A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies
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Abstract
Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 10 11 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.
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SciScore for 10.1101/2020.10.29.361287: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization CDR2 was randomized in stage one, PCR templates at this stage were equal molar mixtures of plasmids carrying DNA encoding frames, including three frame1 versions, one frame2, three frame3 versions and one frame4. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources ) with anti-Flag antibody (Sigma-Aldrich, F1804), then incubating antibody-coated beads with cell lysate or cell media containing 3xFlag tagged target proteins at 4°C for 2 hours. anti-Flagsuggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)F1804suggested: NoneSciScore for 10.1101/2020.10.29.361287: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization CDR2 was randomized in stage one, PCR templates at this stage were equal molar mixtures of plasmids carrying DNA encoding frames, including three frame1 versions, one frame2, three frame3 versions and one frame4. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources ) with anti-Flag antibody (Sigma-Aldrich, F1804), then incubating antibody-coated beads with cell lysate or cell media containing 3xFlag tagged target proteins at 4°C for 2 hours. anti-Flagsuggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)F1804suggested: NoneFor anti-Myc selection, magnetic beads were coated by anti-Myc antibody (ThermoFisher Scientific, 13-2500) only. anti-Mycsuggested: NonePlates were washed three times with wash buffer, HRP conjugated anti-His tag secondary antibody (BioLegend, 652503) diluted 1:2000 in blocking buffer was then added to the plates and incubated at RT for 1 hour. anti-His tag secondary antibody ( BioLegend , 652503suggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T ACE2 were a kind gift of Michael Farzan. HEK293T ACE2suggested: RRID:CVCL_YZ65)Plates were washed once with PBST (PBS, ThermoFisher Scientific, with 0.02% TritonX-100), a 1:1 mixture of HEK293T cell culture media containing secreted RBD-3xFlag and blocking buffer (PBST with 1% nonfat dry milk) was added to the plates and incubated at RT for 1 hour. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Medium was then removed from HEK293T ACE2/TMPRSS2 cells and replaced with 150 μl of the VHH + pseudotyped lentivirus solution. HEK293T ACE2/TMPRSS2suggested: NoneRecombinant DNA Sentences Resources psPAX2 and pCMV-VSV-G were previously described 20. pTRIP-SFFV-EGFP-NLS was previously described 21 (a gift from Nicolas Manel; Addgene plasmid # 86677; http://n2t.net/addgene:86677; RRID:Addgene_86677). cDNA for human TMPRSS2 and Hygromycin resistance gene was obtained by synthesis (IDT). detected: RRID:Addgene_86677)Software and Algorithms Sentences Resources CDR-directed clustering analysis: Computational analysis for CDR-directed clustering was performed using custom python scripts. pythonsuggested: (IPython, RRID:SCR_001658)Percentage GFP was quantified on a Cytoflex LX (Beckman Coulter) and data were analyzed with FlowJo. FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: Thank you for sharing your code.
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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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