epitope shared by the spike proteins of different sarbecoviruses. a The domain-colored cryo-EM structure of GW01 bound with S-ECD is shown in two perpendicular views. The heavy (blue) and light (pink) chains of GW01 bind to two “up” RBDs. Two up RBD monomers are colored orange and light blue, while the down RBD is colored gray. b Structural comparison of epitopes between ACE2 (green) and GW01 bound to RBD. The black circle includes extensive hydrophilic interactions on the interface between GW01 and RBD. c The binding epitope of GW01 bound to the RBD of S-ECD is colored blue, shown on the left, and is observed to overlap with the green-colored epitope of ACE2 bound to RBD on G502 (red ring). d Detailed analysis of the interface between GW01 and RBD. Polar interactions are indicated by red dashed lines. e Epitope of GW01. Residues that decreased neutralization sensitivities (fold change >10) of GW01. Conservation of residues F374, K378, R408, P463, and G504 in SARS-CoV-2 variant sequences (f) and different sarbecoviruses (g). h The RBD epitopes of S2A4 (PDB ID: 7JVC), S304 (PDB ID: 7JW0), and CR3022 (PDB ID: 6W41), which are colored cyan, red, and…

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  Fig. 4. Neutralization of GW01-related bispecific antibodies against the currently circulating SARS-CoV-2 NAb-escape variants and sarbecoviruses. a Neutralization potency and breadth of mAbs against 56 currently circulating variants. Identification of putative NAb-escape variants by screening 56 currently circulating SARS2-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.2, with sarbecovirus NAbs GW01, 6I18, and S309, as well as SARS-CoV-2 NAbs 3D13, 10C2, 22H22, CC12.1, and COVA1-21. b Bispecific antibody sequence and structure diagram. c Neutralization potency and breadth of GW01-related bispecific antibodies against a panel of 20 NAb-resistant variants, SARS-CoV and 2 bat SARSr-CoVs (WIV1 and Rs3367). d Neutralization of G9 against authentic SARS-CoV-2 and its variants B.1.1.7, B.1.351, B.1.617.2, and B.1.525, and authentic GX-pangolin. e Binding affinities of G9, GW01, and S309 binding to RBD proteins of five sarbecoviruses. f Neutralizing mechanism of bispecific antibody G9. Key mutated residues that can affect the neutralizing activity of the antibodies are mapped on the RBD. The purple block is for REGN10989, the blue block is for GW01, and the green block is for both…

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  Fig. 5. Prophylactic and therapeutic effect of G9 against SARS-CoV-2 infection of lung, brain, and associated disease in hACE2-transgenic mice. a Experimental scheme of the animal model. b Bodyweight changes in SARS-CoV-2-infected HFH4-hACE2-transgenic mice in the prevention group (P), treatment group (T), and PBS group of GW01 and G9. The percentage of weight change was calculated on day 5 for all animals. The symbol “+” signifies that the mice died. c, d Viral load as assessed by S gene RNA RT–PCR from lung tissue of the GW01 and G9 groups (c) or brain tissue from the G9 groups (d) on day 5 post infection. e Pathological changes in HFH4-hACE2-transgenic mouse lung on day 5 after SARS-CoV-2 infection. Mouse lung tissue was stained with H&E to observe pathological changes. Scale bars, 200 μm. f Viral antigen was detected by anti-SARS-CoV-2 N protein polyclonal antibody (red) in lung bronchi and alveoli collected 5 DPI. Images were collected using a Pannoramic MIDI system (3DHISTECH, Budapest). Scale bars, 100 μm.