Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants
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Abstract
The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.
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SciScore for 10.1101/2021.04.01.437942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: All donors signed the standard NYBC blood donor consent form that indicates blood may be used for research.
IACUC: The research protocol was approved and performed in accordance with Scripps Research IACUC Protocol #20-0003.Randomization Forty-eight golden Syrian hamsters (Mesocricetus auratus, about 6 weeks old, individually housed) sourced from Envigo, Haslett, MI were randomly assigned to four groups of 12 animals each with equal numbers of males and females and a randomized treatment order. Blinding The study was blinded to the staff who handled the animals. Power Analysis not detected. Sex as a biological variable Forty-eight golden Syrian hamsters … SciScore for 10.1101/2021.04.01.437942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Consent: All donors signed the standard NYBC blood donor consent form that indicates blood may be used for research.
IACUC: The research protocol was approved and performed in accordance with Scripps Research IACUC Protocol #20-0003.Randomization Forty-eight golden Syrian hamsters (Mesocricetus auratus, about 6 weeks old, individually housed) sourced from Envigo, Haslett, MI were randomly assigned to four groups of 12 animals each with equal numbers of males and females and a randomized treatment order. Blinding The study was blinded to the staff who handled the animals. Power Analysis not detected. Sex as a biological variable Forty-eight golden Syrian hamsters (Mesocricetus auratus, about 6 weeks old, individually housed) sourced from Envigo, Haslett, MI were randomly assigned to four groups of 12 animals each with equal numbers of males and females and a randomized treatment order. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibody binding to the beads was detected with 2.5 µg/mL goat anti-human IgG-Alexa Fluor 647 (Jackson Immunoresearch 109-606-170) anti-human IgG-Alexa Fluor 647suggested: NoneFor IgA1 or IgA2 antibodies, VH sequences were also cloned into plasmids containing the IgA1 or IgA2 constant region (Genscript). IgA2suggested: NoneIgA1suggested: NoneThe beads were stained with 10 µg/mL of each antibody for 30 min at room temperature, washed, and stained with 2.5 µg/mL goat anti-human IgG Alexa Fluor 647 (Jackson Immunoresearch 109-606-170). anti-human IgGsuggested: (Jackson ImmunoResearch Labs Cat# 109-606-170, RRID:AB_2337902)Antibody synergy experiments: Five antibodies, CV503, CV521, CV664, CV993 and CV1182, were tested for synergy in neutralizing authentic SARS-CoV-2 (FRNA assay). CV503suggested: NoneCV521suggested: NoneCV664suggested: NoneCV1182suggested: NoneDesign of DVD-IgTM bispecific antibodies: The mAbs CV503, CV521, CV664, CV993, CV1182 and CV1206 were used to create bispecific DVD-IgTM antibodies. CV993suggested: NoneCV1206suggested: NoneStructure modeling: Epitope bins represented by C135 (PDB ID: 7K8Z), S309 (PDB ID: 6WPS), ACE2 (PDB ID: 6M0J), CR3022 (PDB ID: 6W41), as well as NTD-specific antibody 4-8, were modeled onto a SARS-CoV-2 spike protein (PDB ID: 7C2L). ACE2suggested: NoneCR3022suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)Experimental Models: Cell Lines Sentences Resources This assay was performed by incubating a fixed volume of virus [0.5 multiplicity of infection (MOI)] with the antibodies for 1 h at 37 °C prior to adding to Vero E6 cells (BEI, Manassas, VA, USA, NR-596) plated in 96-well plates. Vero E6suggested: NonePseudovirions were generated by co-transfection of HEK293T cells with plasmids encoding MLV-gag/pol, MLV-CMV-Luciferase, and SARS-CoV-2 spike (GenBank: MN908947) with an 18 aa truncation at the C-terminus. HEK293Tsuggested: NoneAfter the incubation, 10,000 Hela-hACE2 cells generated by lentivirus transduction of wild-type Hela cells and enriched by fluorescence-activated cell sorting (FACS) using biotinylated SARS-CoV-2 RBD conjugated with streptavidin-Alexa Fluor 647 (ThermoFisher Scientific, S32357) were added to the mixture with 20 µg/ml Dextran (Sigma, 93556-1G) for enhanced infectivity. Helasuggested: NoneAt 48 hr post incubation, the supernatant was aspirated, and HeLa-hACE2 cells were then lysed in luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100). HeLa-hACE2suggested: Nonetransducing plasmid pHR’ CMV-Luc, S plasmid from SARS-CoV-2 (Wuhan-1, D614G, B.1.1.7, B.1.351) with TMPRSS2 into 293T cells using Fugene 6 transfection reagent (Promega, Madison, WI)54–56. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)B.1.1.7 virus contained the following spike mutations: del-H69–V70, del-Y144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H. B.1.351 virus contained the following spike mutations: L18F, D80A, D215G, del- L242_244, R246I, K417N, E484K, N501Y, D614G, A701V. 293T-ACE2 cells, provided by Dr. Michael Farzan, were plated into 96-well white/black Isoplates (PerkinElmer, Waltham, MA) at 5,000 cells per well the day before transdution of SARS-CoV-2. 293T-ACE2suggested: RRID:CVCL_YZ65)Antibody VH or VL sequences were cloned into plasmids containing an IgG1 or relevant light chain backbone (Genscript) and used to transfect Expi293 cells (ThermoFisher Scientific) Expi293suggested: RRID:CVCL_D615)Software and Algorithms Sentences Resources First, we plotted a standard curve of serially diluted virus (3000, 1000, 333, 111, 37, 12, 4, 1 PFU) versus RLU using four-parameter logistic regression (GraphPad Prism ver. 8) below: (y: RLU, x: PFU, a,b,c and x0 are parameters fitted by standard curve) To convert sample RLU into PFU, use the equation below: (if y < a then x = 0)
Percentage neutralization was calculated by the following equation:
VC =
GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Percent neutralization and neutralization IC50s were calculated using GraphPad Prism 8.0.2. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)The samples were then acquired in a BD LSRFortessa X-50 flow cytometer (BD biosciences) and analyzed using Flowjo (BD biosciences). Flowjosuggested: (FlowJo, RRID:SCR_008520)Residues with a buried surface area (BSA) > 0 Å2, as calculated by the PISA program61, were used for defining the epitopes. PISAsuggested: (PISA, RRID:SCR_015749)RELION 3.070 was used for 2D and 3D classifications and final refinements. RELIONsuggested: (RELION, RRID:SCR_016274)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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