Novel Trispecific Neutralizing Antibodies With Enhanced Potency and Breadth Against Pan‐Sarbecoviruses

Figures

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  FIGURE 1. Construction and characterization of different forms of bispecific antibodies. (A) Virtual structure of the prefusion stabilized Omicron XBB S ectodomain trimer in complex with PW5‐5, PW5‐535, and PW5‐570. Surface representation of RBD showing the buried binding site, including PW5‐5 (cyan), PW5‐535 (blue), and PW5‐570 (green). (B) The SARS‐CoV‐2 WT trimer was captured onto the HIS1K biosensors. Then, PW5‐5 was used to saturate the trimer binding sites and the association of PW5‐535 and PW5‐570 was measured sequentially. (C) Schematic diagram of the molecular configurations of prototype antibodies (PW5‐5, PW5‐535, and PW5‐570) and distinct formats of bsAbs. KiH, the Knob‐into‐Hole design with the S354C and T366 W mutations in the heavy chain CH3 region on the Knob arm, and the Y349C, T366S, L368A, and Y407A mutations in the CH3 region on the Hole arm. SC, (scFv)2‐Fc format, which is composed of two scFv conjugated to the N‐terminal of the Fc. GF, IgG‐(scFv)2, which is composed of one scFv conjugated to the C‐terminal of another antibody IgG1. (D) Schematic presentation of the bsAbs. HC, heavy chain. (E) Heatmap showing the neutralization IC50 values of each Ab with indicated…

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  FIGURE 2. Construction and characterization of novel trispecific antibodies. (A) Schematic diagram of the molecular configurations of trispecific antibodies, Tri‐1 and Tri‐2. Analysis of protein expression by non‐reduced (NR) and reduced (R) SDS‐PAGE. Antibody domains are colored by structural architecture (deep orange and orange, variable heavy and light chain of PW5‐5; deep blue and blue, variable heavy and light chain of PW5‐535; deep green and green, variable heavy and light chain of PW5‐570). (B) Schematic representation of Tri‐1. (C) Purification of indicated antibodies by the size exclusion chromatography (SEC). (D–F) Binding kinetics of trispecific antibodies and prototype antibodies to the RBD of SARS‐CoV (D), SARS‐CoV‐2 WT (E), and XBB.1.16 (F). (G and H) Summary of the data (G) or scatter plot (H) of the affinities (K D), association (K on), and dissociation (K off) of indicated antibodies to the RBDs of SARS‐CoV, SARS‐CoV‐2 WT, and Omicron XBB.1.16, as measured by the bio‐layer interferometry (BLI).

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  FIGURE 3. Binding properties of novel trispecific antibodies. (A) Recombinant human ACE2 was immobilized on HIS1K sensors, followed by the addition of a mixture containing the indicated antibodies and SARS‐CoV‐2 WT spike trimer. As a positive control, the antibody was instead by PBST buffer, and then was loaded onto the human ACE2 immobilized biosensor (gray). (B) The XBB trimer was captured onto the HIS1K biosensors. Then, PW5‐5, PW5‐535, and PW5‐570 were sequentially used to saturate the trimer binding sites, and the association of Tri‐1 or Tri‐2 was subsequently measured. (C) The XBB trimer was initially captured onto the HIS1K biosensors, following the indicated prototype antibody (PW5‐5, PW5‐535, or PW5‐570) was injected onto the RBD‐connected biosensors, which have already saturated with Tri‐1 (left) or Tri‐2 (right). (D and E) The XBB.1.16 RBD was captured onto the HIS1K biosensors at 40, 200, and 1000 ng/mL for 300 s, respectively. Summary of the data (D) or scatter plot (E) of the affinities (K D), association (K on), and dissociation (K off) of Tri‐1‐ and structural‐related mAbs and bsAbs to indicated concentrations RBD.

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  FIGURE 4. Pseudovirus neutralization of novel trispecific antibodies against the current SARS‐CoV‐2 Omicron variants and other sarbecoviruses. (A) Neutralization by indicated antibodies against the current SARS‐CoV‐2 Omicron variants, including BF.7, XBB.1.5, XBB.1.16, XBB.2.3.3, BA.2.86, EG.5.1, HK.3, JN.1, JN.1.7, KP.2, and KP.3.1.1. (B) Neutralization curves of SARS‐CoV‐2‐ (Pangolin CoV‐GD and Bat CoV RaTG13) and SARS‐CoV‐related sarbecoviruses (Bat WIV1 and SHC014) by indicated antibodies. (C and D) Radar charts were drawn based on the IC50 values of indicated antibodies against the current SARS‐CoV‐2 variants (C), and SARS‐CoV‐ or SARS‐CoV‐2‐ related sarbecoviruses (D). All neutralization assays were repeated at least three times and are presented as the mean ± SD.

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  FIGURE 5. Neutralization of novel trispecific antibodies against authentic SARS‐CoV and SARS‐CoV‐2 variants. (A and B) Neutralization curves (A) and IC50 values (B) of trispecific antibodies and their prototype antibodies against authentic SARS‐CoV and major SARS‐CoV‐2 Omicron variants, including BA.1, XBB.1, EG.5.1, JN.1, JN.1.7, KP.2, and KP.3.1.1. The data represent one of at least three independent experiments and are presented as the mean ± SD.