Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants

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1. Version published to 10.1016/j.chempr.2022.07.012

   Oct 1, 2022
2. 

   ScreenIT

   Mar 17, 2022

   SciScore for 10.1101/2022.03.11.483867: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The protein band was detected with HRP-conjugated anti-Hisx6 antibody (Proteintech, #HRP-66005).	anti-Hisx6 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The mixture was diluted 50 times with HBSS and incubated at 37 °C for 1 h, 100 μL of which was subsequently added to 1.5 × 105 ACE2-expressing 293T cells and incubated at 37 °C for 1 h.	293T suggested: None
   The virus stocks were prepared in Vero E6 (ATCC) and titers were determined by plaque assays on Vero-E6 cells.	Vero E6

   SciScore for 10.1101/2022.03.11.483867: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The protein band was detected with HRP-conjugated anti-Hisx6 antibody (Proteintech, #HRP-66005).	anti-Hisx6 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   The mixture was diluted 50 times with HBSS and incubated at 37 °C for 1 h, 100 μL of which was subsequently added to 1.5 × 105 ACE2-expressing 293T cells and incubated at 37 °C for 1 h.	293T suggested: None
   The virus stocks were prepared in Vero E6 (ATCC) and titers were determined by plaque assays on Vero-E6 cells.	Vero E6 suggested: RRID:CVCL_XD71) Vero-E6 suggested: None
   Neutralizing assays were performed in 293T-ACE2 cells (15,000 cells per well) in a white opaque 96-well plates.	293T-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Nanobody expression and purification: Plasmid pBAD-H11D4, pBAD-MR17K99Y, or pBAD-SR was transformed into E. coli BL21(DE3) for wildtype protein expression.	pBAD-SR suggested: None
   Plasmid pBAD-H11D4 (27TAG), pBAD-H11D4 (30TAG), pBAD-H11D4 (100TAG), pBAD-H11D4 (112TAG), pBAD-H11D4 (115TAG), pBAD-H11D4 (116TAG), pBAD-MR17K99Y (99TAG), pBAD-MR17K99Y (101TAG), pBAD-SR4 (37TAG), pBAD-SR4(54TAG), or pBAD-SR4 (57TAG) was co-transformed respectively with plasmid pEVOL-FSYRS into E. coli BL21(DE3) for FSY incorporated protein expression.	pBAD-H11D4 suggested: None pBAD-MR17K99Y suggested: None pBAD-SR4 suggested: None pEVOL-FSYRS suggested: None
   The cells were transfected with 25 μg pcDNA-ACE2-34TAG and 25 μg pMP-FSYRS plasmids according to manufacturer’s protocol.	pcDNA-ACE2-34TAG suggested: None pMP-FSYRS suggested: None

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   A potential caveat is that the cross-linking rate can be negatively impacted when the binding affinity between protein drug and target decreases drastically due to target mutation, such as mNb6(108FFY) and Beta RBD. In rare cases, mutation can even occur at the cross-linking residue, which would abolish cross-linking at that site. However, as we demonstrated here and previously,19 44 PERx is a general strategy that can be applied on diverse nanobodies and each nanobody can have FFY incorporated at multiple sites to target different natural residues on Spike RBD to achieve crosslink (Figure 3). Therefore, other nanobodies able to bind mutated Spike RBD could be similarly engineered, and diverse covalent nanobodies could be used in combination to ensure the cross-linking and inhibition of future potential variants. When in use, the irreversible cross-linking ability together with multi-targeting of these covalent nanobodies should achieve complete viral inhibition and mechanistically prevent drug resistance. Nonetheless, animal tests and clinical trial are warranted to confirm these potential benefits of covalent protein drugs. Fast reaction kinetics would be critical for effective inhibition of viral infection, as it enables covalent crosslinks to promptly form between the protein drug and the target within a shorter contact time, and to reach higher extent for more effective neutralization. By introducing electron withdrawing fluorine substitution we designed and genetically ...

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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: We did not find any issues relating to colormaps.

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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.
   • No funding statement was detected.
   • 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 10.1101/2022.03.11.483867v1 on bioRxiv

   Mar 14, 2022