Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617
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Abstract
COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.
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SciScore for 10.1101/2021.12.21.473733: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen-2018-20068; Chen-2020-20358; Wilen-2018-20198). Sex as a biological variable M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization. Randomization Six to eight-week-old K18-hACE2 littermate-controlled mice, mixed gender (male / female) mice were divided randomly into three groups, and administered with 20 mg/kg (of mice body weight) Clone 2, Clone 6 or placebo / control, via intraperitoneal (IP) injection. Blinding Replication, randomization, blinding and reagent validations: Replicate … SciScore for 10.1101/2021.12.21.473733: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen-2018-20068; Chen-2020-20358; Wilen-2018-20198). Sex as a biological variable M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization. Randomization Six to eight-week-old K18-hACE2 littermate-controlled mice, mixed gender (male / female) mice were divided randomly into three groups, and administered with 20 mg/kg (of mice body weight) Clone 2, Clone 6 or placebo / control, via intraperitoneal (IP) injection. Blinding Replication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study. Power Analysis not detected. Cell Line Authentication Contamination: All cell lines tested negative for mycoplasma. Table 2: Resources
Antibodies Sentences Resources Plasmid construction: The cDNA sequences of the paired variable heavy and light chain region of anti-RBD antibody clones were synthesized as gBlocks (IDT) and cloned by the Gibson assembly (NEB) into human IgG1 heavy chain and light chain expression plasmids, pFUSEss-CHIg-hG1(InvivoGen, pfusess-hchg1) and pFUSE2ss-CLIg-hK (InvivoGen, pfuse2ss-hclk), respectively. pfusess-hchg1suggested: NonepFUSE2ss-CLIg-hKsuggested: None, containing cDNA sequence of variable region of light chain of anti-RBD antibody clones and the regions overlapping with corresponding flanking sequences of EcoRI and BsiWI restriction sites pFUSE2ss-CLIg-hK, were ordered from IDT. anti-RBDsuggested: NoneIgG1 antibodies were expressed in Expi293FTM cells. IgG1suggested: None) bispecific antibody is a human IgG1-like bispecific antibody, generated based on CrossMab-KiH bispecific constructs, including pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1. pFUSE2ss-knobheavy-hG1suggested: NonepFUSE2ss-HoleLight-hKsuggested: NonepFUSE2ss-HoleHeavy-hG1suggested: NoneBLI): Antibody binding kinetics for anti-spike mAbs were evaluated by BLI on an Octet RED96e instrument (FortéBio) at room temperature. anti-spikesuggested: NoneAfter that, the biosensors were associated with indicated concentrations of the antibodies (from 50 nM to 0.78125 nM with 2-fold dilutions, where the kinetic buffer was served as the negative control) for 200 s, then dissociated in the kinetic buffer for 1000 s. (2) 25ng/ul of Clone13A-IgG1 antibodies were captured on a AHC biosensor (ForteBio). Clone13A-IgG1suggested: NoneThe anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. anti-Ssuggested: Noneanti-mouse IgGsuggested: (Millipore Cat# 12-349, RRID:AB_390192)Experimental Models: Cell Lines Sentences Resources 15x106 293FT cells were seeded in a 150 mm plates one day before in 20 ml D10 media. 293FTsuggested: NoneBriefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI. 293Tsuggested: NoneThe pseudovirus neutralization assays were performed on 293T-hACE2 cell line 71. 293T-hACE2suggested: None5x105 Vero-E6 cells were plated per well of a 96-well plate. Vero-E6suggested: NoneAntibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h. Vero-TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)The replication-competent SARS-CoV-2 (USA-WA1/2020) virus was produced in Vero E6 cells, and the titer was determined by plaque assay using WT VeroE6. Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization. C57BL/6Jsuggested: RRID:IMSR_JAX:000664)Clone 5-11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)The K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from the Jackson Laboratory and bred in house using a trio breeding scheme. K18-hACE2suggested: RRID:IMSR_GPT:T037657)B6.Cg-Tg(K18-ACE2)2Prlmn/Jsuggested: RRID:IMSR_JAX:034860)In vivo efficacy testing of humanized Clone13A to authentic SARS-CoV-2 virus: 10-12-week-old littermate-controlled female and male K18hAce2Tg+ mice were pretreated with 20 mg/kg of either control hIgG1 (purchased from BioXCell) or clone 13A mAb (produced by the Chen lab) administered IP in 300 uL of DPBS. K18hAce2Tg+suggested: NoneRecombinant DNA Sentences Resources Parallelly, pFUSE2-CLIg-hK is a cloning plasmid that expresses the constant region of the human kappa light chain and contains multiple cloning sites to enable cloning of the light chain variable region. pFUSE2-CLIg-hKsuggested: NoneFour plasmids were employed: pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1. pFUSE2ss-knobheavy-hG1suggested: NonepFUSE2ss-HoleLight-hKsuggested: NonepFUSE2ss-HoleHeavy-hG1suggested: NoneThe pFUSE2ss-knobLight-hK is pFUSE2ss-CLIg-hK with no further editing. pFUSE2ss-CLIg-hKsuggested: NoneThe gBlock (pPR024), containing constant region of heavy chain with two knob mutations and the regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and then cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone by the Gibson assembly (NEB). pPR024suggested: NoneThe pFUSE2ss-HoleLight- hK was generated by replacing the constant region of Light chain (CL) in pFUSE2ss-CLIg-hK with CH1 region of heavy chain in pFUSEss-CHIg-hG1 vector. pFUSEss-CHIg-hG1suggested: NoneThe gBlock (pPR023), containing cDNA sequence of constant region of light chain, CH2 and CH3 with “hole” mutations, and regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone through Gibson assembly (NEB). pPR023suggested: NoneThe pVP21-SA and pVP28-Indian were generated based on pcDNA3.1-pSARS-CoV-2-S, which was derived by insertion of a synthetic human codon- optimized cDNA (Geneart) encoding a WA1 SARS-CoV-2 S protein. pVP28-Indiansuggested: NonepcDNA3.1-pSARS-CoV-2-Ssuggested: NoneFor pVP28-Indian, four gBlocks, contains mutations in Indian variant regions overlapping with corresponding flanking sequences of NheI and BamHI restriction sites pcDNA3.1-pSARA-CoV-2. pcDNA3.1-pSARA-CoV-2suggested: NoneFor the HIV1-based SARS-CoV-2 spike pseudotyped virus generation, WT pcDNA3.1-pSARS-CoV-2-S, pVP21-SA-variant, and pVP28-Indian variant lacking the C-terminal 19 codons were employed. pVP21-SA-variantsuggested: NoneA pair of forward and reverse primers were utilized to amplify fragments lacking the C- terminal 19 codons with pVP21-SAvariant and pVP28-Indian variant as template separately. pVP21-SAvariantsuggested: NoneThe amplified fragments were gel-purified and cloned into pVP21-SAvariant backbone and pVP28-Indianvariant backbone, digested with BbvCI and BamHI. pVP28-Indianvariantsuggested: NoneRecombinant antibody generation: The top-ranked enriched IgG clones were selected and cDNAs of relative variable region of paired heavy- and light-chain were codon-optimized and cloned separately into human IgG1 heavy chain and light chain expression vectors, containing the human IgG1 constant regions (pFuse plasmids) pFusesuggested: NoneThe variable region of Clone 6 heavy chain was cloned into pFUSE2ss-knobheavy-hG1vector. pFUSE2ss-knobheavy-hG1vectorsuggested: NoneThe variable region of Clone 6 light chain was cloned into pFUSE2ss-knobLight-hK vector. pFUSE2ss-knobLight-hKsuggested: NonePlasmid expression a C-terminally truncated SARS-CoV-2 S protein (pSARS-CoV-2Δ19) was obtained from Dr Bieniasz’ lab. pSARS-CoV-2Δ19suggested: NoneBriefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI. pCCNanoLuc2AEGFPsuggested: NoneIt was inserted into a piggybac transposon (Matt Wilson of Baylor College of Medicine, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; resultant plasmid was named pT-PB-SARS-CoV-2-Spike- IRES-Blasti. pCMV-piggybacsuggested: NonepT-PB-SARS-CoV-2-Spike- IRES-Blastisuggested: NoneThis too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES- Blasti. pT-PB-SARS-CoV-2-UKsuggested: NonehACE2 was subsequently introduced by VSV G-mediated HIV-based transduction using pHIV-CMV-hACE2-IRES-Puro to produce HOS-3734, which cell lines maintained in selection using 10 μg/mL puromycin (Sigma-Aldrich). pHIV-CMV-hACE2-IRES-Purosuggested: NoneTZMbl cells stably expressing wild type S/UK variant S were created by co-transfecting TZMbl cells with pT-PB-SARS-CoV-2- Spike-IRES-Blasti or pT-PB-SARS-CoV- 2-UK Spike-IRES-Blasti, respectively, along with pCMV-piggybac and resistant cells selected with 10 μg/mL blasticidin (Invivogen). pT-PB-SARS-CoV-2-suggested: NonepT-PB-SARS-CoV-suggested: NoneThe control TZMbl cell line not expressing S was generated by co-transfecting pCMV- piggybac with pT-pB-IRES-Blasti and selecting for blasticidin-resistant TZMbl cells. pCMV-suggested: RRID:Addgene_99510)pT-pB-IRES-Blastisuggested: NoneSoftware and Algorithms Sentences Resources Antibody humanization: In order to humanize the antibody, we first determine the six CDR loops from murine variable domains by using the online free program “IGBLAST” (https://www.ncbi.nlm.nih.gov/igblast/). https://www.ncbi.nlm.nih.gov/igblast/suggested: (IgBLAST, RRID:SCR_002873)The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.) GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Data were analyzed with non-linear regression using GraphPad Prism to determine the neutralization curve and the IC50 values calculated. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Image processing and 3D reconstruction using cryoSPARC 78 produced similar results. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)The final map of each body was corrected for K3 detector modulation and sharpened by a negative B-factor estimated by RELION 80, and then merged in Chimera for deposition. RELIONsuggested: (RELION, RRID:SCR_016274)The initial models were subsequently manually rebuilt in COOT 83, followed with iterative cycles of refinement in Refmac5 84 and PHENIX 85. COOTsuggested: (Coot, RRID:SCR_014222)The cryo-EM structures and homology models were analyzed in Pymol. Pymolsuggested: (PyMOL, RRID:SCR_000305)Genomic sequencing raw data are deposited to Gene Expression Omnibus (GEO) with the accession code (GSE174635). Gene Expression Omnibussuggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)Results from OddPub: Thank you for sharing your code and data.
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