Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617

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1. Version published to 10.1038/s41467-022-29288-3

   Mar 28, 2022
2. 

   ScreenIT

   Dec 30, 2021

   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-hchg1 suggested: None pFUSE2ss-CLIg-hK suggested: 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-RBD suggested: None
   IgG1 antibodies were expressed in Expi293FTM cells.	IgG1 suggested: 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-hG1 suggested: None pFUSE2ss-HoleLight-hK suggested: None pFUSE2ss-HoleHeavy-hG1 suggested: None
   BLI): Antibody binding kinetics for anti-spike mAbs were evaluated by BLI on an Octet RED96e instrument (FortéBio) at room temperature.	anti-spike suggested: None
   After 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-IgG1 suggested: None
   The 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-S suggested: None anti-mouse IgG suggested: (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.	293FT suggested: None
   Briefly, 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.	293T suggested: None
   The pseudovirus neutralization assays were performed on 293T-hACE2 cell line 71.	293T-hACE2 suggested: None
   5x105 Vero-E6 cells were plated per well of a 96-well plate.	Vero-E6 suggested: None
   Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h.	Vero-TMPRSS2 suggested: 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 E6 suggested: 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/6J suggested: RRID:IMSR_JAX:000664)
   Clone 5-11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice.	BALB/c suggested: 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-hACE2 suggested: RRID:IMSR_GPT:T037657) B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: 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: None
   Recombinant 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-hK suggested: None
   Four plasmids were employed: pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1.	pFUSE2ss-knobheavy-hG1 suggested: None pFUSE2ss-HoleLight-hK suggested: None pFUSE2ss-HoleHeavy-hG1 suggested: None
   The pFUSE2ss-knobLight-hK is pFUSE2ss-CLIg-hK with no further editing.	pFUSE2ss-CLIg-hK suggested: None
   The 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).	pPR024 suggested: None
   The 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-hG1 suggested: None
   The 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).	pPR023 suggested: None
   The 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-Indian suggested: None pcDNA3.1-pSARS-CoV-2-S suggested: None
   For 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-2 suggested: None
   For 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-variant suggested: None
   A 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-SAvariant suggested: None
   The amplified fragments were gel-purified and cloned into pVP21-SAvariant backbone and pVP28-Indianvariant backbone, digested with BbvCI and BamHI.	pVP28-Indianvariant suggested: None
   Recombinant 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)	pFuse suggested: None
   The variable region of Clone 6 heavy chain was cloned into pFUSE2ss-knobheavy-hG1vector.	pFUSE2ss-knobheavy-hG1vector suggested: None
   The variable region of Clone 6 light chain was cloned into pFUSE2ss-knobLight-hK vector.	pFUSE2ss-knobLight-hK suggested: None
   Plasmid expression a C-terminally truncated SARS-CoV-2 S protein (pSARS-CoV-2Δ19) was obtained from Dr Bieniasz’ lab.	pSARS-CoV-2Δ19 suggested: None
   Briefly, 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.	pCCNanoLuc2AEGFP suggested: None
   It 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-piggybac suggested: None pT-PB-SARS-CoV-2-Spike- IRES-Blasti suggested: None
   This too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES- Blasti.	pT-PB-SARS-CoV-2-UK suggested: None
   hACE2 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-Puro suggested: None
   TZMbl 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: None pT-PB-SARS-CoV- suggested: None
   The 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-Blasti suggested: None
   Software 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.)	GraphPad suggested: (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 Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Image processing and 3D reconstruction using cryoSPARC 78 produced similar results.	cryoSPARC suggested: (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.	RELION suggested: (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.	COOT suggested: (Coot, RRID:SCR_014222)
   The cryo-EM structures and homology models were analyzed in Pymol.	Pymol suggested: (PyMOL, RRID:SCR_000305)
   Genomic sequencing raw data are deposited to Gene Expression Omnibus (GEO) with the accession code (GSE174635).	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   Results from OddPub: Thank you for sharing your code and data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 54. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.12.21.473733v1 on bioRxiv

   Dec 24, 2021