Structure-guided bispecific antibody engineering confers broad protection against KP.3.1.1 and sarbecoviruses

Figures

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  Figure 1. Binding properties and affinities of bsAbs in different formats (A and B) Binding of 7F3 and GW01 to the RBD proteins of SARS-CoV-2 variants. (C) Schematic diagrams of the structures of bispecific antibodies. (D) Molecular size analysis of bsAbs was conducted using cross-linking SDS-PAGE and traditional SDS-PAGE techniques. (E) Binding of bsAbs to the RBD proteins of SARS-CoV-2 variants, including EG.5 and BA.2.86, as assessed through ELISA. S309 served as a control antibody. (F) The binding affinities (KD values) of bsAbs to the RBDs of SARS-CoV-2 variants were quantified using biolayer interferometry (BLI), the parental antibodies 7F3 and GW01, as well as S309 were used as reference controls. See also Figures S1 and S2.

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  Figure 2. Potency and breadth of bsAbs (A) Neutralization activity of bsAbs and their parental antibodies 7F3 and GW01 against seven pseudoviruses. S309 was included as a positive control. (B) Neutralization breadth and potency of bispecific antibodies G7-Fc and 7G-Fc against the SARS-CoV-2 variants and sarbecoviruses. S309, parental antibodies 7F3 and GW01 as well as their mixture were used as controls. (C) Neutralization of authentic EG.5 viruse by G7-Fc. Data are represented as mean ± SEM.

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  Figure 3. Negative-Stain (NS) micrographs and cryo-EM structures of BA.2.86 S trimer in complex with the bispecific antibodies (A) NS micrographs of BA.2.86 S trimer in complex with the bispecific antibodies G7-Fc, 7G-Fc, G-Fc-7, and 7-Fc-G. Scale bar, 100 nm (B) G7-Fc IgG binding to BA.2.86 S trimer, forming a trimer dimer. S trimers are depicted as gray, with GW01 scFv and 7F3 scFv shown in orange and cyan, respectively. IgG Fc region is illustrated as a gray box. One complete G7-Fc IgG molecule is circled with a green dashed line. (C) Schematic diagram of bispecific antibody G7-Fc. (D) The interaction of BA.2.86 S Trimer 1 with three G7-Fc molecules. Three S monomers are colored in different shades of gray. BA.2.86 RBD mutations relative to BA.2 and XBB.1.5 are colored in red and purple. Three G7-Fcs are depicted as transparent volumes, and the linkers are shown as black lines. The epitopes of GW01 and 7F3 are colored in yellow and cyan, respectively. Mutations W452, K481, and K484 (purple) are involved in the 7F3 epitopes. (E) mAbs binding to the RBD of WT in competition with ACE2 was assessed through BLI. (F) 7F3 binding to the RBD of WT in competition with other antibodies. The HIV-1…

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  Figure 4. Epitope mapping, interaction network, and mutation validation of GW01 scFv and 7F3 scFv (A) Structure of the BA.2.86 RBD complex with one GW01 scFv and one 7F3 scFv. The RBD is depicted as a gray volume, with the RBM colored in pink. Epitopes, classified by Barnes RBD-targeting antibodies, are highlighted with curved lines. GW01 scFv and 7F3 scFv are shown in transparent orange and cyan, respectively. (B) Hydrogen bonding network between GW01 scFv and the RBD. Six hydrogen bonds and their corresponding residues are labeled. (C) Hydrophobic interaction network between GW01 scFv and the RBD. The hydrophobic surface is colored in yellow, with the residues involved labeled accordingly. (D) Hydrogen bonding network between 7F3 scFv and the RBD. Sixteen hydrogen bonds and their corresponding residues are labeled. (E) Hydrophobic interaction network bewteen 7F3 scFv and the RBD. The hydrophobic surface is colored in yellow, with the residues involved labeled accordingly. (F) Neutralization of G7-Fc against the BA.2.86 single mutants. The residues of BA.2.86 spike involved in hydrogen bonding interaction were mutated to alanine, individually. (G and H) BsAbs binding to the RBDs of EG.5…

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  Figure 5. Structure-guided optimization of G7-Fc overcomes KP.3.1.1 immune evasion (A) Neutralization of KP.3.1.1 by G7-Fc. (B) Close-up view of the hydrophobic interaction between Y229 of 7F3 and the BA.2.86 RBD. (C) Close-up view of a potential steric clash between Y229 of 7F3 and the JN.1 RBD, caused by the L455S mutation (indicated by a red dashed line). (D) Surface electrostatic potential maps of RBDs from BA.2.86, JN.1, KP.2, and KP.3.1.1, with the 7F3 scFv shown as a sky-blue ribbon. (E) Predicted contacts between L455S and Q493E mutations in KP.3.1.1 and the 7F3 scFv, with atomic pairs connected by green dashed lines (distance ≤4.0 Å). (F) Predicted contacts between the L455S mutation and the Y229N mutation of G7-Fc (yellow dashed lines). (G) Predicted interactions between the Q493E mutation and the A227S or A227T mutations in G7-Fc. Single point mutations based on the BA.2.86 RBD structure in C–E were predicted by Dunbrack rotamers.37 (H) Neutralization activity of G7-Fc variants harboring A227S/T and Y229N mutations against KP.3.1.1.

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  Figure 6. Prophylactic and therapeutic efficacy of G7-Fc via various administration routes (A) Schematic representations illustrating the preventive (P) and therapeutic (T) applications of G7-Fc against EG.5 infection. G7-Fc was administered either intraperitoneally (i.p.) or intranasally (i.n.) in K18-hACE2 mice (n = 5 for prophylactic and therapeutic groups, and n = 4 for PBS group). (B) EG.5 viral titers in lung tissues, measured as focus-forming units (FFU), were assessed 48 h post-infection. Each data point corresponds to an individual mouse within the respective groups. Statistical significance was determined using unpaired t-tests in GraphPad Prism software (version 9), and p < 0.001 is denoted as ∗∗∗, p < 0.0001 is denoted as ∗∗∗∗. Data are represented as mean ± SEM.