Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3

Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3 Jianwei Zhu, Yunji Liao, Haoneng Tang, Hang Ma, Huifang Zong, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7686776/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Given the continuously mutating nature of SARS-CoV-2, the sensitivity of most monoclonal therapeutic antibodies has decreased to the mutants. Bispecific antibodies (BsAbs), with their unique antiviral mechanisms that enable simultaneous binding to two epitopes, offer a distinct advantage in combating the continuous viral mutation by preventing immune escape and enhancing viral neutralization. In this study, we synthesized 67 bsAbs based on the epitope distribution from antibodies isolated using single B-cell cloning from convalescent patients and phage display, 11 of which showed superior neutralization of WA1/2020 compared to their parent antibodies. One bispecific antibody (9A6-6C3), exhibiting 100-fold greater neutralizing activity than its parent antibodies, efficiently neutralized various SARS-CoV-2 variants (IC50 < 100ng/mL). Structural analysis indicates that 9A6 binds to the H-RBD epitope, encountering spatial conflict with the NTD of neighboring S monomer, while 6C3 is capable of binding to a conserved loop on S2. In vitro evidence demonstrates that 9A6-6C3 promotes the disassembly of the S protein, exposing S2, which likely contributes to its broad-spectrum neutralizing activity. In summary, we discovered a potential broad-spectrum mechanism and presented an epitope design strategy for bsAbs, offering valuable insights for the design and development of bsAbs in the fight against COVID-19. Biological sciences/Immunology/Infectious diseases/Viral infection Biological sciences/Structural biology/Molecular modelling Biological sciences/Drug discovery/Biologics/Antibody therapy neutralizing antibody SARS-CoV-2 bispecific antibody Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Although the symptoms of COVID-19 infection have significantly alleviated compared to the early stages, about 10%~20% of patients still experience post-infection sequelae three months after infection( 1 – 3 ). Studies indicated that reinfection with SARS-CoV-2 may cause further damage to the human body( 4 ). Therefore, the development of drugs against COVID-19 remains of great value and significance. Among all treatment methods, neutralizing antibodies (nAbs) have significant advantages due to their specificity, effective binding capabilities, pharmacokinetics studied, and the ability to be produced on a large scale( 5 – 7 ). The spike (S) protein of SARS-CoV-2 controls the virus’s invasion process, making it an ideal target for developing neutralizing antibodies( 8 , 9 ). The development of neutralizing antibodies against SARS-CoV-2 has been remarkably successful, with several antibodies showing great antiviral effects and being approved by regulatory agents for the treatment and prevention of the virus( 6 , 10 , 11 ). However, as a single-stranded RNA virus, SARS-CoV-2 continues to mutate, causing many monoclonal antibodies to lose their antiviral activity( 12 – 14 ). One solution to this problem is to combine monoclonal antibodies targeting different epitopes to produce antibody cocktails or bsAbs( 15 , 16 ). To date, several bsAbs have been developed to combat SARS-CoV-2, most of these bsAbs simultaneously target different epitopes on the receptor-binding domain (RBD) to maximize contact with residual residues( 17 – 21 ). Yuan et al. developed a bispecific antibody that can simultaneously target both RBD and S2, exhibiting superior neutralization breadth( 22 ). Cho et al. developed a bispecific antibody targeting RBD and the N-terminal domain (NTD), but it did not demonstrate improved neutralization activity( 23 ). The antiviral mechanisms of bsAbs remain incompletely characterized, particularly in terms of their interactions with distinct subunits of the S protein. Additionally, the potential synergistic effects between the two parent antibodies used to create these bsAbs remain unclear. In this study, based on previous research progresses, we classified the binding epitopes of 33 antibodies isolated using single B-cell cloning from convalescent patients and phage display into four types using competitive ELISA. Additionally, we identified one S2-binding antibody (6C3) with potent neutralizing activity, which served as a parent antibody for bispecific design. Sixty seven bsAbs were prepared through Bispecific Antibody by Protein Trans-splicing (BAPTS) platform ( 24 , 25 ), from which 11 have enhanced neutralizing capabilities, including 9A6-6C3. 9A6-6C3 effectively neutralized various SARS-CoV-2 subtypes, including the widely spread Alpha, Beta, Gamma, Delta, and Omicron BA.1 variants. 9A6 binds to H-RBD epitope( 26 ), in both “up” and “down” conformations of the RBD. This binding site would spatially clash with the NTD domain of an adjacent S protomer, which may lead to, we propose, the degradation of the S trimer protein and thus exposing S2. Exposing S2 may promote the binding of 6C3 to the conserved loop in the S2 subunit. The antiviral mechanism of 9A6-6C3 was investigated using Western blot and cell-based assays. In Jurkat-S cells, 9A6-6C3 was able to promote extensive abnormal cleavage of the S protein, leading to the release of the S1 subunit, while 9A6 also caused degradation of the full-length S protein. Unlike the parent antibodies, 9A6-6C3 promoted the exposure of S2. In the assay system using pseudoviruses, 9A6-6C3 could lead to extensive cleavage of the S protein on the virus surface. These results suggested that designing bsAbs that simultaneously bind to H-RBD epitope and S2 may effectively induce the cleavage of the viral S protein and promote the exposure of S2, providing a reference for the design of bsAbs against SARS-CoV-2. Materials and Methods Epitope Binning Assay To evaluate the epitope correlation between two antibodies, a competition enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, the first antibody was coated onto plates (BEAVER) at a concentration of 2 μg/mL and incubated overnight at 4°C. After washing off the excess antibody with PBS, the plates were blocked with 3% skim milk. SARS-CoV-2 S1 protein (Sino Biological) was biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (ThermoFisher) and then incubated with 50 μg/mL of the second antibody or PBS as a control. The mixture was incubated at 37°C for 1 hour. Following incubation, the plates were washed three times with PBS, and diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma) (1:2000) was added. After a second incubation at 37°C for 1 hour, the binding of S1 protein to the coated first antibody was detected using TMB Single-Component Substrate Solution (Solarbio). Absorbance was measured at 450 nm wavelength using the Infinite M200 PRO Multimode Microplate Reader (TECAN). The competitive binding percentage of the two antibodies was calculated by comparing to the PBS control. Bispecific Antibody Production BsAbs were designed and generated using the Bispecific Antibody by BAPTS platform, as previously described. Fragments A and B were selected from two antibodies with minimal epitope clash. To produce fragment A, three plasmids (pCDNA3.4) encoding the full-length heavy chain A, light chain A, and the Fc region of heavy chain B fused to the C-terminal half-intein were co-transfected into ExpiCHO-S cells. For fragment B, two plasmids (pCDNA3.4) containing the light chain B and Fab region of heavy chain B fused to the N-terminal half-intein were co-transfected into the cells. Fragment A and fragment B were purified using HiTrap KappaSelect or HiTrap LambdaSelect columns (Cytiva), using glycine (100 mM, pH 2.5) or acetic acid (100 mM, pH 3.0) as elution buffers. To generate bsAbs, fragments A and B were mixed in a molar ratio of 1:1.6, in the presence of 2 mM Tris-(2-carboxyethyl)-phosphine (TCEP), and incubated for 4 hours at room temperature. The reaction was terminated by adding 2 mM dehydroascorbic acid. The bsAbs were initially purified using Protein A resin (GE Healthcare) and further purified by HiTrap Capto MMC ImpRes (Cytiva). A linear gradient elution was used for Capto MMC purification, transitioning from 10 mM phosphate + 10 mM Tris + 50 mM NaCl (pH 6.0) to 10 mM phosphate + 10 mM Tris (pH 9.0). The final bsAbs, with purity greater than 98% as confirmed by size exclusion chromatography, were concentrated and stored at −80°C. Monoclonal Antibody Production Plasmids containing the full-length of antibody heavy chain or light chain were amplified in E. coli DH5α and extracted by using PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen). Antibodies were expressed using the ExpiCHO™ expression system (Thermo Fisher). The cell culture process was implemented following the manufacturer's recommendations. The cell culture medium was collected 12 days post transfection and centrifuged at 300×g for 10 min to remove the cells. After further centrifugation at 7000×g for 30 min for removing the cell debris, the supernatant was subjected to MabSelect Sure (Cytiva) antibody affinity purification. In an AKTA avant protein separation and purification system (Cytiva), the MabSelect Sure column was equilibrated with 2 column volumes of 20 mM PB + 150 mM NaCl (pH 7.2). Then the medium samples were loaded and washed with 10 column volumes of 100 mM citric acid (pH 5.0). Antibodies were eluted with 5 column volumes of 100 mM citric acid (pH 3.0), followed by storage in a cocktailed buffer containing 10 mM Histidine-HCl, 9% trehalose, and 0.01% polysorbate 80. Binding ELISA ELISA was applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems). Antigens were diluted with ELISA Coating Buffer (Solarbio) to 1.0 μg/mL and immobilized onto High Binding ELISA 96-Well Plate (BEAVER) with 100 μL per well overnight at 4°C. Plates were washed four times with PBST (Solarbio) and blocked with 3% skim milk for 1 h at 37°C. Then, serially diluted antibodies were added 100 μL per well and incubated at 37°C for 1 h. After pipetting off the unbound antibodies, plates were washed four times with PBST and further incubated with 100 μL per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1:5000 dilution, Sigma) for 1 h at 37°C. After a final four times washing with PBST, the binding of antibodies with SARS-CoV-2 antigens were visualized by adding 100 μL peroxidase substrate TMB Single-Component Substrate solution (Solarbio) and incubating for 15 min in dark. The reaction was terminated by adding 50 μL stop buffer (Solarbio) and the plates were immediately submitted to an ELISA microplate reader (TECAN Infinite M200 Pro) to measure the optical density (OD) at 450 nm. Data were analyzed with GraphPad Prism Version 9.0.0 and EC 50 values were determined using a four-parameter nonlinear regression. ACE2 competition ELISA For experiments involving competitive binding of antibodies to SARS-CoV-2 RBD or the S trimer, the recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher), following the manufacturer’s instruction. SARS-CoV-2 RBD (Sino Biological), the S-trimer (AcroBiosystems), mutated RBD (Sino Biological) and mutated S-trimer (AcroBiosystems) were coated on high binding ELISA 96-well plates (BEAVER). To obtain the optimal hACE2-Fc concentration for this experiment, concentration-dependent binding of biotinylated hACE2-Fc to encapsulated SARS-CoV-2 antigen was measured by performing a conventional receptor-binding ELISA. 80% maximum effective concentration (EC 80 ) of biotinylated hACE2-Fc was calculated by a four-parameter nonlinear fit. The antibody was serially diluted in 1% BSA (Sigma) and 50 μL was added to the plate. The EC 80 concentration of biotinylated hACE2-Fc was subsequently pipetted in. After incubation for 1 h at 37°C, the plates were washed four times with PBST (136.9 mM NaCl，2.68 mM KCl, 10 mM Na 2 HPO 4 •12H 2 O, 2 mM KH 2 PO 4 , 0.1% (v/v) Tween20, pH7.4) (Solarbio) and incubated with 100 μL of 1:2000 dilution of hypersensitive streptavidin-oxidase polymer (Sigma). After further washing, 100 μL of TMB (3, 3′,5 ,5′-Tetramethylbenzidine) (Solarbio) was added and then bound hACE2 was detected in a microplate reader. four-parameter nonlinear regression fitting was applied in GraphPad Prism version 9.0.0 for analysis of the results. Generating 293T-ACE2 cells Human ACE2 was stably expressed on the surface of HEK-293T cells (ATCC, CRL-3216) to obtain the 293T-ACE2 cells. In this work, a three-plasmid lentivirus transfection system was used. The ACE2 gene was inserted into the pHIV-puro plasmid, and prepared together with the packaging plasmids psPAX2 and VSV-G using an Endo-free Plasmid Mini Kit (Omega). HEK-293T cells were seeded into a 10 cm dish and cultured at 37 °C under 5% CO 2 condition. After the cells reached 70%-80% confluence, plasmids were co-transfected into cells using Lipofectamine 3000 reagent (Thermo Fisher). The cell medium was replaced with fresh DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) 6h later. After further culture for 48 h, the medium supernatant was collected and centrifuged at 3000×g for 10 min. The supernatant was transferred to a 15 mL centrifuge tube and mixed with the Lentivirus Concentration Reagent (Genomeditech) for overnight incubation. The next day, the mixture was centrifuged at 3220×g for 30 min, followed by resuspending the viral particles with 200 μL DMEM medium. For infecting HEK-293T cells, 5 × 10 5 cells were seeded into a 6-well plate and incubated overnight at 37 °C with 5% CO 2 . Then 10 μL of concentrated virus was added. 48 h later, cells were transferred to a 10 cm dish, and puromycin (Beyotime Biotechnology) at final concentration of 10 μg/mL was added. Fluorescence activated cell sorting was used to further select the cells with high expression of ACE2. Using S1-mFc recombinant protein (Sino Biological), a DNA sequence encoding the SARS-CoV-2 (2019-nCoV) spike protein S1 Subunit (YP_009724390.1) (Val16-Arg685) was expressed with the Fc region of mouse IgG1 at the C-terminus, as the primary antibody and FITC-labeled goat anti-mouse IgG antibody (Jackson) as the secondary antibody, the cells in the top 1% of fluorescence intensity were obtained on a BD FACSJazz cell sorter (BD). The top 1% cells were expanded for pseudovirus neutralization experiments. SPR Assay The antigen was diluted in acetate (Cytiva) at pH 5.0 and covalently coupled to the chip using an amine coupling kit (Cytiva). After reaching a coupling level of 15 RU, excess antigen is washed off and unbound sites are blocked with ethanolamine. The antibodies were serially diluted 2-fold from 1.250 to 0.039 μg/mL with HBS-EP buffer (Cytiva) and then injected into the chip at 30 μL/min for 120 seconds. Afterwards, the conjugates were dissociated with HBS-EP buffer for 300 or 600 seconds, followed by chip regeneration with glycine (Cytiva) at pH 1.5. Parameters including Ka, Kd, and KD values were calculated using the monovalent analyte model of BIAevaluation software. Pseudovirus packaging and neutralization The S sequences of SARS-CoV-2 (GenBank: QHD43416.1) or variants with intracellular 21 residue deletion were inserted into the pMD2.G plasmid. A luciferase gene was constructed into plasmid pCDH-luc1 as a reporter. Pseudoviruses were obtained as the lentivirus packaging method described above. For testing pseudovirus neutralizing activity by antibodies, 293T-ACE2 cells were seeded into a white 96-well plate (Corning) at a density of 1 × 10 4 per well, and cultured overnight at 37 °C with 5% CO 2 . Serial 10-fold dilutions of antibodies from 200 μg/mL were mixed with equal volume of diluted pseudoviruses in DMEM medium (with 10% FBS). Medium containing the same amount of pseudovirus but without antibody was used as infection control. After incubation at 37 °C for 30 min, the medium of the 293T-ACE2 cells was replaced with 100 μL of antibody-pseudovirus mixture. All operations involving pseudoviruses were performed in a biosafety level 2 laboratory of the School of Pharmacy at Shanghai Jiao Tong University. After further culturing for 48 hours, 50 μL of ONE-Glo™ Luciferase Assay System substrate (Promega) was added to each well, and the fluorescence intensity was immediately measured in a microplate reader (TECAN). Data were analyzed using GraphPad Prism version 9.0.0 software, and the IC 50 values were calculated using a four-parameter nonlinear regression function. Structural Modeling The structure of 9A6 in complex with the SARS-CoV-2 RBD was predicted using Swiss-model (https://swissmodel.expasy.org/). The 6C3 and S2 (amino acids 699-1053) complex structure was predicted using AlphaFold3 (https://alphafold.ebi.ac.uk/). All structural visualizations were generated using Chimera (https://www.cgl.ucsf.edu/chimera/). Overlapping Peptide Assay To identify the binding region of 6C3, a set of overlapping 15-mer peptides, each biotinylated at the N-terminal, covering the S2 exposed regions of SARS-CoV-2 spike protein, were synthesized (Genscript). Recombinant streptavidin (Thermo Fisher) was coated onto a 96-well plate at a concentration of 4 μg/mL overnight. The following day, after blocking and washing, each overlapping peptide was added to the wells at a concentration of 4 μg/mL and incubated for 1 hour at 37°C. After four washes, mAb 6C3 was added at a concentration of 10 μg/mL and incubated for another hour at 37°C. Following incubation, plates were washed and incubated with 100 μL of goat anti-human IgG (Fc-specific)-peroxidase secondary antibody (Sigma) diluted 1:5000 for 1 hour at 37°C. After a final wash, TMB substrate solution was added and incubated for 15 minutes in the dark, followed by the addition of stop buffer to terminate the reaction. The optical density at 450 nm was measured using a microplate reader (TECAN Infinite M200 Pro). Study on the Antiviral Mechanism of 9A6-6C3 by Western Blot To investigate the antiviral mechanism of 9A6-6C3, Western blotting was employed to analyze the degradation of the SARS-CoV-2 spike protein on the surface of S-Jurkat cells. S-Jurkat cells were incubated with 50 μg/mL of 9A6, 6C3, or 9A6-6C3 for 12 hours or 6 days, and the levels of the S1 and S2 subunits of the S protein were determined. Cell lysates were harvested, and protein concentrations were quantified using a BCA assay. Equal amounts of protein were loaded and separated by SDS-PAGE, followed by transferring to PVDF membranes. Membranes were then probed with primary antibodies against S1 and S2 subunits, followed by HRP-conjugated secondary antibodies. Signal detection was performed using the ECL detection system, and the protein bands were quantified using ImageJ software. Additionally, the ability of 9A6-6C3 to cleave the S protein during pseudovirus infection was assessed. S-Jurkat cells were exposed to pseudovirus in the presence of 9A6-6C3, and the levels of cleaved S protein were detected by Western blot. The effect of TMPRSS2-mediated degradation of the S protein on 9A6-6C3-mediated cleavage was also evaluated by pre-treating S-Jurkat cells with a TMPRSS2 inhibitor, followed by incubation with 9A6-6C3, and assessing the degradation of the S protein as described. Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay Jurkat cells stably expressing the SARS-CoV-2 spike protein were seeded into 96-well plates on the day of the experiment. Antibodies were prepared at serially diluted concentrations and added to the wells, followed by incubation for 30 minutes at 37°C. Peripheral blood mononuclear cells (PBMCs) were then added as effector cells at an effector-to-target (E:T) ratio of 25:1. The co-culture was incubated for either 8 or 21 hours to allow for cytotoxicity induction. Cytotoxicity was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Kit (Promega) according to the manufacturer's protocol. OKT3 was included as a positive control in all assays. Antibody-Dependent Cellular Phagocytosis (ADCP) Assay To differentiate macrophages, CD14 + monocytes were cultured in the presence of M-CSF for 7 days. Differentiated macrophages were subsequently stained with Violet dye (PB450). In parallel, Jurkat-S cells were labeled with CFSE dye and incubated with serial dilutions of the test antibodies for 15 minutes at 4°C. Following this pre-incubation, the antibody-bound Jurkat-S cells were added to the macrophages and co-incubated at 37°C for 30 minutes to allow phagocytosis to occur. After incubation, the macrophages were digested, fixed, and analyzed using flow cytometry. Macrophages that had phagocytosed Jurkat-S cells were identified as PB450 + FITC + populations. The percentage (%) of ADCP was calculated as the proportion of PB450 + FITC + cells relative to the total PB450 + macrophages. Complement-Dependent Cytotoxicity (CDC) Assay Jurkat cells stably expressing the SARS-CoV-2 spike protein were seeded into 96-well plates on the day of the experiment. Serial dilutions of antibodies were prepared and added to the cells, followed by the addition of normal human complement serum to a final concentration of 25%. The plates were incubated at 37°C for 18 hours. After incubation, the remaining viable cells were quantified using the CellTiter-Glo Reagent Kit. Alternatively, cell death was assessed by staining with propidium iodide (PI) and analyzing the samples using flow cytometry to determine the percentage of dead cells. Role of Funders Funders did not have any role in the study design, data collection, data analyses, interpretation, or writing of this report. Results The bsAbs were designed by BAPTS platform In a previous study, several neutralizing antibodies were isolated from patients infected with the WA1/2020 virus, and an additional set of neutralizing antibodies was obtained through phage display screening(10,11,27,28). From these, 32 monoclonal antibodies with neutralizing activity were selected. Of these, 30 were derived from patient B cells, while two antibodies (R35 and R32) were sourced from the phage display library. Epitope classification of these antibodies was conducted using competitive ELISA, resulting in the identification of four distinct epitope categories. Specifically, 7G10, 8G4, 9E12, 9E2, 9D11, 7B12, 8E12, 5C12, 11F7, R32, 12F8, 8F6 and 8C12 were classified as the first category; 9A6, 5H10, 10D4, 8D8, 7D10 and 7B9 as the second category; R35, 8G9, 8D4, 9A8, 2G1 and 9B10 as the third category; 9B12, 13A12, 9C4, 11D3, 2B8, 3A4 and 5B2 as the fourth category (Fig.1a). Within each category, antibodies with superior binding activity were selected. Ultimately, antibody 11F7, 8F6, 8G4, 9D11 and 9E12 from the first category, 9D4, 7B9, 9A6 from the second category, 2G1, 8G9 and R35 from the third category, and 13A12, 2B8 and 3A4 from the fourth category were selected as the parent antibodies (Fig. 1b and 1c). Additionally, 6C3, which showed binding ability to the S2 subunit, was also selected as a parent antibody (Fig. 1d). Based on the identified epitope distribution, each parent antibody was combined with antibodies from different categories to generate novel bsAbs. Utilizing the BAPTS platform, a total of 67 bsAbs were produced within one month (Fig. 2a). The majority of the bsAbs exhibit good affinity for WA1/2020 RBD (indicated by the unmarked red curves in the graph) (Fig. 2b). However, the affinity of the bsAbs derived from 6C3 is weaker, with the exception of 8G9-6C3, 7B9-6C3, and 9A6-6C3. The competitive binding ability of these three bsAbs against ACE2 was evaluated, using the potent antibody 2G1 previously studied in our laboratory as a positive control(11). These three bsAbs did not compete with RBD for binding to various strains of the RBD or S trimer (Fig. 3a). The bsAbs with superior affinity were further evaluated for their neutralizing activity, with most displaying excellent neutralization potency (IC 50 < 100ng/ml) (Fig. 3b). Among these bsAbs, 11 had IC 50 values lower than the optimal values of the two parent antibodies, with 9A6-6C3 showing an IC 50 value that was 100 times more potent compared to the parent antibody 9A6 (Fig. 3c). Based on these findings, antibody 9A6-6C3 has been prioritized for further investigation. 9A6-6C3 is a broad neutralizer against SARS-CoV-2 infection To further validate the broad-spectrum activity of 9A6-6C3, surface plasmon resonance (SPR) analysis was performed for affinity measurement of the bsAb to SARS-CoV-2 variants. The results demonstrated that 9A6-6C3 exhibited superior affinity for various variants, with KD values predominantly in the picomolar range, particularly for the Cluster 5 variant, where a KD of 2.82 femtomoles was achieved (Fig. 4a). Omicron BA.1 S trimer was immobilized on a CM5 chip to assess whether both arms of the bispecific antibody could simultaneously bind to the S protein. After the injection of high concentrations of 9A6 or 6C3, 9A6-6C3 was still able to bind, indicating that both arms of the antibody could engage the S protein trimer simultaneously (Fig. 4b and 4c). Furthermore, when 9A6 was injected first, the RU changes caused by 9A6 and 9A6-6C3 were not significantly different (Fig. 4b). However, in the group where 6C3 was injected first, the RU change caused by 6C3 was significantly less than that caused by 9A6-6C3 (Fig. 4c), suggesting that the 6C3 epitope may be relatively inaccessible for binding under these conditions. One possible inference from these findings is that in actual binding, 9A6 may bind to the S protein first, followed by 6C3, and the binding of 9A6 would facilitate the binding of 6C3. The neutralizing potency of 9A6-6C3 against SARS-CoV-2 variants infection was evaluated using the S-pseudotyped lentivirus neutralization assay. For WA1/2020, B.1.351, B.1.1.7, P.1, Cluster 5, and Delta variants, 9A6-6C3 exhibited excellent neutralizing activity (IC 50 < 100ng/ml), especially for Beta type and Cluster 5 type, IC 50 is less than 5ng/ml (Fig. 5a-f). The neutralizing activity of 9A6-6C3 against these strains improved by 10 to 100 times compared to 9A6 alone. 6C3 did not possess neutralizing activity, but this was remedied when combined with 9A6 to form a bispecific antibody. This suggests that 9A6-6C3 may have a unique antiviral mechanism different from the parent antibodies. 9A6-6C3 binds to H-RBD epitope and the conserved loop of S2 To characterize the binding epitope of 9A6, we first used Swiss-model to simulate its binding to RBD. The simulation revealed that 9A6 binds to the side of RBD without competing with ACE2 (Fig. 6a). Subsequently, competitive ELISA was performed with several neutralizing antibodies known to bind specific epitopes, along with 9A6. The results indicated that 9A6 partially overlaps with the epitopes of 35B5 and S2H97 (Fig. 6b and 6c), which is consistent with the predicted binding mode from the Swiss-model simulation (Fig. 6a). The epitope bound by 9A6, previously reported by Chi et al., is referred to as the H-RBD epitope(26). When 9A6 binds to the Omicron S trimer, it can spatially clash with NTD, regardless of whether 9A6 binds to the “up” or “down” RBD (Fig. 6d). Peptides covering the S2 protein were used to detect binding with the 6C3 antibody, 6C3 exhibited strong binding to the “IKQYGDCLGDIAARD” peptide on the S2 protein and also binds to the “GDCLGDIAARDLICA” peptide (Fig. 6e). The epitope predicted by Alphafold3 are consistent with the experimental results (Fig. 6f). This epitope, deeply buried within the normal S protein trimer (Fig. 6g), is likely inaccessible for direct binding by neutralizing antibodies. Cheng et al. have indicated that the “IKQYGDCLGDIAARD” peptide is conserved throughout the evolution of SARS-CoV-2(29). Considering the SPR data, these results suggested that the binding of 9A6 may lead to the disassembly of the S trimer, thereby exposing the 6C3 epitope on the S2 subunit and facilitating its binding. The combined effect of 9A6-mediated S trimer disassembly and 6C3 binding to the exposed S2 epitope collectively enhances the neutralizing activity. This mechanism likely explains why the bispecific antibody exhibits significantly better neutralizing activity than its parent antibodies. 9A6-6C3 promotes the abnormal degradation of viral S protein In order to further validate whether 9A6-6C3 can promote degradation of the S protein, and to explore the differences in antiviral mechanisms between 9A6-6C3 and its parent antibodies, a series of Western blot experiments were designed. 9A6-6C3 were co-cultured with Jurkat cells overexpressing the S protein, and after 6 hours, the cells were harvested for Western blot analysis to measure the levels of S, S1, and S2 proteins (Fig. 7a and 7b). The results showed that the S2 fragment levels in the 9A6-6C3 group were significantly higher than in other groups (Fig. 7b), while the S1 fragment levels were lower (Fig. 7a). This suggests that 9A6-6C3 promote the cleavage of the S protein, leading to the shedding of S1 and the exposure of S2 protein. No significant differences in the levels of the S protein were observed, likely due to a dynamic equilibrium between the cleavage of the S protein on the cell surface and the synthesis of the S protein inside the cells. The same experimental method was used for six-day incubation, and similar results were obtained (Fig. 7c and 7d). To verify whether 9A6-6C3 could promote the cleavage of the S protein during infection, 9A6-6C3 were incubated with pseudoviruses and added to HEK293T cells overexpressing ACE2. After 6 hours, the cell-containing medium was collected for Western blotting. The amount of the S protein in the 9A6-6C3 group was significantly lower than in other groups (Fig. 7e), confirming our hypothesis. Additional experiments were designed to investigate whether 9A6-6C3 would affect the TMPRSS2-mediated cleavage process. HEK293T cells overexpressing ACE2 and TMPRSS2 were incubated with the S protein, resulting in cleavage of a small amount of the S protein compared to cells expressing ACE2 alone (Fig. 7f). The addition of TMPRSS2 inhibitors Camostat Mesilate or Bromhexine blocked this cleavage. However, when 9A6-6C3 was added in the presence of these inhibitors, the amount of the S protein still decreased sharply (Fig. 7f). This indicates that 9A6-6C3 promotes the S protein reduction independently of TMPRSS2 activity. ADCC, ADCP, and CDC functions of 9A6-6C3 To assess the ADCC function of 9A6-6C3, Jurkat cells expressing the S protein were employed by adding the antibodies with various concentrations to the cells, followed by the addition of PBMCs, with OKT3 used as a positive control. After incubating with the cells for 8 hours, the ADCC effect of 9A6-6C3 was weak (Fig. 8a). Whereas after 21 hours of incubation, 9A6-6C3 exhibited a strong ADCC effect (Fig. 8a), indicating that 9A6-6C3 can facilitate the clearance of infected cells. To evaluate the ADCP function, we labeled Jurkat-S cells and monocyte-derived macrophages with two fluorescent dyes, CFSE and violet dye, respectively. Subsequently, a series of concentrations of 9A6-6C3 were added. Flow cytometry analysis of the percentage of violet-positive macrophages containing both dyes revealed a certain phagocytic effect of macrophages on Jurkat-S cells (Fig. 8c). 9A6-6C3 exhibited measurable ADCC and ADCP functions. This suggests that it can bind to virus-infected cells and engage immune effector mechanisms. Furthermore, while the S1 protein spontaneously degrades during this process, 9A6-6C3 appears capable of persisting on the cell surface long enough to mediate these functions. This contrasts with many anti-RBD neutralizing antibodies that may fail to persist on the cell surface due to S1 shedding, potentially explaining their reduced ADCC and ADCP activity. To evaluate the CDC activity of the antibodies, different concentrations of antibodies and normal human complement serum were added to Jurkat-S cells. It was found that 9A6-6C3 possessed a weak complement activation ability (Fig. 8d and 8e). In summary, 9A6-6C3 has good ADCC and ADCP functions, as well as a weak CDC function, capable of effectively activating the human immune system to clear or kill cells infected by viruses. Discussion In the treatment of various diseases, bsAbs have shown considerable advantages, especially in cancer therapy(30–33). BsAbs have unique therapeutic mechanisms due to their ability to bind different epitopes and their potential for extensive cross-linking(34–37). Interacting with SARS-CoV-2, the bsAbs targeting the S protein can bind to distinct sites, either within a single monomer or across adjacent monomers(18,38). This dual-binding capability enables cross-linking of S proteins, which confers therapeutic advantages through novel mechanisms. However, for COVID-19, research on the antiviral mechanisms of bsAbs is limited, far less than the structural studies and antiviral mechanism research of monoclonal neutralizing antibodies. Furthermore, most currently developed bsAbs target exclusively the RBD domain and lack in-depth mechanistic investigations. Consequently, the full therapeutic potential of bsAbs remains unrealized. In this study, based on a comprehensive evaluation of monoclonal neutralizing antibodies, we have developed for the bsAbs, which can effectively accelerate the generation of the bsAbs against SARS-CoV-2. Among these bsAbs, 9A6-6C3 exhibits broad-spectrum neutralizing activity far superior to its parent antibodies. Mechanistically, it binds epitopes that induce steric hindrance with the NTD of adjacent S monomers within the trimer, while simultaneously engaging a conserved loop on the S2 subunit. Multiple experimental results indicate that the binding of 9A6 can lead to the degradation of the S protein and facilitate the binding of 6C3 to the conserved epitope on S2. Research by Chi et al. has shown that co-incubating the H-RBD antibody with the S trimer protein results in the disassembly of the S protein trimer under cryo-EM(26). Similarly, other researchers have found that antibodies with spatial hindrance to NTD of adjacent S monomer can also induce the disassembly of S trimer, even for BQ.1.1 and XBB as well(18,39,40). On the other hand, the binding site of 6C3 is deeply buried within the S trimer, which is advantageous for resisting viral mutations and also beneficial for stimulating ADCC or ADCP functions. The ‘hidden’ epitopes have always been targets for nAbs, as these more concealed epitopes are usually located inside the S trimer and are less likely to mutate. However, at the same time, most hidden epitopes are difficult for neutralizing antibodies to bind to. 9A6-6C3 does not directly block the binding of ACE2 and RBD but induces protein degradation to expose a concealed epitope for binding. Similarly, designing a bispecific antibody or cocktail therapy that can bind to the H-RBD epitope and another hidden epitope may also produce the same effect. Our research provides a new approach for the design of bsAbs against SARS-CoV-2 and also offers a new mechanistic understanding for the treatment of COVID-19. Rao et al. have demonstrated that the development of a bispecific antibody, which include one arm binding to the H-RBD epitope, can substantially restore the neutralizing efficacy of another antibody(21). This suggests that the design of bsAbs targeting the H-RBD epitope may offer a broadly applicable strategy. Highly effective monoclonal antibodies isolated from memory B cells serve as a primary source for antibody development to against never ended SARS-CoV-2 variants. However, the repeated process of isolating these antibodies is costly, time-consuming, and may not keep pace with the rapid mutation rate of the virus. In this context, leveraging existing monoclonal antibody research to develop antibodies with novel neutralizing mechanisms represents an ideal strategy. Here, we present the rapid development of a series of bsAbs through a rational approach, along with the proposal of a new antiviral mechanism, which could significantly contribute to ongoing research on SARS-CoV-2. Declarations Contributors Conceptualization: JWZ, LH Methodology: LH, YJL, HM, HFZ, YSY, MYW Investigation: YJL, HNT, HM, HFZ, LW, YK Formal analysis: YJL, HNT, HM Visualization: YJL, HM, HNT Writing - Original Draft: YJL Writing - Review & Editing: JWZ, YQX, HJ, LH Supervision: JWZ Project administration, BHZ, YLB Data sharing statement All data supporting the findings of this study are available in the paper. Declaration of interests Authors declare no competing interests. Acknowledgments We thank Jingli Hou from Institute of Translational Medicine, Shanghai Jiao Tong University, China for assistance in SPR affinity analysis. 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02:33:51","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120291,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSBIO2592760structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/2b2058ec9ba308565cf14134.xml"},{"id":93541583,"identity":"7ea73d60-5501-448d-b5aa-63095d9dc32a","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132722,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/4f2b96c893c7ed5f8f254d5a.html"},{"id":93541569,"identity":"29bfbf85-e746-4cd7-b716-b73f8b084bd1","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":925981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of parental antibodies for bispecific antibodies. a\u003c/strong\u003e Epitope analysis of 32 monoclonal antibodies derived from B cells of patients infected with WA1/2020. \u003cstrong\u003eb, c\u003c/strong\u003e Binding affinity of 14 monoclonal antibodies to RBD proteins of WA1/2020 was measured by ELISA experiments. \u003cstrong\u003ed\u003c/strong\u003e Binding affinity of 6C3 to RBD protein of WA1/2020 was measured by ELISA experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/cf737e5b54a6935fc586b98e.png"},{"id":93541566,"identity":"36b4f885-a367-43a0-a99f-dbc8f9d2596c","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":538093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and binding ability of bsAbs against WA1/2020.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic diagram of bsAbs generated by BAPTS platform. \u003cstrong\u003eb \u003c/strong\u003eScreening for bsAbs bivalent binding to SARS-CoV-2 spike trimer. WA1/2020 spike trimer was coated overnight, and serial concentrations of bsAbs and their parental mAbs were added to the plates. After incubating for 1 h at 37℃, secondary antibody was added and finally absorbance at OD450nm was read.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/ed5fccc8e88f2980e5d0636a.png"},{"id":93541568,"identity":"4184b344-4f7c-4e7e-8b02-38679266dda2","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":702676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking and neutralization of bsAbs against various SARS-CoV-2 variants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003emAbs, BsAb and antibody cocktails blocked the ACE2 binding to single point mutant RBD proteins and various S trimers. \u003cstrong\u003eb, c \u003c/strong\u003eScreening of bsAbs based on pseudovirus neutralization method \u003cstrong\u003e(b)\u003c/strong\u003e, antibodies with enhanced neutralizing activity are listed in \u003cstrong\u003e(c).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/1c057d7613d88c0f365b966f.png"},{"id":93541576,"identity":"4cfb7e16-1f26-4512-b449-9bd29ed4b178","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":536482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAffinity of 9A6-6C3 to several SARS-CoV-2 variants by SPR. a\u003c/strong\u003e Affinity of 9A6-6C3 against SARS-CoV-2 spike mutants. SARS-CoV-2 spike trimers or RBDs were immobilized onto CM5 chip through amino coupling. Serial diluted concentrations of 9A6-6C3 BsAb were flowed through and the response unit (RU) was detected by Biacore 8K. The kinetic parameters were calculated by Biacore Insight Evaluation Software.\u003cstrong\u003e b, c\u003c/strong\u003e 9A6-6C3 simultaneously binds two distinct epitopes on the S trimer. The immobilized S trimer was incubated with 9A6 (\u003cstrong\u003eb\u003c/strong\u003e) or 6C3 (\u003cstrong\u003ec\u003c/strong\u003e) until saturation and then incubated with 9A6-6C3, response unit (RU) was detected by Biacore 8K.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/2e83d604c54365fb5e6c9769.png"},{"id":93541572,"identity":"f8d1735c-c61d-44f0-b5e4-6ca95f2305a0","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutralization of 9A6-6C3 against SARS-CoV-2 variants.\u003c/strong\u003e Neutralization of 9A6-6C3 against WA1/2020 (\u003cstrong\u003ea\u003c/strong\u003e), Alpha (\u003cstrong\u003eb\u003c/strong\u003e), Beta (\u003cstrong\u003ec\u003c/strong\u003e), Gamma (\u003cstrong\u003ed\u003c/strong\u003e), Cluster 5 (\u003cstrong\u003ee\u003c/strong\u003e), and Delta (\u003cstrong\u003ef\u003c/strong\u003e). Pseudoviruses with active titer higher than 1 × 10\u003csup\u003e7\u003c/sup\u003e TU/mL were employed in this study. Concentration-dependent neutralization of 9A6-6C3 was quantified by detecting the fluorescence from the luciferase reporter. Data in duplicate are displayed as means ± SD.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/cf95231a54e157e2fab98d3f.png"},{"id":93541581,"identity":"c0d3fe4e-2b70-480c-8663-6324deb6ea4f","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2534675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComputational simulation reveals the binding between 9A6-6C3 and the SARS-CoV-2 S trimer.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Prediction of the interaction between 9A6 and RBD using Swiss-model. \u003cstrong\u003eb\u003c/strong\u003e Epitope analysis of 9A6, 35B5, S2X259, Sotrovimab, Imdevimab and S2H97 with RBD. \u003cstrong\u003ec\u003c/strong\u003eCompetitive ELISA binding experiments of 9A6, 35B5, S2X259, Sotrovimab, Imdevimab and S2H97. \u003cstrong\u003ed\u003c/strong\u003e Binding of 9A6 to the ‘up’ RBD and ‘down’ RBD in the Omicron S trimer. \u003cstrong\u003ee\u003c/strong\u003e Binding affinity of 6C3 with 24 peptide segments covering S2 by ELISA. \u003cstrong\u003ef\u003c/strong\u003e Binding affinity of 6C3 to the IKQY peptide segment. \u003cstrong\u003eg\u003c/strong\u003e The binding site of 6C3 in the S trimer.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/658e1a5e88b98aabea2b4f04.png"},{"id":93544019,"identity":"e488d8df-54db-4704-9006-624beb4108c6","added_by":"auto","created_at":"2025-10-15 02:49:52","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":462914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy on the Antiviral Mechanism of 9A6-6C3 by Western Blot. a-d\u003c/strong\u003e Degradation of S protein on the surface of S-Jurkat cells by 9A6-6C3. The levels of S1 (\u003cstrong\u003ea\u003c/strong\u003e) and S2 (\u003cstrong\u003eb\u003c/strong\u003e) were measured after incubating S-Jurkat cells with 50 μg/ml of 9A6, 6C3 and 9A6-6C3 for 12 hours. The levels of S1 (\u003cstrong\u003ec\u003c/strong\u003e) and S2 (\u003cstrong\u003ed\u003c/strong\u003e) were measured after incubating S-Jurkat cells with 50 μg/ml of 9A6, 6C3 and 9A6-6C3 for 6 days. \u003cstrong\u003ee\u003c/strong\u003e Ability of 9A6-6C3 to cleave the S protein during pseudovirus infection. \u003cstrong\u003ef \u003c/strong\u003eTMPRSS2-mediated degradation of S protein does not impact 9A6-6C3-mediated S protein cleavage.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/506dfb8df0fb7c287f289a88.jpeg"},{"id":93541584,"identity":"7e75c1ef-8a46-456c-9ede-6e209dd36913","added_by":"auto","created_at":"2025-10-15 02:33:51","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":367871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADCC, ADCP, and CDC functions of 9A6-6C3. a, b \u003c/strong\u003eADCC function of 9A6-6C3. Serial diluted concentrations of antibodies were added to S-Jurkat cells for 30 min at 37℃, following by adding PBMC at E:T=25:1. After incubation for 8h (\u003cstrong\u003ea\u003c/strong\u003e) or 21h (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec \u003c/strong\u003eADCP function of 9A6-6C3. Macrophages were differentiated from CD14\u003csup\u003e+\u003c/sup\u003e cells, were combined with antibody-treated Jurkat-S cells. Following incubation, flow cytometry identified phagocytosing macrophages, and the percentage of phagocytosis (%ADCP) was calculated. \u003cstrong\u003ed, e\u003c/strong\u003e S-Jurkat cells were incubated with antibodies and complement serum in a 96-well plate. After 18 hours, cell viability was assessed using a luminescent reagent (\u003cstrong\u003ed\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/601ebc0af0f4e548a07e4ab8.jpeg"},{"id":93544756,"identity":"56982b9c-f35a-41e6-899b-385d5fb5e23a","added_by":"auto","created_at":"2025-10-15 02:57:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6756602,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7686776/v1/e08a2277-419b-433a-a04c-6cab0763e9e5.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlthough the symptoms of COVID-19 infection have significantly alleviated compared to the early stages, about 10%~20% of patients still experience post-infection sequelae three months after infection(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Studies indicated that reinfection with SARS-CoV-2 may cause further damage to the human body(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Therefore, the development of drugs against COVID-19 remains of great value and significance. Among all treatment methods, neutralizing antibodies (nAbs) have significant advantages due to their specificity, effective binding capabilities, pharmacokinetics studied, and the ability to be produced on a large scale(\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The spike (S) protein of SARS-CoV-2 controls the virus\u0026rsquo;s invasion process, making it an ideal target for developing neutralizing antibodies(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The development of neutralizing antibodies against SARS-CoV-2 has been remarkably successful, with several antibodies showing great antiviral effects and being approved by regulatory agents for the treatment and prevention of the virus(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, as a single-stranded RNA virus, SARS-CoV-2 continues to mutate, causing many monoclonal antibodies to lose their antiviral activity(\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). One solution to this problem is to combine monoclonal antibodies targeting different epitopes to produce antibody cocktails or bsAbs(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). To date, several bsAbs have been developed to combat SARS-CoV-2, most of these bsAbs simultaneously target different epitopes on the receptor-binding domain (RBD) to maximize contact with residual residues(\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Yuan et al. developed a bispecific antibody that can simultaneously target both RBD and S2, exhibiting superior neutralization breadth(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Cho et al. developed a bispecific antibody targeting RBD and the N-terminal domain (NTD), but it did not demonstrate improved neutralization activity(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The antiviral mechanisms of bsAbs remain incompletely characterized, particularly in terms of their interactions with distinct subunits of the S protein. Additionally, the potential synergistic effects between the two parent antibodies used to create these bsAbs remain unclear.\u003c/p\u003e\u003cp\u003eIn this study, based on previous research progresses, we classified the binding epitopes of 33 antibodies isolated using single B-cell cloning from convalescent patients and phage display into four types using competitive ELISA. Additionally, we identified one S2-binding antibody (6C3) with potent neutralizing activity, which served as a parent antibody for bispecific design. Sixty seven bsAbs were prepared through Bispecific Antibody by Protein Trans-splicing (BAPTS) platform (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), from which 11 have enhanced neutralizing capabilities, including 9A6-6C3. 9A6-6C3 effectively neutralized various SARS-CoV-2 subtypes, including the widely spread Alpha, Beta, Gamma, Delta, and Omicron BA.1 variants. 9A6 binds to H-RBD epitope(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), in both \u0026ldquo;up\u0026rdquo; and \u0026ldquo;down\u0026rdquo; conformations of the RBD. This binding site would spatially clash with the NTD domain of an adjacent S protomer, which may lead to, we propose, the degradation of the S trimer protein and thus exposing S2. Exposing S2 may promote the binding of 6C3 to the conserved loop in the S2 subunit. The antiviral mechanism of 9A6-6C3 was investigated using Western blot and cell-based assays. In Jurkat-S cells, 9A6-6C3 was able to promote extensive abnormal cleavage of the S protein, leading to the release of the S1 subunit, while 9A6 also caused degradation of the full-length S protein. Unlike the parent antibodies, 9A6-6C3 promoted the exposure of S2. In the assay system using pseudoviruses, 9A6-6C3 could lead to extensive cleavage of the S protein on the virus surface. These results suggested that designing bsAbs that simultaneously bind to H-RBD epitope and S2 may effectively induce the cleavage of the viral S protein and promote the exposure of S2, providing a reference for the design of bsAbs against SARS-CoV-2.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEpitope Binning Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the epitope correlation between two antibodies, a competition enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, the first antibody was coated onto plates (BEAVER) at a concentration of 2 \u0026mu;g/mL and incubated overnight at 4\u0026deg;C. After washing off the excess antibody with PBS, the plates were blocked with 3% skim milk. SARS-CoV-2 S1 protein (Sino Biological) was biotinylated using the EZ-Link\u0026trade; Sulfo-NHS-LC-LC-Biotin kit (ThermoFisher) and then incubated with 50 \u0026mu;g/mL of the second antibody or PBS as a control. The mixture was incubated at 37\u0026deg;C for 1 hour. Following incubation, the plates were washed three times with PBS, and diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma) (1:2000) was added. After a second incubation at 37\u0026deg;C for 1 hour, the binding of S1 protein to the coated first antibody was detected using TMB Single-Component Substrate Solution (Solarbio). Absorbance was measured at 450 nm wavelength using the Infinite M200 PRO Multimode Microplate Reader (TECAN). The competitive binding percentage of the two antibodies was calculated by comparing to the PBS control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBispecific Antibody Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBsAbs were designed and generated using the Bispecific Antibody by BAPTS platform, as previously described. Fragments A and B were selected from two antibodies with minimal epitope clash. To produce fragment A, three plasmids (pCDNA3.4) encoding the full-length heavy chain A, light chain A, and the Fc region of heavy chain B fused to the C-terminal half-intein were co-transfected into ExpiCHO-S cells. For fragment B, two plasmids (pCDNA3.4) containing the light chain B and Fab region of heavy chain B fused to the N-terminal half-intein were co-transfected into the cells. Fragment A and fragment B were purified using HiTrap KappaSelect or HiTrap LambdaSelect columns (Cytiva), using glycine (100 mM, pH 2.5) or acetic acid (100 mM, pH 3.0) as elution buffers. To generate bsAbs, fragments A and B were mixed in a molar ratio of 1:1.6, in the presence of 2 mM Tris-(2-carboxyethyl)-phosphine (TCEP), and incubated for 4 hours at room temperature. The reaction was terminated by adding 2 mM dehydroascorbic acid. The bsAbs were initially purified using Protein A resin (GE Healthcare) and further purified by HiTrap Capto MMC ImpRes (Cytiva). A linear gradient elution was used for Capto MMC purification, transitioning from 10 mM phosphate + 10 mM Tris + 50 mM NaCl (pH 6.0) to 10 mM phosphate + 10 mM Tris (pH 9.0). The final bsAbs, with purity greater than 98% as confirmed by size exclusion chromatography, were concentrated and stored at \u0026minus;80\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonoclonal Antibody Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmids containing the full-length of antibody heavy chain or light chain were amplified in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eDH5\u0026alpha; and extracted by using PureLink\u0026trade; HiPure Plasmid Miniprep Kit (Invitrogen). Antibodies were expressed using the ExpiCHO\u0026trade; expression system (Thermo Fisher). The cell culture process was implemented following the manufacturer\u0026apos;s recommendations. The cell culture medium was collected 12 days post transfection and centrifuged at 300\u0026times;g for 10 min to remove the cells. After further centrifugation at 7000\u0026times;g for 30 min for removing the cell debris, the supernatant was subjected to MabSelect Sure (Cytiva) antibody affinity purification. In an AKTA avant protein separation and purification system (Cytiva), the MabSelect Sure column was equilibrated with 2 column volumes of 20 mM PB + 150 mM NaCl (pH 7.2). Then the medium samples were loaded and washed with 10 column volumes of 100 mM citric acid (pH 5.0). Antibodies were eluted with 5 column volumes of 100 mM citric acid (pH 3.0), followed by storage in a cocktailed buffer containing 10 mM Histidine-HCl, 9% trehalose, and 0.01% polysorbate 80.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBinding ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eELISA was applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems). Antigens were diluted with ELISA Coating Buffer (Solarbio) to 1.0\u0026nbsp;\u0026mu;g/mL and immobilized onto High Binding ELISA 96-Well Plate (BEAVER) with 100\u0026nbsp;\u0026mu;L per well overnight at 4\u0026deg;C. Plates were washed four times with PBST (Solarbio) and blocked with 3% skim milk for 1 h at 37\u0026deg;C. Then, serially diluted antibodies were added 100\u0026nbsp;\u0026mu;L per well and incubated at 37\u0026deg;C for 1 h. After pipetting off the unbound antibodies, plates were washed four times with PBST and further incubated with 100\u0026nbsp;\u0026mu;L per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1:5000 dilution, Sigma) for 1 h at 37\u0026deg;C. After a final four times washing with PBST, the binding of antibodies with SARS-CoV-2 antigens were visualized by adding 100\u0026nbsp;\u0026mu;L peroxidase substrate TMB Single-Component Substrate solution (Solarbio) and incubating for 15 min in dark. The reaction was terminated by adding 50\u0026nbsp;\u0026mu;L stop buffer (Solarbio) and the plates were immediately submitted to an ELISA microplate reader (TECAN Infinite M200 Pro) to measure the optical density (OD) at 450 nm. Data were analyzed with GraphPad Prism Version 9.0.0 and EC\u003csub\u003e50\u003c/sub\u003e values were determined using a four-parameter nonlinear regression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACE2 competition ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor experiments involving competitive binding of antibodies to SARS-CoV-2 RBD or the S trimer, the recombinant hACE2-Fc protein\u0026nbsp;was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher), following the manufacturer\u0026rsquo;s instruction. SARS-CoV-2 RBD (Sino Biological), the S-trimer (AcroBiosystems), mutated RBD (Sino Biological) and mutated S-trimer (AcroBiosystems) were coated on high binding ELISA 96-well plates (BEAVER). To obtain the optimal hACE2-Fc concentration for this experiment, concentration-dependent binding of biotinylated hACE2-Fc to encapsulated SARS-CoV-2 antigen was measured by performing a conventional receptor-binding ELISA. 80% maximum effective concentration (EC\u003csub\u003e80\u003c/sub\u003e) of biotinylated hACE2-Fc was calculated by a four-parameter nonlinear fit. The antibody was serially diluted in 1% BSA (Sigma) and 50 \u0026mu;L was added to the plate. The EC\u003csub\u003e80\u003c/sub\u003e concentration of biotinylated hACE2-Fc was subsequently pipetted in. After incubation for 1 h at 37\u0026deg;C, the plates were washed four times with PBST (136.9 mM NaCl，2.68 mM KCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026bull;12H\u003csub\u003e2\u003c/sub\u003eO, 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.1% (v/v) Tween20, pH7.4) (Solarbio) and incubated with 100 \u0026mu;L of 1:2000 dilution of hypersensitive streptavidin-oxidase polymer (Sigma). After further washing, 100 \u0026mu;L of TMB (3, 3\u0026prime;,5 ,5\u0026prime;-Tetramethylbenzidine) (Solarbio) was added and then bound hACE2 was detected in a microplate reader. four-parameter nonlinear regression fitting was applied in GraphPad Prism version 9.0.0 for analysis of the results.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenerating 293T-ACE2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman ACE2 was stably expressed on the surface of HEK-293T cells (ATCC,\u0026nbsp;CRL-3216) to obtain the 293T-ACE2 cells. In this work, a three-plasmid lentivirus transfection system was used. The ACE2 gene was inserted into the pHIV-puro plasmid, and prepared together with the packaging plasmids psPAX2 and VSV-G using an Endo-free Plasmid Mini Kit (Omega). HEK-293T cells were seeded into a 10 cm dish and cultured at 37 \u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e condition. After the cells reached 70%-80% confluence, plasmids were co-transfected into cells using Lipofectamine 3000 reagent (Thermo Fisher). The cell medium was replaced with fresh DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) 6h later. After further culture for 48 h, the medium supernatant was collected and centrifuged at 3000\u0026times;g for 10 min. The supernatant was transferred to a 15 mL centrifuge tube and mixed with the Lentivirus Concentration Reagent (Genomeditech) for overnight incubation. The next day, the mixture was centrifuged at 3220\u0026times;g for 30 min, followed by resuspending the viral particles with 200 \u0026mu;L DMEM medium.\u003c/p\u003e\n\u003cp\u003eFor infecting HEK-293T cells, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were seeded into a 6-well plate and incubated overnight at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Then 10 \u0026mu;L of concentrated virus was added. 48 h later, cells were transferred to a 10 cm dish, and puromycin (Beyotime Biotechnology) at final concentration of 10 \u0026mu;g/mL was added. Fluorescence activated cell sorting was used to further select the cells with high expression of ACE2. Using S1-mFc recombinant protein (Sino Biological), a DNA sequence encoding the SARS-CoV-2 (2019-nCoV) spike protein S1 Subunit (YP_009724390.1) (Val16-Arg685) was expressed with the Fc region of mouse IgG1 at the C-terminus, as the primary antibody and FITC-labeled goat anti-mouse IgG antibody (Jackson) as the secondary antibody, the cells in the top 1% of fluorescence intensity were obtained on a BD FACSJazz cell sorter (BD). The top 1% cells were expanded for pseudovirus neutralization experiments.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSPR Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antigen was diluted in acetate (Cytiva) at pH 5.0 and covalently coupled to the chip using an amine coupling kit (Cytiva). After reaching a coupling level of 15 RU, excess antigen is washed off and unbound sites are blocked with ethanolamine. The antibodies were serially diluted 2-fold from 1.250 to 0.039 \u0026mu;g/mL with HBS-EP buffer (Cytiva) and then injected into the chip at 30 \u0026mu;L/min for 120 seconds. Afterwards, the conjugates were dissociated with HBS-EP buffer for 300 or 600 seconds, followed by chip regeneration with glycine (Cytiva) at pH 1.5. Parameters including Ka, Kd, and KD values were calculated using the monovalent analyte model of BIAevaluation software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePseudovirus packaging and neutralization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe S sequences of SARS-CoV-2 (GenBank: QHD43416.1) or variants with intracellular 21 residue deletion were inserted into the pMD2.G plasmid. A luciferase gene was constructed into plasmid pCDH-luc1 as a reporter. Pseudoviruses were obtained as the lentivirus packaging method described above. For testing pseudovirus neutralizing activity by antibodies, 293T-ACE2 cells were seeded into a white 96-well plate (Corning) at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e per well, and cultured overnight at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Serial 10-fold dilutions of antibodies from 200 \u0026mu;g/mL were mixed with equal volume of diluted pseudoviruses in DMEM medium (with 10% FBS). Medium containing the same amount of pseudovirus but without antibody was used as infection control. After incubation at 37 \u0026deg;C for 30 min, the medium of the 293T-ACE2 cells was replaced with 100 \u0026mu;L of antibody-pseudovirus mixture. All operations involving pseudoviruses were performed in a biosafety level 2 laboratory of the School of Pharmacy at Shanghai Jiao Tong University. After further culturing for 48 hours, 50 \u0026mu;L of ONE-Glo\u0026trade; Luciferase Assay System substrate (Promega) was added to each well, and the fluorescence intensity was immediately measured in a microplate reader (TECAN). Data were analyzed using GraphPad Prism version 9.0.0 software, and the IC\u003csub\u003e50\u003c/sub\u003e values were calculated using a four-parameter nonlinear regression function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural Modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structure of 9A6 in complex with the SARS-CoV-2 RBD was predicted using Swiss-model (https://swissmodel.expasy.org/). The 6C3 and S2 (amino acids 699-1053) complex structure was predicted using AlphaFold3 (https://alphafold.ebi.ac.uk/). All structural visualizations were generated using Chimera (https://www.cgl.ucsf.edu/chimera/).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverlapping Peptide Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the binding region of 6C3, a set of overlapping 15-mer peptides, each biotinylated at the N-terminal, covering the S2 exposed regions of SARS-CoV-2 spike protein, were synthesized (Genscript). Recombinant streptavidin (Thermo Fisher) was coated onto a 96-well plate at a concentration of 4 \u0026mu;g/mL overnight. The following day, after blocking and washing, each overlapping peptide was added to the wells at a concentration of 4 \u0026mu;g/mL and incubated for 1 hour at 37\u0026deg;C. After four washes, mAb 6C3 was added at a concentration of 10 \u0026mu;g/mL and incubated for another hour at 37\u0026deg;C. Following incubation, plates were washed and incubated with 100 \u0026mu;L of goat anti-human IgG (Fc-specific)-peroxidase secondary antibody (Sigma) diluted 1:5000 for 1 hour at 37\u0026deg;C. After a final wash, TMB substrate solution was added and incubated for 15 minutes in the dark, followed by the addition of stop buffer to terminate the reaction. The optical density at 450 nm was measured using a microplate reader (TECAN Infinite M200 Pro).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy on the Antiviral Mechanism of 9A6-6C3 by Western Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the antiviral mechanism of 9A6-6C3, Western blotting was employed to analyze the degradation of the SARS-CoV-2 spike protein on the surface of S-Jurkat cells. S-Jurkat cells were incubated with 50 \u0026mu;g/mL of 9A6, 6C3, or 9A6-6C3 for 12 hours or 6 days, and the levels of the S1 and S2 subunits of the S protein were determined. Cell lysates were harvested, and protein concentrations were quantified using a BCA assay. Equal amounts of protein were loaded and separated by SDS-PAGE, followed by transferring to PVDF membranes. Membranes were then probed with primary antibodies against S1 and S2 subunits, followed by HRP-conjugated secondary antibodies. Signal detection was performed using the ECL detection system, and the protein bands were quantified using ImageJ software. Additionally, the ability of 9A6-6C3 to cleave the S protein during pseudovirus infection was assessed. S-Jurkat cells were exposed to pseudovirus in the presence of 9A6-6C3, and the levels of cleaved S protein were detected by Western blot. The effect of TMPRSS2-mediated degradation of the S protein on 9A6-6C3-mediated cleavage was also evaluated by pre-treating S-Jurkat cells with a TMPRSS2 inhibitor, followed by incubation with 9A6-6C3, and assessing the degradation of the S protein as described. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody-Dependent Cellular Cytotoxicity (ADCC) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJurkat cells stably expressing the SARS-CoV-2 spike protein were seeded into 96-well plates on the day of the experiment. Antibodies were prepared at serially diluted concentrations and added to the wells, followed by incubation for 30 minutes at 37\u0026deg;C. Peripheral blood mononuclear cells (PBMCs) were then added as effector cells at an effector-to-target (E:T) ratio of 25:1. The co-culture was incubated for either 8 or 21 hours to allow for cytotoxicity induction. Cytotoxicity was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Kit (Promega) according to the manufacturer\u0026apos;s protocol. OKT3 was included as a positive control in all assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody-Dependent Cellular Phagocytosis (ADCP) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo differentiate macrophages, CD14\u003csup\u003e+\u003c/sup\u003e monocytes were cultured in the presence of M-CSF for 7 days. Differentiated macrophages were subsequently stained with Violet dye (PB450). In parallel, Jurkat-S cells were labeled with CFSE dye and incubated with serial dilutions of the test antibodies for 15 minutes at 4\u0026deg;C. Following this pre-incubation, the antibody-bound Jurkat-S cells were added to the macrophages and co-incubated at 37\u0026deg;C for 30 minutes to allow phagocytosis to occur. After incubation, the macrophages were digested, fixed, and analyzed using flow cytometry. Macrophages that had phagocytosed Jurkat-S cells were identified as PB450\u003csup\u003e+\u003c/sup\u003e FITC\u003csup\u003e+\u003c/sup\u003e populations. The percentage (%) of ADCP was calculated as the proportion of PB450\u003csup\u003e+\u003c/sup\u003e FITC\u003csup\u003e+\u003c/sup\u003e cells relative to the total PB450\u003csup\u003e+\u003c/sup\u003e macrophages.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplement-Dependent Cytotoxicity (CDC) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJurkat cells stably expressing the SARS-CoV-2 spike protein were seeded into 96-well plates on the day of the experiment. Serial dilutions of antibodies were prepared and added to the cells, followed by the addition of normal human complement serum to a final concentration of 25%. The plates were incubated at 37\u0026deg;C for 18 hours. After incubation, the remaining viable cells were quantified using the CellTiter-Glo Reagent Kit. Alternatively, cell death was assessed by staining with propidium iodide (PI) and analyzing the samples using flow cytometry to determine the percentage of dead cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of Funders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunders did not have any role in the study design, data collection, data analyses, interpretation, or writing of this report.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThe bsAbs were designed by BAPTS platform\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a previous study, several neutralizing antibodies were isolated from patients infected with the WA1/2020 virus, and an additional set of neutralizing antibodies was obtained through phage display screening(10,11,27,28). From these, 32 monoclonal antibodies with neutralizing activity were selected. Of these, 30 were derived from patient B cells, while two antibodies (R35 and R32) were sourced from the phage display library. Epitope classification of these antibodies was conducted using competitive ELISA, resulting in the identification of four distinct epitope categories. Specifically, 7G10, 8G4, 9E12, 9E2, 9D11, 7B12, 8E12, 5C12, 11F7, R32, 12F8, 8F6 and 8C12 were classified as the first category; 9A6, 5H10, 10D4, 8D8, 7D10 and 7B9 as the second category; R35, 8G9, 8D4, 9A8, 2G1 and 9B10 as the third category; 9B12, 13A12, 9C4, 11D3, 2B8, 3A4 and 5B2 as the fourth category (Fig.1a). Within each category, antibodies with superior binding activity were selected. Ultimately, antibody 11F7, 8F6, 8G4, 9D11 and 9E12 from the first category, 9D4, 7B9, 9A6 from the second category, 2G1, 8G9 and R35 from the third category, and 13A12, 2B8 and 3A4 from the fourth category were selected as the parent antibodies (Fig. 1b and 1c). Additionally, 6C3, which showed binding ability to the S2 subunit, was also selected as a parent antibody (Fig. 1d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the identified epitope distribution, each parent antibody was combined with antibodies from different categories to generate novel bsAbs. Utilizing the BAPTS platform, a total of 67 bsAbs were produced within one month (Fig. 2a). The majority of the bsAbs exhibit good affinity for WA1/2020 RBD (indicated by the unmarked red curves in the graph) (Fig. 2b). However, the affinity of the bsAbs derived from 6C3 is weaker, with the exception of 8G9-6C3, 7B9-6C3, and 9A6-6C3. The competitive binding ability of these three bsAbs against ACE2 was evaluated, using the potent antibody 2G1 previously studied in our laboratory as a positive control(11). These three bsAbs did not compete with RBD for binding to various strains of the RBD or S trimer (Fig. 3a). The bsAbs with superior affinity were further evaluated for their neutralizing activity, with most displaying excellent neutralization potency (IC\u003csub\u003e50\u003c/sub\u003e \u0026lt; 100ng/ml) (Fig. 3b). Among these bsAbs, 11 had IC\u003csub\u003e50\u003c/sub\u003e values lower than the optimal values of the two parent antibodies, with 9A6-6C3 showing an IC\u003csub\u003e50\u003c/sub\u003e value that was 100 times more potent compared to the parent antibody 9A6 (Fig. 3c). Based on these findings, antibody 9A6-6C3 has been prioritized for further investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9A6-6C3 is a broad neutralizer against SARS-CoV-2 infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the broad-spectrum activity of 9A6-6C3, surface plasmon resonance (SPR) analysis was performed for affinity measurement of the bsAb to SARS-CoV-2 variants. The results demonstrated that 9A6-6C3 exhibited superior affinity for various variants, with KD values predominantly in the picomolar range, particularly for the Cluster 5 variant, where a KD of 2.82 femtomoles was achieved (Fig. 4a). Omicron BA.1 S trimer was immobilized on a CM5 chip to assess whether both arms of the bispecific antibody could simultaneously bind to the S protein. After the injection of high concentrations of 9A6 or 6C3, 9A6-6C3 was still able to bind, indicating that both arms of the antibody could engage the S protein trimer simultaneously (Fig. 4b and 4c). Furthermore, when 9A6 was injected first, the RU changes caused by 9A6 and 9A6-6C3 were not significantly different (Fig. 4b). However, in the group where 6C3 was injected first, the RU change caused by 6C3 was significantly less than that caused by 9A6-6C3 (Fig. 4c), suggesting that the 6C3 epitope may be relatively inaccessible for binding under these conditions. One possible inference from these findings is that in actual binding, 9A6 may bind to the S protein first, followed by 6C3, and the binding of 9A6 would facilitate the binding of 6C3. The neutralizing potency of 9A6-6C3 against SARS-CoV-2 variants infection was evaluated using the S-pseudotyped lentivirus neutralization assay. For WA1/2020, B.1.351, B.1.1.7, P.1, Cluster 5, and Delta variants, 9A6-6C3 exhibited excellent neutralizing activity (IC\u003csub\u003e50\u003c/sub\u003e \u0026lt; 100ng/ml), especially for Beta type and Cluster 5 type, IC\u003csub\u003e50\u003c/sub\u003e is less than 5ng/ml (Fig. 5a-f). The neutralizing activity of 9A6-6C3 against these strains improved by 10 to 100 times compared to 9A6 alone. 6C3 did not possess neutralizing activity, but this was remedied when combined with 9A6 to form a bispecific antibody. This suggests that 9A6-6C3 may have a unique antiviral mechanism different from the parent antibodies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9A6-6C3 binds to H-RBD epitope and the conserved loop of S2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the binding epitope of 9A6, we first used Swiss-model to simulate its binding to RBD. The simulation revealed that 9A6 binds to the side of RBD without competing with ACE2 (Fig. 6a). Subsequently, competitive ELISA was performed with several neutralizing antibodies known to bind specific epitopes, along with 9A6. The results indicated that 9A6 partially overlaps with the epitopes of 35B5 and S2H97 (Fig. 6b and 6c), which is consistent with the predicted binding mode from the Swiss-model simulation (Fig. 6a). The epitope bound by 9A6, previously reported by Chi et al., is referred to as the H-RBD epitope(26). When 9A6 binds to the Omicron S trimer, it can spatially clash with NTD, regardless of whether 9A6 binds to the \u0026ldquo;up\u0026rdquo; or \u0026ldquo;down\u0026rdquo; RBD (Fig. 6d). Peptides covering the S2 protein were used to detect binding with the 6C3 antibody, 6C3 exhibited strong binding to the \u0026ldquo;IKQYGDCLGDIAARD\u0026rdquo; peptide on the S2 protein and also binds to the \u0026ldquo;GDCLGDIAARDLICA\u0026rdquo; peptide (Fig. 6e). The epitope predicted by Alphafold3 are consistent with the experimental results (Fig. 6f). This epitope, deeply buried within the normal S protein trimer (Fig. 6g), is likely inaccessible for direct binding by neutralizing antibodies. Cheng et al. have indicated that the \u0026ldquo;IKQYGDCLGDIAARD\u0026rdquo; peptide is conserved throughout the evolution of SARS-CoV-2(29). Considering the SPR data, these results suggested that the binding of 9A6 may lead to the disassembly of the S trimer, thereby exposing the 6C3 epitope on the S2 subunit and facilitating its binding. The combined effect of 9A6-mediated S trimer disassembly and 6C3 binding to the exposed S2 epitope collectively enhances the neutralizing activity. This mechanism likely explains why the bispecific antibody exhibits significantly better neutralizing activity than its parent antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9A6-6C3 promotes the abnormal degradation of viral S protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further validate whether 9A6-6C3 can promote degradation of the S protein, and to explore the differences in antiviral mechanisms between 9A6-6C3 and its parent antibodies, a series of Western blot experiments were designed. 9A6-6C3 were co-cultured with Jurkat cells overexpressing the S protein, and after 6 hours, the cells were harvested for Western blot analysis to measure the levels of S, S1, and S2 proteins (Fig. 7a and 7b). The results showed that the S2 fragment levels in the 9A6-6C3 group were significantly higher than in other groups (Fig. 7b), while the S1 fragment levels were lower (Fig. 7a). This suggests that 9A6-6C3 promote the cleavage of the S protein, leading to the shedding of S1 and the exposure of S2 protein. No significant differences in the levels of the S protein were observed, likely due to a dynamic equilibrium between the cleavage of the S protein on the cell surface and the synthesis of the S protein inside the cells. The same experimental method was used for six-day incubation, and similar results were obtained (Fig. 7c and 7d). To verify whether 9A6-6C3 could promote the cleavage of the S protein during infection, 9A6-6C3 were incubated with pseudoviruses and added to HEK293T cells overexpressing ACE2. After 6 hours, the cell-containing medium was collected for Western blotting. The amount of the S protein in the 9A6-6C3 group was significantly lower than in other groups (Fig. 7e), confirming our hypothesis. Additional experiments were designed to investigate whether 9A6-6C3 would affect the TMPRSS2-mediated cleavage process. HEK293T cells overexpressing ACE2 and TMPRSS2 were incubated with the S protein, resulting in cleavage of a small amount of the S protein compared to cells expressing ACE2 alone (Fig. 7f). The addition of TMPRSS2 inhibitors Camostat Mesilate or Bromhexine blocked this cleavage. However, when 9A6-6C3 was added in the presence of these inhibitors, the amount of the S protein still decreased sharply (Fig. 7f). This indicates that 9A6-6C3 promotes the S protein reduction independently of TMPRSS2 activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADCC, ADCP, and CDC functions of 9A6-6C3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the ADCC function of 9A6-6C3, Jurkat cells expressing the S protein were employed by adding the antibodies with various concentrations to the cells, followed by the addition of PBMCs, with OKT3 used as a positive control. After incubating with the cells for 8 hours, the ADCC effect of 9A6-6C3 was weak (Fig. 8a). Whereas after 21 hours of incubation, 9A6-6C3 exhibited a strong ADCC effect (Fig. 8a), indicating that 9A6-6C3 can facilitate the clearance of infected cells. To evaluate the ADCP function, we labeled Jurkat-S cells and monocyte-derived macrophages with two fluorescent dyes, CFSE and violet dye, respectively. Subsequently, a series of concentrations of 9A6-6C3 were added. Flow cytometry analysis of the percentage of violet-positive macrophages containing both dyes revealed a certain phagocytic effect of macrophages on Jurkat-S cells (Fig. 8c). 9A6-6C3 exhibited measurable ADCC and ADCP functions. This suggests that it can bind to virus-infected cells and engage immune effector mechanisms. Furthermore, while the S1 protein spontaneously degrades during this process, 9A6-6C3 appears capable of persisting on the cell surface long enough to mediate these functions. This contrasts with many anti-RBD neutralizing antibodies that may fail to persist on the cell surface due to S1 shedding, potentially explaining their reduced ADCC and ADCP activity. To evaluate the CDC activity of the antibodies, different concentrations of antibodies and normal human complement serum were added to Jurkat-S cells. It was found that 9A6-6C3 possessed a weak complement activation ability (Fig. 8d and 8e). In summary, 9A6-6C3 has good ADCC and ADCP functions, as well as a weak CDC function, capable of effectively activating the human immune system to clear or kill cells infected by viruses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the treatment of various diseases, bsAbs have shown considerable advantages, especially in cancer therapy(30\u0026ndash;33). BsAbs have unique therapeutic mechanisms due to their ability to bind different epitopes and their potential for extensive cross-linking(34\u0026ndash;37). Interacting with SARS-CoV-2, the bsAbs targeting the S protein can bind to distinct sites, either within a single monomer or across adjacent monomers(18,38). This dual-binding capability enables cross-linking of S proteins, which confers therapeutic advantages through novel mechanisms. However, for COVID-19, research on the antiviral mechanisms of bsAbs is limited, far less than the structural studies and antiviral mechanism research of monoclonal neutralizing antibodies. Furthermore, most currently developed bsAbs target exclusively the RBD domain and lack in-depth mechanistic investigations. Consequently, the full therapeutic potential of bsAbs remains unrealized.\u003c/p\u003e\n\u003cp\u003eIn this study, based on a comprehensive evaluation of monoclonal neutralizing antibodies, we have developed for the bsAbs, which can effectively accelerate the generation of the bsAbs against SARS-CoV-2. Among these bsAbs, 9A6-6C3 exhibits broad-spectrum neutralizing activity far superior to its parent antibodies. Mechanistically, it binds epitopes that induce steric hindrance with the NTD of adjacent S monomers within the trimer, while simultaneously engaging a conserved loop on the S2 subunit. Multiple experimental results indicate that the binding of 9A6 can lead to the degradation of the S protein and facilitate the binding of 6C3 to the conserved epitope on S2. Research by Chi et al. has shown that co-incubating the H-RBD antibody with the S trimer protein results in the disassembly of the S protein trimer under cryo-EM(26). Similarly, other researchers have found that antibodies with spatial hindrance to NTD of adjacent S monomer can also induce the disassembly of S trimer, even for BQ.1.1 and XBB as well(18,39,40). On the other hand, the binding site of 6C3 is deeply buried within the S trimer, which is advantageous for resisting viral mutations and also beneficial for stimulating ADCC or ADCP functions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u0026lsquo;hidden\u0026rsquo; epitopes have always been targets for nAbs, as these more concealed epitopes are usually located inside the S trimer and are less likely to mutate. However, at the same time, most hidden epitopes are difficult for neutralizing antibodies to bind to. 9A6-6C3 does not directly block the binding of ACE2 and RBD but induces protein degradation to expose a concealed epitope for binding. Similarly, designing a bispecific antibody or cocktail therapy that can bind to the H-RBD epitope and another hidden epitope may also produce the same effect. Our research provides a new approach for the design of bsAbs against SARS-CoV-2 and also offers a new mechanistic understanding for the treatment of COVID-19. Rao et al. have demonstrated that the development of a bispecific antibody, which include one arm binding to the H-RBD epitope, can substantially restore the neutralizing efficacy of another antibody(21). This suggests that the design of bsAbs targeting the H-RBD epitope may offer a broadly applicable strategy.\u003c/p\u003e\n\u003cp\u003eHighly effective monoclonal antibodies isolated from memory B cells serve as a primary source for antibody development to against never ended SARS-CoV-2 variants. However, the repeated process of isolating these antibodies is costly, time-consuming, and may not keep pace with the rapid mutation rate of the virus. In this context, leveraging existing monoclonal antibody research to develop antibodies with novel neutralizing mechanisms represents an ideal strategy. Here, we present the rapid development of a series of bsAbs through a rational approach, along with the proposal of a new antiviral mechanism, which could significantly contribute to ongoing research on SARS-CoV-2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JWZ, LH\u003c/p\u003e\n\u003cp\u003eMethodology: LH, YJL, HM, HFZ, YSY, MYW\u003c/p\u003e\n\u003cp\u003eInvestigation: YJL, HNT, HM, HFZ, LW, YK\u003c/p\u003e\n\u003cp\u003eFormal analysis: YJL, HNT, HM\u003c/p\u003e\n\u003cp\u003eVisualization: YJL, HM, HNT\u003c/p\u003e\n\u003cp\u003eWriting - Original Draft: YJL\u003c/p\u003e\n\u003cp\u003eWriting - Review \u0026amp; Editing: JWZ, YQX, HJ, LH\u003c/p\u003e\n\u003cp\u003eSupervision: JWZ\u003c/p\u003e\n\u003cp\u003eProject administration, BHZ, YLB\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jingli Hou from Institute of Translational Medicine, Shanghai Jiao Tong University, China for assistance in SPR affinity analysis. This work was supported by National Natural Science Foundation of China (81773621, 82073751 to JWZ), National Science and Technology Major Project \u0026ldquo;Key New Drug Creation and Manufacturing Program\u0026rdquo; of China (No.2019ZX09732001-019 to JWZ), Key R\u0026amp;D Supporting Program (Special Support for Developing Medicine for Infectious Diseases) from the Administration of Chinese and Singapore Tianjin Eco-city to Jecho Biopharmaceuticals Ltd. Co., Shanghai Jiao Tong University \u0026ldquo;Crossing Medical and Engineering\u0026rdquo; grant (20X190020003 to JWZ).\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShah MM, Spencer BR, James-Gist J, Haynes JM, Feldstein LR, Stramer SL, et al. Long-Term Symptoms Associated With SARS-CoV-2 Infection Among Blood Donors. 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Clin Cancer Res Off J Am Assoc Cancer Res. 2021 Oct 15;27(20):5457\u0026ndash;64. \u003c/li\u003e\n\u003cli\u003eNiu M, Yi M, Wu Y, Lyu L, He Q, Yang R, et al. Synergistic efficacy of simultaneous anti-TGF-\u0026beta;/VEGF bispecific antibody and PD-1 blockade in cancer therapy. J Hematol OncolJ Hematol Oncol. 2023 Aug 12;16(1):94. \u003c/li\u003e\n\u003cli\u003eTian Z, Liu M, Zhang Y, Wang X. Bispecific T cell engagers: an emerging therapy for management of hematologic malignancies. J Hematol OncolJ Hematol Oncol. 2021 May 3;14(1):75. \u003c/li\u003e\n\u003cli\u003ePan Z, Chen J, Xiao X, Xie Y, Jiang H, Zhang B, et al. Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement. Acta Pharm Sin B. 2022 Apr;12(4):1928\u0026ndash;42. \u003c/li\u003e\n\u003cli\u003eMa H, Zhang X, Zeng W, Zhou J, Chi X, Chen S, et al. A bispecific nanobody dimer broadly neutralizes SARS-CoV-1 \u0026amp; 2 variants of concern and offers substantial protection against Omicron via low-dose intranasal administration. Cell Discov. 2022 Dec 9;8(1):132. \u003c/li\u003e\n\u003cli\u003eHao A, Song W, Li C, Zhang X, Tu C, Wang X, et al. Defining a highly conserved cryptic epitope for antibody recognition of SARS-CoV-2 variants. Signal Transduct Target Ther. 2023 July 8;8(1):269. \u003c/li\u003e\n\u003cli\u003eWang X, Hu A, Chen X, Zhang Y, Yu F, Yue S, et al. A potent human monoclonal antibody with pan-neutralizing activities directly dislocates S trimer of SARS-CoV-2 through binding both up and down forms of RBD. Signal Transduct Target Ther. 2022 Apr 5;7(1):114. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"neutralizing antibody, SARS-CoV-2, bispecific antibody","lastPublishedDoi":"10.21203/rs.3.rs-7686776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7686776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGiven the continuously mutating nature of SARS-CoV-2, the sensitivity of most monoclonal therapeutic antibodies has decreased to the mutants. Bispecific antibodies (BsAbs), with their unique antiviral mechanisms that enable simultaneous binding to two epitopes, offer a distinct advantage in combating the continuous viral mutation by preventing immune escape and enhancing viral neutralization. In this study, we synthesized 67 bsAbs based on the epitope distribution from antibodies isolated using single B-cell cloning from convalescent patients and phage display, 11 of which showed superior neutralization of WA1/2020 compared to their parent antibodies. One bispecific antibody (9A6-6C3), exhibiting 100-fold greater neutralizing activity than its parent antibodies, efficiently neutralized various SARS-CoV-2 variants (IC50\u0026thinsp;\u0026lt;\u0026thinsp;100ng/mL). Structural analysis indicates that 9A6 binds to the H-RBD epitope, encountering spatial conflict with the NTD of neighboring S monomer, while 6C3 is capable of binding to a conserved loop on S2. \u003cem\u003eIn vitro\u003c/em\u003e evidence demonstrates that 9A6-6C3 promotes the disassembly of the S protein, exposing S2, which likely contributes to its broad-spectrum neutralizing activity. In summary, we discovered a potential broad-spectrum mechanism and presented an epitope design strategy for bsAbs, offering valuable insights for the design and development of bsAbs in the fight against COVID-19.\u003c/p\u003e","manuscriptTitle":"Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 02:33:46","doi":"10.21203/rs.3.rs-7686776/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6ba688d5-f4f9-49d2-8e7e-480f27761beb","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55589344,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"},{"id":55589345,"name":"Biological sciences/Structural biology/Molecular modelling"},{"id":55589346,"name":"Biological sciences/Drug discovery/Biologics/Antibody therapy"}],"tags":[],"updatedAt":"2025-10-28T18:06:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 02:33:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7686776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7686776","identity":"rs-7686776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}