Dear Dr. Jonas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Yalong Dang

Academic Editor

PLOS ONE

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Jost B. Jonas, Songhomitra Panda-Jonas: European patent EP 3 271 392, JP 2021-119187, and US 2021 0340237 A1: “Agents for use in the therapeutic or prophylactic treatment of myopia or hyperopia; European patent application 23196899.1 “EGFR Antagonists for the treatment of diseases involving unwanted migration, proliferation, and metaplasia of retinal pigment epithelium (RPE) cells“. All other authors: None

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The sample size of 8 rabbits (4 per time point) may be limiting for statistical analysis. A larger sample size and blinding could improve the study's robustness. Using Ringer's solution instead of ranibizumab as the control makes it challenging to attribute effects specifically to panitumumab. The Wilcoxon test with 4 animals per group may have limited power to detect differences.

Suggestions for Improvement:

IOP monitoring: Repeated measurements over time would provide better safety assessment.

Statistical analysis: The adding of analysis of variance components may be favorable.

Clinical discussion: Expanding on potential clinical implications would strengthen the manuscript.

Reviewer #2: Dear Editor,

This manuscript is a very simple study on the potential toxicity of multiple antibody injections in a rabbit model. Overall, the manuscript is well written, and needs only minor copy-editing for grammar. E.g., line 177 “injections were performed in anesthesia” should be corrected to “injections were performed under anesthesia”. Line 169: “Examinations of the anterior chamber neither revealed signs of intraocular inflammation” needs minor correction for clarity. Statistical analysis is appropriate.

Although straightforward, and generally well designed, this study is missing data would greatly strengthen the authors case that these treatments are safe. This is particularly important since the n for each experimental time point is quite low (n=4, 8 rabbits total). Namely:

- Including representative images for the control eyes, a positive control, and a negative control for the TUNEL labeling. Include labels of the retinal layers on the figures.

- Including representative images of the H&E stained retinas – both the control and the antibody-injected eyes for comparison. Include labels for the retinal layers.

- Including representative images for the control eyes for the fundus and OCT data. Labeling the retina layers.

- Including imaging data for the anterior segment of the eye (line 169).

As for data availability, the authors have opted to not share the data – i.e., they selected No, some restrictions will apply. I don’t understand why, as it’s a very simple study and it should be quite easy to make all of the data, for only 8 rabbits, available. In fact, sharing this data would strengthen the merits and claims of this study.

I think that this study should not be published until more data are shown and the figures reflecting these data amended. I chose “minor revisions” because these data should exist, and I am not requesting more experiments, just showing more data from the experiments that were done. A simple additional experiment that could be performed would be to label eyes for microglial infiltration (Iba1), which would potentially signal early biological signs of inflammation.

Reviewer #3: The manuscript examines the potential effect of combined intraocular injection of panitumumab and ranibizumab on the rabbit retina. Such a “safety” study is valuable given the potential benefit of adding an EGF receptor antibody (panitumumab) to the well-established effect of VEGF antibodies (e.g. ranibizumab) as a treatment for exudative maculopathies. Whereas VEGF antibodies reduce neovascularization, they do not reverse the formation of an irreversible subretinal fibrous scar due to RPE proliferation. In vitro studies have suggested that EGF promotes RPE proliferation, an effect that can be blocked by EGF receptor blockers. Clinical studies have suggested that intraocular EGF levels are elevated in exudative age-related macular degeneration and myopic macular neovascularization. An obvious idea would be to employ a combined treatment, with blockers of VEGF and EGF receptors used concurrently. This current manuscript suggests that an important first step is to test the safety of such a combined treatment using an animal model (rabbit).

The study employs four measures to compare rabbit eyes injected with either the combination of panitumumab and ranibizumab (right eye) or saline (left eye): 1) histological measures of retinal layer composition (cell counts) and thickness; 2) intraocular pressure (IOP); 3) TUNEL-labelling to detect apoptosis; and 4) fundus photography and OCT.

Taken as a whole, the results suggest that the combination of panitumumab and ranibizumab does not produce any signs of retinal damage: there were no statistically significant differences between the treated (right) and control (left) eye with respect to retinal layer thickness, mcell number or IOP; there was no evidence of TUNEL-labelling in treated or control retina; there were no signs of changes visualized using fundus photography and OCT.

The results, as presented, have several weaknesses that limit the suitability of the manuscript:

1. No photomicrographs of the retinal sections are provided to allow the reader to evaluate the quality of the histology.

2. Given the large variation inherent in the histological measurements and the sample size, it is not clear if the sensitivity is sufficient. At the very least, some discussion about what would constitute a meaningful change in the parameters would be informative.

3. In Table 1, two P-values of 0.04 are reported. Why were these not considered significant, given that alpha was set at 0.05?

4. The TUNEL data is weak without a positive control, demonstrating that the method employed by the authors is capable of detecting TUNEL+ cells.

5. Figure 2 is inadequate. What features in these images were considered important, and there should be examples comparing these parameters in treated vs. untreated eyes. The description of the results related to the figure is inadequate. What signs of “intraretinal inflammation”? What “other changes”? Why not use OCT to perform retina thickness measurements that would add to the histology?

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Safety of íntravitreal panitumumab combined with intravitreal ranibizumab in rabbits

PONE-D-25-38703R1

Dear Dr. Jonas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yalong Dang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors well addresseed the concerns raised by the three reviewer, I think it would be acceptable in its current form for publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: The manuscript is now in strong shape. The reasoning is clear, and the study presents meaningful scientific implications. The revised statistical analyses are more robust, especially with the inclusion of the new figures—such as the TUNEL-stained images, the positive TUNEL control, and the anterior ocular segment photograph—which enhance the reliability and clarity of the results. The added statistical details further strengthen the overall rigor of the work.

The authors have also appropriately discussed the study’s limitations and outlined directions for future research, making the manuscript as comprehensive as the experimental conditions reasonably allow while paving the way for relevant follow-up investigations. As all datasets now align with PLOS ONE’s requirements, the paper is suitable for publication.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

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