Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows effective anti-tumor activity

Kaewta Rattanapisit; Pipob Suwanchaikasem; Christine Joy I. Bulaon; Shiying Guo; Waranyoo Phoolcharoen

doi:10.1371/journal.pone.0316790

Peer Review History

Original SubmissionSeptember 11, 2024
29 Nov 2024 Decision Letter - Michael C Burger, Editor PONE-D-24-40114Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows improvement of anti-tumor activityPLOS ONE Dear Dr. Suwanchaikasem, 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 modify your manuscript according to the suggestions brought forward by the reviewers. Please discuss the reasons where this might not be justifyable. Please submit your revised manuscript by Jan 13 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. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper describes the design, expression, purification and testing of a fusion of the Pembrolizumab antibody with the IL15-IL15 complex. Comments: - Interesting idea, especially if individual co-administration studies have shown enhanced effect in tumour reduction. - The novelty of the approach is in expressing the complex in plants and harvesting the same through farming. Major Comments - The functionality of the fused IL15alpha and IL15 is not described. In the absence of data on this part of the fusion, the entire hypothesis is questionable. - ELISA on Pembrolizumab and the fusion binding- it was interesting to see that indeed the individual binding graphs has similiar slopes- a direct head to head competition assay would have thrown more light on the affinities - The 'purified' molecule had multiple bands in the commassie blue staining but shows a single band on western. What are the bands then? Is this a purification artefact? - The animal model- without details on the exposure data, PK (minimum) it is very difficult to conclude on the comparative efficacies- additionally without a dose response study a comparison of the two moieties would be incorrect. - Wonder if the fusion protein is immunogenic- presence of antibody in the treated animals would have shed light on these aspects. Reviewer #2: The manuscript entitled "Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows improvement of anti-tumor activity" by Rattanapisit et al. is very interesting but I cannot fully agree with the conclusions based on the data. First of all, the graphics don't show significant differences in binding or toxicity between the test molecule and keytruda. Binding did not include error bars, thus it is not possible to decide whether the curves are significantly different. In fact, it is seen that the binding is a bit faster for keytruda, and maybe a little stronger for the test molecule, but without error bars and significance calculations it is not possible to make any conclusion. Yet, I would say that there is no difference in binding. Second, tumor size and tumor volume don't show significant differences, while body weight seems a bit better for keytruda, although no real differences are seen at day 27. I think Figure 7 shows that the activity of both molecules is identical within the potential statistical deviations or experimental errors. In conclusion, this is a good manuscript and the authors demonstrated that IL-15-conjugated Pembrolizumab can be successfully produced in a plant-based system, and as they have shown in their prior publications, the methods are sound and viable, and that conclusion is acceptable; but the conclusion that this conjugated molecule is better than the control molecule in controlling tumor growth is not supported by the data. I would conclude that the manuscript can be accepted if the authors include all the statistical calculations for all the experiments, and either show evidence of their claim of better efficacy, or change their conclusion and the title, indicating that this construct is not significantly better than the test molecule; ie, keytruda. ****** 6. 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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. https://doi.org/10.1371/journal.pone.0316790.r001
Revision 1
16 Dec 2024 Author Response Responses to Reviewers’ comments Reviewer #1: The paper describes the design, expression, purification and testing of a fusion of the Pembrolizumab antibody with the IL15-IL15 complex. Comments: - Interesting idea, especially if individual co-administration studies have shown enhanced effect in tumour reduction. - The novelty of the approach is in expressing the complex in plants and harvesting the same through farming. Response: Thank you for your positive feedback on this project. Within plant molecular farming platforms, we believe that this manuscript has demonstrated an important successful expression of antibody-cytokine fusion molecule using plants. The compound was produced steadily, and it showed acceptable anti-tumor activity, not different from the commercial Pembrolizumab. Major Comments: - The functionality of the fused IL15alpha and IL15 is not described. In the absence of data on this part of the fusion, the entire hypothesis is questionable. Response: Thank you for your suggestion. We have included the functional description and importance of IL-15Rα and IL-15 parts in the introduction, line no. 42-45 and 64-66, and discussion, line no. 324-327. Basically, fusing IL-15Rα with IL-15 allows the compound to interact with IL-15Rβ and IL-15Rγ, located on the T and NK cells, without primarily binding to the antigen-presenting cells. - ELISA on Pembrolizumab and the fusion binding- it was interesting to see that indeed the individual binding graphs has similar slopes- a direct head to head competition assay would have thrown more light on the affinities Response: Thank you for your comment. We agreed that the head-to-head competition assay will assist direct comparison between the efficacy of unconjugated and IL-15-conjugated pembrolizumab. It will provide useful information on which compound would perform better in terms of PD-L1 protein binding. We have included this suggestion in the discussion, line no. 390-392, and aim to conduct this experiment in the next phase of product development. We have also revised the ELISA plot (Figure 4) to make it easier to understand. - The 'purified' molecule had multiple bands in the Coomassie blue staining but shows a single band on western. What are the bands then? Is this a purification artefact? Response: The bands that were observed at approximately 200-250 kDa on the SDS-PAGE gel, stained with Coomassie blue, but unfound on the Western blot analysis could be partial or cleaved pembrolizumab heavy chains. They might be co-purified with a complete form of pembrolizumab-IL-15Rα-IL-15 fusion by protein A affinity chromatography. After purification, these products appeared in the drug product and were detected with Coomassie blue staining due to protein being. However, they were not detected in the Western blot analysis with anti-Gamma and anti-Kappa antibodies because of incomplete formation of heavy chain and light chain antibody, lacking binding epitope for anti-Gamma and anti-Kappa antibodies to detect. We have incorporated this explanation in the discussion, line no. 361-367. - The animal model- without details on the exposure data, PK (minimum) it is very difficult to conclude on the comparative efficacies- additionally without a dose response study a comparison of the two moieties would be incorrect. Response: Thank you for your recommendation. We agreed that pharmacokinetics study and dose response curves would provide more insight into this study. We aim to conduct this pharmacokinetic study along with immunogenicity and extended efficacy in the next phase of product development. In this study, our primary goal is to demonstrate success of the design and production of Pembrolizumab fusion with IL-15Rα-IL-15 complex using the plant molecular farming system. We have added this suggestion to the discussion part, line no. 409-413. - Wonder if the fusion protein is immunogenic- presence of antibody in the treated animals would have shed light on these aspects. Response: Thank you for your valid suggestion. Yes, immunogenic data is crucial data to guarantee safety of this molecule. This part of the experiment will be carried out in the next phase of product development. In this study, we intended to show the design of the Pembrolizumab-IL-15Rα-IL-15 fusion and capability of plant molecular platform to produce such molecule. We have included this clarification in the discussion part, line no. 409-413. Reviewer #2: The manuscript entitled "Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows improvement of anti-tumor activity" by Rattanapisit et al. is very interesting but I cannot fully agree with the conclusions based on the data. Response: Thank you for your feedback. We have revised the conclusion of this manuscript per your suggestion. We, therefore, summarized that anti-tumor efficacy of unconjugated Pembrolizumab and Pembrolizumab-IL-15Rα-IL-15 was comparable in mouse colorectal cancer model. Nonetheless, the conjugated molecule showed a good sign of tumor-growth inhibition and should be further investigated for its potential and safety in different cancer models using dose response study. First of all, the graphics don't show significant differences in binding or toxicity between the test molecule and keytruda. Binding did not include error bars, thus it is not possible to decide whether the curves are significantly different. In fact, it is seen that the binding is a bit faster for keytruda, and maybe a little stronger for the test molecule, but without error bars and significance calculations it is not possible to make any conclusion. Yet, I would say that there is no difference in binding. Response: Thank you for pointing this out. Yes, we agreed that, with error bar and statistical data, the binding plot will improve data interpretation to discern the difference between the conjugated Pembrolizumab-IL-15Rα-IL-15 and Keytruda. We have edited Figure 4 by including error bars and statistical test for each timepoint. The tabular data was also supplied in Supplementary Table S2. We used two-way ANOVA with Sidak’s multiple comparison test to examine PD-1 binding efficacy between Pembrolizumab-IL-15Rα-IL-15 and Keytruda. Binding properties of both molecules were relatively similar. The only difference was observed at the concentration of 0.15 μg/ml, where Keytruda was slightly better bound to PD-1 protein. The calculated EC50 was not significant difference. Therefore, we concluded that the affinity to PD-1 protein of both molecules was relatively similar. We have included this point to the result, line no. 245-247. Second, tumor size and tumor volume don't show significant differences, while body weight seems a bit better for keytruda, although no real differences are seen at day 27. I think Figure 7 shows that the activity of both molecules is identical within the potential statistical deviations or experimental errors. Response: Thank you for your comment. We agreed that anti-tumor efficacy of unconjugated Pembrolizumab and Pembrolizumab-IL-15Rα-IL-15 were not significantly different. We toned down the efficacy results of Pembrolizumab-IL-15Rα-IL-15 and added suggestion for further studies to investigate its potential in different tumor models. In the manuscript, we have modified the abstract, line no 24-25, the result, line no. 294-295, the discussion, line no. 392-396 and 409-417 and the conclusion, line no. 422, to summarize that Pembrolizumab-IL-15Rα-IL-15 showed comparable tumor growth inhibition to commercial Keytruda. In conclusion, this is a good manuscript and the authors demonstrated that IL-15-conjugated Pembrolizumab can be successfully produced in a plant-based system, and as they have shown in their prior publications, the methods are sound and viable, and that conclusion is acceptable; but the conclusion that this conjugated molecule is better than the control molecule in controlling tumor growth is not supported by the data. I would conclude that the manuscript can be accepted if the authors include all the statistical calculations for all the experiments, and either show evidence of their claim of better efficacy, or change their conclusion and the title, indicating that this construct is not significantly better than the test molecule; ie, keytruda. Response: Thank you for your appreciation of our study. We have revised the manuscript title to “Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows effective anti-tumor activity”. We have edited the discussion and conclusion to imply that this molecule showed acceptable anti-tumor efficacy and needs the following studies to understand its safety and efficacy profiles. In this manuscript, we highlight more on the design and production strategy of this molecule using plant molecular farming system. Moreover, we have included statistical data to Figure 4 as suggested. Statistical data for the other experiments was all provided. Attachments Attachment Submitted filename: 20241214_Response to Reviewers01.docx https://doi.org/10.1371/journal.pone.0316790.r002
17 Dec 2024 Decision Letter - Michael C Burger, Editor Plant-derived Pembrolizumab in conjugation with IL-15Rα-IL-15 complex shows effective anti-tumor activity PONE-D-24-40114R1 Dear Dr. Suwanchaikasem, 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Michael C Burger, M.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0316790.r003
Formally Accepted
23 Dec 2024 Acceptance Letter - Michael C Burger, Editor PONE-D-24-40114R1 PLOS ONE Dear Dr. Suwanchaikasem, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Michael C Burger Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0316790.r004

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