Dear Dr. Liu,

The data presented in the manuscript need to be extended and updated to support the authors hypothesis (see the Editor's comments below)

Please submit your revised manuscript by Jun 20 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.Thank you for stating the following financial disclosure: [Shanghai Natural Science Foundation of China (21ZR1407900)].

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: [We thank the Center of Cryo-Electron Microscopy, Core Facility of Shanghai Medical College, Fudan University for the supports on cryo-EM data collection and data analyses. This work was supported by grants from the Shanghai Natural Science Foundation of China (21ZR1407900).]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.]

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments:

The manuscript described generation and characterization of the novel monoclonal anti-HER-2 – specific antibodies. Two identified antibodies, murine m66 and rabbit r40, demonstrated inhibitory activity on growth of breast cancer cells and are shown to target different epitopes than clinically accepted trastuzumab and pertuzumab. Monoclonal rabbit r40 antibody could be developed further for testing in the clinical application.

The data presented in the manuscript are not quite sufficient to support the authors conclusions, and there are a few concerns that the authors need to address.

1. Antibody generation:
2. What formats were the recombinant antibodies produced using the selected sequences from B cells?
3. Protocols for ELISA and “pull-down” screening assays have to be provided.
4. How did the authors confirmed selectivity and specificity of the antibodies?
5. Competition assay
6. What is “supersaturating IgG” [concentration] – how it was determined? What was the concentration of His-tagged IgG? There was no single dose-response binding curve for any reported antibody in the whole manuscript.
7. Why mouse antibodies were tested via ELISA, and rabbit – via flow cytometry?
8. Flow cytometry competitive experiment was not described adequately in the Results section. If the authors used transient transfection, how was the efficiency of the transfection tested? What was the concentrations for all used antibodies? How were these concentrations determined?
9. Inhibition breast cancer cell proliferation assay
10. There was no figure that allows to compare the levels of HER2 expression in three cell line used. Binding dose-response curves with tested antibodies (including clinical references trastuzumab and pertuzumab) are required for each of the cell lines before the other experiments are performed. The data presented on Figure 3 are not sufficient do describe the tested antibodies binding abilities.
11. What concentration of the tested antibodies did induce the inhibitory effect and what were the EC50 s for the inhibition? What criteria did the authors use to compare the inhibitory effect for all 4 antibodies? It looks that the effective concentrations for the tested antibodies were way too high (100µg/mL equal 67nM)
12. Why the data presented in a different manner for different cells (Figure 2).
13. Why NRG1-independent and NRG1-dependent proliferation experiment was performed only in MCF7 cells?
14. HER2 signaling
15. Again, what was the rationale to use one or another cell line in different experiments? The authors either need a good rationale to use one cell liner, or use all three and show the results for all three.
16. What was a rationale for using 50µg/mL of the antibodies? This is unusually high concentration for an antibody, especially for the antibodies with the reported high affinity – in this manuscript, the novel antibodies exhibit sub-nanomolar (r40) and low nanomolar (m66) affinity.
17. Western blot images need quantification.
18. Interestingly, r40 affects the AKT pathway in SK-BR-3 cell line but not in BT-474 (Figure 4). Is it a cell-line dependent effect, or there is a rational reason for it?
19. For the ligand-dependent signaling, r40 affected AKT pathway in BT-474, and, to least extent, in SK-BR-3 but not in MCF7. Did the authors consider this difference?
20. Finally, pharmacological inhibitors of the AKT and ERK are needed as controls for these type of the experiments

[Note: HTML markup is below. Please do not edit.]

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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

Reviewer #1: These authors sought to identify a novel antibody specific to HER2-expressing cancers that could combat treatment resistance and cancer recurrence. They found that r40 could inhibit several key pathways, including cell proliferation, by modulating several cellular pathways such as PI3K/AKT and MAPK.

I found this paper very interesting to read and I congratulate the authors on their novel work. My main comments are around sentence structure and grammatical/spelling errors. I found that the authors would often switch between past and present tense when discussing their results. For clarity and readability, it is important to pick one tense and stick to it throughout. I also found that the authors sometimes abbreviated terms with the acronym coming before the term. For example:

"The EGFR (human epidermal growth factor receptor) family comprises four members..."

However, this should read:

"The human epidermal growth factor receptor (EGFR) family comprises four members..."

Other times the authors correctly put the acronym in brackets afterwards. Please ensure this is consist across the manuscript. I have attached my revisions in a word doc. Again, I would like to congratulate the authors on a novel study that address an urgent gap in clinical care for patients with HER2 cancers.

Reviewer #2: This manuscript demonstrated the selection of mouse antibody m66 and rabbit antibody r40, which target distinct sites on HER2 that are different from those bound by trastuzumab and pertuzumab. It also examined their effects on inhibiting HER2-dependent signaling pathways.

Unfortunately, this manuscript does not include data from cancer cell proliferation inhibition assays using these antibodies in combination. As a result, the insights gained from the structural analysis of the antibody-HER2 complexes are not fully utilized, diminishing the impact of the report.

Additionally, while the experiments were conducted properly and yielded new findings, the results were presented in a manner that could lead to erroneous conclusions.

Consequently, it has been determined that, in its current form, this manuscript is insufficient for publication.

Throughout the manuscript, the initial citations of figures are not appropriately placed. Figures should be referenced within specific explanatory contexts. For example, Fig 1A should be cited in the sentence: "As expected, each non-His-tagged IgG significantly blocked...".

Similar citation issues appear throughout the manuscript.

There are numerous parts of the text that are insufficiently explained. The authors should seek feedback from colleagues by having them review the manuscript.

Below are some examples:

- In the Abstract and Introduction sections, both r40 and m66 should be mentioned, along with a clarification that they are monoclonal antibodies derived from rabbit and mouse, respectively.

- The sentence, "The binding kinetics of trastuzumab, pertuzumab…" should specify the binding target, possibly HER2.

- A reference for PDB entry #7MN5 should be provided.

- Figure legends should provide more specific details to allow the experiments to be understood without referring to the Materials and methods section.

This manuscript requires extensive proofreading, as it contains numerous spelling mistakes and typographical errors. Ensure that the co-authors thoroughly review the content.

For example, in the sentence: "The final structural models were built with the HER2-trastuzumab-...", the reference for PDB entry #60GE may be [Ref. 10] rather than [Ref. 25].

About the Figures:

Fig 2C: There is no statistically significant difference between r40 and trastuzumab in the NRG1 ligand treatment of MCF7 cells. Therefore, statements such as "Antibody r40 shows a more potent effect than trastuzumab..." should be avoided to prevent misinterpretation.

Fig 3: The horizontal axes should be aligned to facilitate vertical comparisons, as demonstrated in Fig 1B. Additionally, the peak intensity of r40 in BT-474 appears to be very low. What could be the possible explanation?

Figs 4 and 5: Blots should be digitally quantified and, if possible, represented as graphs. Of course, images of the blots should be incorporated into the graphs. Ideally, statistical analysis should also be conducted, and the discussion should be based on those results.

Fig 6: It would be better to adjust the display order of the figures. Following the sequence in the Results section, the results for m66 and r40 should be presented separately for each, in the order of trimer, tetrameric complex, and superimposed structure.

Reviewer #3: A nice in vitro study of selecting a new antibodies to HER2. Study is methodologically sound, data are well presented and manuscript as a whole is written in logical manner. Cryo EM part of study is especially commendable.

**********

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: Yes: Benjamin Benzon

**********

[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.

Attachments

Attachment

Submitted filename: PONE-D-25-13465_rev.docx

Dear Dr. Liu,

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.

Please submit your revised manuscript by Oct 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

There are a few discrepancies between the dose response curves and tabulated EC50 values (Fig. 2 and 6). Please check the numbers, values etc to make sure there is a correspondence in the data.

expi293 cells do not express HER2 naturally, please clarify the experimental details and conclusions. regarding the saturation concentration experiments, ususally the EC50 binding values are considered to be  saturating.

The western blots images must be quantified and normalized to the levels of house keeping proteins (in particular, because the levels of the latters are not even).

It is not quite clear why the authors didn't see the dose response effect when tested HER2 signaling - it may indicate that the effect is not induced by the antibodies. The authors should re-test the dose response to cover the bigger concentration range (i.e. 1: 5 dilution, 6-8 points). Could it also be that the effect is time-dependent, and was not tested at the optimal time point? Also, why the levels of HER2 are so different for untreated MCF-7 (Fig.4)?

Figure 2 is confusing. What is plotted on the X axis - concentration of the antibody or the logarithm of concentration?

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

Reviewer #1: All feedback from previous reviews was successfully implemented. I found some spelling/grammatical errors in this round, and have highlighted these (green) in the manuscript. Other than this, I found this work to be very interesting and well communicated, and believe it is ready for publication once these errors have been corrected. Congratulations to the authors

Reviewer #2: I acknowledge the addition of new data in the revised manuscript, which contributes to strengthening the overall conclusions.

However, the data presentation remains confusing and poorly structured, making the manuscript, in its current form, unsuitable for publication.

To improve clarity and impact, revisions are needed.

The following are examples of suggestions for improvement.

1. Antibody screening should be described in the Materials and Methods section.

- To Supplemental data: ELISA (no data provided?), pull-down assay (Response Letter Fig. 1), and FCM using Expi293 cells (Fig. S2A)

- To M&M: T&P saturation data (Fig. 1B) should be explained as the basis for determining T&P concentrations at 267 nM.

2. The Results section should describe the selection process in comparison to T&P.

- Data: mouse antibody ELISA (Fig. 1A), rabbit antibody FCM using Expi293 cells (Fig. S2B), and FCM using MCF7 cells (Fig. 1C–E*, S2C)

*The r35 data in Fig. 1E is not required

*Legends should be standardized to “non-saturated cells (or MCF7)” and “T&P-, T-, or P-saturated cells (or MCF7)”

3. Emphasize the results of the CCK-8 assay (Fig. 2A, B*) and HER2 signaling analysis (Figs. 4, 5**).

*Including EC50 values (Response Letter Table 1) would be helpful, although the graph curves do not appear to support reliable EC50 estimation.

**Consider placing the HER2 blot at the top of the figure panel

4. Summarize the characteristics of m66 and r40.

- To Results or Supplemental data: SPR (Fig. 2C), FCM (Fig. 3*)

*Cite the paper (doi: 10.1155/2012/486568, Response Letter Figure 1) to support the low HER2 expression in MCF-7 cells

*Include units and labels on the y-axis

5. In Fig. 6, divide the panels into A (m66 group) and B (r40 group), and label each panel with consistent headings such as:

"Cryo-EM map", "High-resolution model", "Superimposed with HER2-T-P“, and "Superimposed with HER2-HER3-NPG1".

6. The manuscript should be carefully reviewed to ensure internal consistency across all parts. Several inconsistencies remain between the revised main text and other sections such as the Materials and Methods and figure captions.

Examples:

- The ELISA EC50 data (Response Letter Fig. 1) are not presented, yet the Materials and Methods section still describes them

- The caption for Fig. 6 does not correspond to the actual figure content

- The statement "When cells were pre-incubated with trastuzumab and pertuzumab, the mean fluorescence intensity (MFI) of the rabbit monoclonal antibody remained comparable to control cells incubated with the rabbit antibody alone" lacks supporting data

- In Fig. 5C, the claim that "m66 inhibited p-AKT (S473) more potently than r40" is not clearly supported by the band intensity shown

- The term "AlphaFold2" is used in the Results section, whereas "AlphaFold" appears in the Materials and Methods section—this should be clarified

7. A thorough proofreading is required. Numerous typographical and grammatical errors are present throughout the manuscript. Please ensure that basic language editing is completed prior to submission.

Examples:

- "While antibody m75 blocked pertuzumab and pertuzumab blocked m75 (Fig 1A)." — This sentence is grammatically incomplete and unclear.

- "..., which explained why m66 and pertuzumab didn’t not bind HER2 simultaneously."

- "..., and then a of serial dilutions of HER2 (1-128 nM) in PBS ..."

- "In summary, we screened a panel of human HER2 antibodies and ..." — This sentence may give the misleading impression that the authors screened human antibodies.

To avoid confusion, it should be revised to: "a panel of (mouse and rabbit) antibodies targeting human HER2."

**********

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

**********

[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.

Attachments

Attachment

Submitted filename: PONE-D-25-13465_R1_rev.pdf

Dear Dr. Liu,

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.

Please submit your revised manuscript by Dec 10 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: I am satisfied that all previous suggestions have been implemented into the manuscript and I have no further recommendations. I believe this manuscript is ready for acceptance. Congratulations to the authors on their interesting work

Reviewer #2: The revised manuscript is significantly more readable and easier to follow. I would like to express my appreciation for the authors' dedicated efforts and commend their hard work.

Below are five points of concern regarding the revision.

If these are addressed, I believe the manuscript will be suitable for publication.

1. The modification of the two mouse antibodies into the human IgG format is not clearly explained. Since the purification of HER2 is described in the Materials and Methods section, it would be beneficial to include a comparable description of the antibody preparation, including whether the antibodies are in human IgG format and whether they are His-tagged.

2. The citation of Figure 3 in the following sentence appears to be unnecessary:

"The m66 antibody, which incorporates variable regions on a human IgG1 backbone, can be detected using an antihuman secondary antibody, similar to trastuzumab and pertuzumab."

3. The methods used for quantification and normalization (as explained in this Response Letter) of the Western blot bands should be included in the Materials and Methods section. Additionally, the legends for Figures 4 and 5 should clarify that the numerical values shown in the figures represent normalized quantitative data.

4. For the description of ECD I, III, and the III/IV interface, appropriate references should be added in the following sentences:

- In Introduction: "...ECD I plays a key role in forming a binding pocket for the dimerization arm of other EGFR family members. ECD III, located centrally within the extracellular domain, serves as a critical structural bridge between ECD II and ECD IV and interacts directly with ECD I."

- In Discussion: "...the ECD III/IV interface, a region where structural rearrangements are more likely to induce HER2 internalization."

5. In the following sentence, the citation [41] should be placed immediately after the term "AlphaFold 2":

"...the HER2-trastuzumab module and the AlphaFold 2[41] predicted structure of m66..."

Response to the authors' inquiry:

>> Would it be appropriate to include descriptions of these assays in the Materials and Methods section? Additionally, would you recommend including these two figures in the Supplementary Materials?

There is no issue as long as the methods and results of the ELISA and pull-down assays are properly described in the supplementary materials.

However, it is essential to cite them explicitly in the main text, specifically in the following sentence:

"...and evaluated for binding affinity using enzyme-linked immunosorbent assay (ELISA) and pull-down assays."

Furthermore, descriptions of ELISA methods, including EC50 and competitive assays, should be harmonized throughout the manuscript.

>> However, as Expi293 cells are not widely recognized for cell surface HER2 protein expression and are not commonly used in breast cancer research, shall we remove the corresponding experimental data and figures regarding HER2 antibody epitope determination in Expi293 cells from the revised manuscript?

This dataset provides evidence of an antibody selection process targeting epitopes distinct from those of T&P, and therefore should not be removed. Although the suitability of using Expi293 cells may be debatable, the experimental conditions are clearly described in the revised manuscript, so no further changes are necessary.

**********

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

**********

[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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Inhibition of HER2 signaling and breast cancer cell growth with a novel antibody targeting HER2 ECD III/IV

PONE-D-25-13465R3

Dear Dr. Liu,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

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

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2: (No Response)

**********

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 #2: No

**********