Review of Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Shane Miersch, Zhijie Li, Reza Saberianfar, Mart Ustav, James Brett Case, Levi Blazer, Chao Chen, Wei Ye, Alevtina Pavlenco, Maryna Gorelik, Julia Garcia Perez, Suryasree Subramania, Serena Singh, Lynda Ploder, Safder Ganaie, Rita E. Chen, Daisy W. Leung, Pier Paolo Pandolfi, Giuseppe Novelli, Giulia Matusali, Francesca Colavita, Maria R. Capobianchi, Suresh Jain, J.B. Gupta, Gaya K. Amarasinghe, Michael S. Diamond, James Rini, Sachdev S. Sidhu. bioRxiv 2020.10.31.362848; doi: https://doi.org/10.1101/2020.10.31.362848 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: In this pre-print, James Rini and Sachdev Sidhu's team applied a modular strategy to develop synthetic tetravalent antibodies against SARS-CoV2. Iterative changes improved the antibody specificity and binding affinity while retaining advantageous biophysical properties for drug development. Furthermore, the authors demonstrated that the antibody was effective against potential escape mutants. Thus, the authors provided a workflow for synthetic antibodies that can be readily applied against COVID-19 and other emerging viral threats. OVERALL ASSESSMENT: STRENGTHS: Overall, we think that the authors have designed a potent antibody candidate using the processes described in this manuscript. The manuscript is written concisely and describes design principles that can guide the engineering of synthetic antibodies with strong neutralizing capabilities against SARS-CoV2. The authors identified residues that confer improved antibody binding affinities and elucidated mechanism of action using electron microscopy and X-ray crystallography. Overall, a framework for rapid therapeutic antibody development was well described in this manuscript. WEAKNESSES: In more than one instance, we thought that analyses could be strengthened by the inclusion of a control antibody. The authors may consider an examination of the potency and biophysical properties of antibodies with specificity against SARS-COV2 (references are made to Bamlanivimab, Casirivimab, and Imdevimab in the introduction). The reader would benefit from a more detailed description of the construction of IgG1 antibodies 15031 – 15033 and the variants of IgG 15033 (data is only provided for 15033 and variants). DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 1.5 ● Figure by figure, do experiments, as performed, have the proper controls? ● [Note: in this section, the class discusses proper controls, but also uses the 'figure-by-figure' opportunity to discuss rationale and approaches] o Figure 1: We discussed at length how these experiments would be stronger by including an existing anti-SARS-CoV2 as a control, to provide a direct head-to-head comparison. Without controls, it was more challenging to assess performance of synthetic antibodies. ▪ Would Figure 1B be better placed as the last panel for clarity? It was unclear to us what 15033-7 was referring to until we had reached the last paragraph in the first Results subsection. ▪ Why did the authors choose to recombine only the third CDR of the light chain together with the CDRs of the heavy chain? Please revise for clarity. ▪ In Figure 1D, the authors examined ACE2 binding to S-protein in the presence of three antibody candidates. It was a bit unclear to us how the signal was normalized – do the bars represent the percentage of the total signal seen with just control IgG? It may be useful to include a dotted line at Y = 1.0. o Figure 2: No issues. o Figure 3: No issues, though the class was unsure why the control antibody Trastuzumab was omitted in Figure 3C. o Figure 4: The class noted that this figure favoured Fab-IgG heavily. Are there representative images for IgG-Fab binding to the viral protein? Similarly, the diagrams all show the viral protein in the 'up' conformation. We were very keen to see how these interactions might change in the 'down' conformation'. o Figure 5: No issues. Are specific analyses performed using methods that are consistent with answering the specific question? o We had reservations about the alanine scanning mutagenesis experiment described in Figure 5. We understand this technique has been used to mimic viral escape mutations, however some of the mutations modelled would have required a change in two nucleotides (e.g. in Phe486). ● Is there the appropriate technical expertise in the collection and analysis of data presented? o Yes, ample technical expertise was demonstrated. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o N/A – there are no statistics featured in the manuscript. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o As mentioned above, we would have appreciated opportunities to assess affinities, potencies, and biophysical properties of existing antibodies against SARS-CoV2 to properly contextualize the results. 2. Quality: Completeness (1–3 scale) SCORE = 2 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o The data broadly support the conclusions made in the title and abstract o The class expressed some doubts about the thermostability results presented in supplemental figures. Some of the antibody candidates do not look as thermostable as Trastuzumab. Would this be worth addressing in the Discussion? ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o The authors may wish to examine antibody cytotoxicity. Non-cytotoxic concentrations should be used in their in vitro experiment (i.e. Figure 5), to exclude the possibility that the decrease in viral infection was due to treatment-related cell death. o As mentioned above, we had reservations about the thermostability of the synthetic antibodies compared to Trastuzumab. 3. Quality: Reproducibility (1–3 scale) SCORE = 2 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o It was unclear to us whether some of the figures showed results from two representative samples or whether only two replicates were performed in total per cohort (e.g. Figure 5A). o We could not find where in the manuscript the authors had specified the total number of biological and technical replicates performed per experiment. ● Is there sufficient raw data presented to assess rigor of the analysis? o We thought it would be useful for readers to examine the viral infection assay in greater detail. Can the authors provide the raw data for Figure 5B (in RLU) as supplemental information? o Line 133 mentions that 15033-7 saw significantly improved avidity compared to the other sixteen mutant 15033 antibodies, but we couldn't locate the data that supports this conclusion. o Lines 121-124 in the manuscript also mention that antibody 15033 "exhibited the highest avidity" compared against 15031 and 15032. Can the authors include this data in Table 1 or perhaps in a Supplemental Table? Are methods for experimentation and analysis adequately outlined to permit reproducibility? o It was unclear to us where the Fab sequences the authors used to construct their phage-display library were sourced. The authors reference a 2013 paper that describes how to construct such a library, however the manuscript does not describe how the sequences were obtained or where they were deposited. Could the authors provide any clarification in the Methods? o There is no mention in the Methods re: how the CDRs of the heavy and light chain regions were recombined in the 15033 antibody. The authors call this library of mutant antibodies the "light chain library" – was this library only created from swapping out CDRs in the light chain and not the heavy chain? o How were full-length IgG1 molecules created from the antibody fragments? We could not find how this was done in the Methods ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o The authors talk about vaccine hesitancy and they note that "how well they will be adopted by the public at large remains an unknown". However, there are vaccine hesitancy reports that include surveys on COVID-19 vaccines that the authors could cite and discuss: ▪ StatsCan Survey: https://www150.statcan.gc.ca/n1/pub/45-28-0001/2020001/article/00073-eng.htm ▪ Nature Medicine: https://www.nature.com/articles/s41591-020-1124-9. o The authors may wish to discuss some relevant COVID19 variants such as the South African and Brazilian variants in the context of their viral escape mutation experiment (Figure 5). ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Are there any relevant papers that discuss how valency can affect an antibody's avidity? Are these two qualities related and complementary or is there a corresponding trade-off that the authors could discuss? o Our class also discussed how we would like the authors to include background and/or insight into the feasibility and challenges of delivering synthetic abs into a physiological system (i.e. animal model, people). Are there any issues with immunogenicity and lower half-lives (which was partially addressed in Figure 1E)? ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant bases for decisions. o Generally, the manuscript was written clearly and concisely. o We recommend a colour-coded legend for Figure 2 so readers will not have to flip back to the Figure Legends to understand the annotations. o In Figures 3B/D the colours for the lines are hard to resolve from one another, most especially the yellow lines against the white background or where the red lines are superimposed on top of the yellow lines. o It was unclear what the solid black lines that are superimposed on the coloured lines in Figure 3D represent. o There is a typo in the caption for Figure 3D (6.7 nM vs 67 nM in the Methods section) o We noticed that line 274 mentions 44 VLP variants, but lines 276-277 suggests that 49 variants were created. Is this a typo? MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.5 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o The class agreed that the most interesting finding in this manuscript is the fabrication of the synthetic antibody and the subsequent workflow for engineering enhanced performance against SARS-CoV-2. o Owing to the present pandemic and the media attention given to the safety of proposed and existing treatments for COVID19, the class agreed that this study is interesting and important, and can be readily understood by both the public and the broader scientific community. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o This manuscript outlines an antibody optimization workflow that can be readily followed and reproduced by other teams. If the antibodies arising from this pipeline work just as well in vivo as they did in vitro, then this paper is an important advance for the field. o The class agreed that this approach could be broadly applied to other viral infections. o The antibodies generated for this study were sourced from a library described in Persson, et al. (2013). Some students thought it challenging to consider the extensibility of this present manuscript without a more thorough description of this library in the Discussion 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o N/A – antibody efficacies were only evaluated in an in vitro model. o Though this is an N/A score, the class would like to note that we are very keen to see how well these synthetic antibodies perform in an in vivo infection model