High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme
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Abstract
There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC 50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.
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SciScore for 10.1101/2020.07.24.219857: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Immunization of Llamas: An adult male 16-year-old llama, named Cormac, was immunized under contract at Triple J Farms (Kent Laboratories, Bellingham, WA). Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The assay plate was washed and 100 µL peroxidase conjugated goat anti-alpaca VHH domain specific antibody (#128-035-232, Jackson ImmunoResearch) transferred into each well and incubated for 1 hour at room temperature. anti-alpaca VHH domain specific antibody (#128-035-232suggested: NoneExperimental Models: Cell Lines Se… SciScore for 10.1101/2020.07.24.219857: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Immunization of Llamas: An adult male 16-year-old llama, named Cormac, was immunized under contract at Triple J Farms (Kent Laboratories, Bellingham, WA). Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The assay plate was washed and 100 µL peroxidase conjugated goat anti-alpaca VHH domain specific antibody (#128-035-232, Jackson ImmunoResearch) transferred into each well and incubated for 1 hour at room temperature. anti-alpaca VHH domain specific antibody (#128-035-232suggested: NoneExperimental Models: Cell Lines Sentences Resources The resulting phage elution was used to infect TG-1 cells and additional phage amplification and immunoselections performed. TG-1suggested: RRID:CVCL_0P34)SARS-CoV-2 pseudotyped lentivirus-based transduction fluorescence inhibition assay: All lentiviruses were propagated in HEK293T/17 cells (ATCC # CRL-11268) according to published Current Protocols in Neuroscience.[41] Briefly, 293T cells were transiently transfected with plasmids expressing SARS-CoV-2 spike protein (GenScript MC_0101081, human codon optimized, ER retention signal removed), psPAX2 (Addgene #12260), and a lentiviral transfer vector CD512-EF1a-RFP (System Biosciences CD512B-1) using Lipofectamine 2000. HEK293T/17suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)293Tsuggested: NoneSoftware and Algorithms Sentences Resources Data analysis was performed with ProteOn Manager 2.1 software, corrected by subtraction of an uncoated column as well as interspot correction. ProteOn Managersuggested: NoneSanger Dideoxy Sequencing: Clones enriched by phage display and positively selected by ELISA assay and bio-layer interferometry measurement were sequenced using a universal Lac-promoter primer (Lac-fwd: 5’-CGTATGTTGTGTGGAATTGTGAGC-3’) with standard Sanger dideoxy sequencing at Genewiz. Genewizsuggested: (GENEWIZ, RRID:SCR_003177)Sequences were trimmed to include only the VHH coding region and the protein coding sequences aligned using the Clustal Omega algorithm[40] included in SnapGene software (GSL Biotech LLC). Clustal Omegasuggested: (Clustal Omega, RRID:SCR_001591)SnapGenesuggested: (SnapGene, RRID:SCR_015052)SARS-CoV-2 pseudotyped lentivirus-based transduction fluorescence inhibition assay: All lentiviruses were propagated in HEK293T/17 cells (ATCC # CRL-11268) according to published Current Protocols in Neuroscience.[41] Briefly, 293T cells were transiently transfected with plasmids expressing SARS-CoV-2 spike protein (GenScript MC_0101081, human codon optimized, ER retention signal removed), psPAX2 (Addgene #12260), and a lentiviral transfer vector CD512-EF1a-RFP (System Biosciences CD512B-1) using Lipofectamine 2000. Neuroscience.suggested: NoneResults 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: We detected the following sentences addressing limitations in the study:There are several limitations of the current results. First, the characterization of NIH-CoVnb-112 and the other nanobodies is not yet complete. Under the current circumstances, we have opted to move forward with dissemination of our findings earlier than might otherwise be typical. Second, a potential new therapeutic such as this nanobody would, even under the best circumstances, come to market relatively slowly compared to a repurposed approved drug. Thus, it is possible that if an approved drug is found to be effective against SARS-CoV-2, it will be available long before this or any other nanobody candidate therapy. Third, it is possible that NIH-CoVnb-112 and other nanobodies may provoke host immune responses. NIH-CoVnb-112 is a llama protein fragment that is similar to human proteins yet cannot be fully humanized and maintain desirable biophysical characteristics. Fourth, NIH-CoVnb-112 and other nanobodies may have unexpected toxicity. There is only one United States Food and Drug Administration-approved nanobody therapeutic, Caplacizumab, and there has not been exposure to a sufficient number of human patients to fully characterize the safety of this class of therapeutics. Despite these limitations, the novel nanobodies that bind to the spike protein RBD have potential widespread utility in the battle against the SARS-CoV-2 pandemic.
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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