PONE-D-23-29661Characterization of Two Affinity Matured Anti-Yersinia pestis F1 Human Antibodies with Medical Countermeasure PotentialPLOS ONE

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"This research was supported by the Los Alamos National Laboratory (LANL) Directed Research

and Development Program (LANL LDRD 20180005DR), and the LANL Richard P. Feynman

Center for Innovation Strategic Investment Initiative. This work was performed, in part, at the

Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S.

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affirmative action equal opportunity employer, is managed by Triad National Security, LLC for

the U.S. Department of Energy’s NNSA, under contract 89233218CNA000001. JPEO-CBRND

through chemical biological defense funding supported part of this work in an effort to analyze

prediscovered antibodies for efficacy against plague.

We thank BEI Resources, NIAID, NIH, for supplying the various forms of Yersinia pestis F1V

fusion protein and Abel Ordonez Luna and Dr. Rekha Panchal for providing us with RAW264.7 cells.

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not

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Additional Editor Comments:

The authors should adequately address the concerns expressed by the reviewers related to the explanation lacking the selection of F1 protein as a target for a therapeutic antibody as it is not a required virulence factor, bioweapons are designed to be F1 negative, and the areas where antibiotic-resistant strains are used do not have the resources to employ mAb therapy. None of these important points are discussed in the manuscript. Also, adequate explanations are needed to justify the system used and the deficiencies in the flow system used to select for binding as a function of the yeast cells that express the antibody.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors present a study on the further development of anti-F1 monoclonal antibodies for use as countermeasures against Yersinia pestis infection. The concept of using F1 as a target is questionable because, while it is a strong immunogen, it is not a required virulence factor for the pathogen, and the general consensus is that weaponized strains will be F1negative as the F1 protein is a principal component of western vaccine development. Additionally, the regions where naturally occurring antibiotic resistant strains are found are among the most resource limited areas in the world; areas where a very expensive technology, like a therapeutic monoclonal antibody could not be employed due to cost. These are items that really need to be discussed in the paper and are absent from it. Additional issues with the study are a general overestimation of the beneficial effects of their affinity maturation system, which is burdensome and had a negligible outcome on survival and affinity as determined by SPR, which is really the gold standard here. The writing in many areas is unclear, specific examples are given below. The way they analyze their binding data by flow cytometry doesn’t not appear to be a good reflection of the ability of the antibody to bind to the antigen, but rather a measure of binding as a function of expression level. If the cell could express the antibody at a high rate, then it bound a lot of antigen. However, that doesn’t mean that an antibody expressed at a lower rate doesn’t have a higher affinity. The exression level of the scFv in in yeast is irrelevant to antibody production because the CDRs are put into a different expression system later on for production of functional antibody. The metrics for defining what was considered good binding are not defined. It’s not really apparent that there were any. It seems that the investigators just drew a gate at the top of the dot plot cluster, but there doesn’t appear to be a defined cutoff.. Furthermore, the whole antibody production design is poorly explained in the paper. There are numerous systems that exist not where CDRs can be cloned into human IgGs that are optimized with glycosylation for Fc receptor interactions and increased uptake. The affinity of the binding domain is only one small part of the functional capacity of an antibody, and it really doesn’t seem like the whole process here improved on antibody binding anything more marginally. All of this reduces enthusiasm for the manuscript. More specific issues are given below.

The intro is wordy and can be shortened. There is a lot of superfluous information there. Mab therapy for COVID was relatively ineffectual unless given very early during infection, and efficacy dropped off through mutation of the spike protein. They did not play a significant role in the pandemic. Furthermore, going on about massive mutations of a functional antigen not being likely to happen doesn’t really have relevance to the current study, as F1 is not an antigen required for virulence, and F1 negative strains cause natural human disease and are the most likely kind of strain to be used in a bioterrorist attack to get around vaccines that have been developed.

Line 78, define F1V for the reader.

Line 118-120: What was considered a high signal, was there a specific MFI cutoff? Also, wouldn’t the PE signal be directly related to the Alexa 633 signal in any case where there is binding? MFI is a characteristic of the amount of fluorescence on a given particle, therefore by definition a high -633 signal would require a high PE signal. You shouldn’t have one without the other. In a perfect world you should have linearity between your 633 signal and your PE signal if the mutated antibody were binding to the F1 at saturation. Thus a clone that has higher expression would artificially be promoted as a clone with better binding, even if the actual affinity could be lower when those CDRs are put into an antibody. It’s also unclear to this reviewer why higher expression in a yeast system would be reflective of any characteristic for the antibody later on given that the variable regions were inserted into a cassette system later on? Perhaps expanded on this discussion a bit further.

Supplementary figure 1 should be in the paper with figure 2. Just showing the MFI in figure 2 alone doesn’t give great information because it is doesn’t account for differences in expression. The Y axis of figure 2 should be labelled MFI, not antigen binding.

The specific binding equation in the methods and materials needs some additional clarification. Throughout this the authors refer to antigen binding as a fluorescence value (633 fluorescence), which presumably is MFI though this is not clearly stated. Not sure if it is the value calculated using this equation. In the equation, AB max is maximum antigen/antibody binding, but what is that? Is that the highest MFI at the highest concentration of antigen? Is that just the MFI from a particular experiment? [ab] = antibody concentration, but concentration of what antibody? You don’t know the concentration of the yeast antibody so it must be the MFI of the SV tag antibody, which is not concentration. This is all unclear. Kd apparent dissociation constant using Kaleidagraph software. Better description of how this is calculated should be provided. It also needs to be defined, exactly where this equation was used because it isn’t apparent.

Line 159-161: ‘not the observed affinity improvements’ rather ‘the magnitude of the difference in the affinity measured’

Line 198-202: I would suggest that the difference is likely native vs. recombinant fusion protein. This idea should be expanded upon.

Line 203-211: SPR is the gold standard for affinity measurement. This should come before the ELISA data.

Line 216: by phagocytes, not just macrophages.

Line 222-223: move to discussion. In addition, have you considered the possibility that your antibodies induced a higher rate of intracellular killing of the Yersinia compared to the control. That would also read out as reduced bacteria following cell lysis.

237-239: You cannot extrapolate a human MaB therapy dose from mice, it’s not worth bothering doing so. They have different kinetics and metabolism of antibodies. Not only that, but you put a human antibody into a mouse, which will enhance it’s clearance. There are mab therapies that go into 150-200mgs in humans.

I’d much rather see survival curves than a table.

Your whole paper lacks even a mention of F1 negative pestis. It is very relevant here, and really needs to be included. F1 is a great antigen, it is fully protective at very low doses in a single shot in animal studies, but hasn’t gained approval because of the African Green Monkey studies at USAmRIID and the fact that it is completely ineffective against fully lethal F1 negative strains. Bioweapons will likely be F1 negative because it is a prominent vaccine target. The other issue, is the fact that mAb therapy is out of reach, cost-wise in the regions where antibiotic resistant bacteria are present. (Madagascar/central Africa). That will greatly limit usage. Its nice that you did the study showing that they are stable at 37C and 45C for long periods of time, but the overall impact of that finding is limited by the fact that they are serum proteins. IgG evolved through nature to exist at 37C for months.

Line 271: The technology is academically interesting, but the drawbacks haven’t been fully highlighted. It is labor intensive, and of the antibodies generated one showed no appreciable increase in affinity via SPR, the gold standard, and the one that did show an appreciable increase, worked worse in vivo. That’s not a great selling point.

Reviewer #2: This manuscript describes the maturation and characterization of mAbs as a potential therapeutic for pneumonic plague. As Y. pestis is categorized as a Category A agent, continuing research into mechanisms of pathogenicity as well as new treatment strategies is important to our defense.

Overall, the manuscript was clear. Please find my suggestions below, organized by Line:

87- replace ] with ) after technologies

172 – This sentence describes the direct output of your DLS measurements, specifically single peaks. However, that is not what is shown in Figure 3, which is referenced. Either consider adding your DLS scan output or move the reference to Figure 3 to the end of the sentence in Line 174.

179-180 – Why weren’t both IgGs subjected to the same DLS temperature scan? AM2 was analyzed at only 3 temperatures, whereas AM8 was analyzed at 7.

220 – There is a random e after the word either.

225 – Language should be modified for publication. Hours should also be spelled out not abbreviated h.

Conclusion – In general, the conclusion could be strengthened with specific result details and references that support why they are favorable findings. It is a bit bold to declare dose dependency of your mAb protection when your LD50s vary significantly across your experiments.

Figure 2 – Does this data represent combined experiments (if so how many) or is it representative (if so of how many)?

Figure 3 – Your methods state that the hydrodynamic radius of a control antibody was also measured. That should be included in figure 3 to demonstrate the variability in your IgGs over time is standard or better than standard.

Figure 5 – Statistical stars should be centered above data.

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Reviewer #1: No

Reviewer #2: No

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Characterization of Two Affinity Matured Anti-Yersinia pestis F1 Human Antibodies with Medical Countermeasure Potential

PONE-D-23-29661R1

Dear Dr. Lillo,

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Kind regards,

Chandra Shekhar Bakshi, DVM, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have comprehensively addressed all the concerns from the previous review.

Reviewers' comments: