Targeting prostate cancer by new bispecific monocyte engager directed to prostate-specific membrane antigen

Gargi Das; Jakub Ptacek; Jana Campbell; Xintang Li; Barbora Havlinova; Satish kumar Noonepalle; Alejandro Villagra; Cyril Barinka; Zora Novakova

doi:10.1371/journal.pone.0307353

Peer Review History

Original SubmissionJuly 3, 2024
3 Jul 2024 Author Response https://doi.org/10.1371/journal.pone.0307353.r001
25 Jul 2024 Decision Letter - Pier Carlo Ricci, Editor PONE-D-24-27288Targeting prostate cancer by new bispecific monocyte engager directed to prostate-specific membrane antigenPLOS ONE Dear Dr. Novakova, 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 Sep 08 2024 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. 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Additional Editor Comments: Dear Dr. Zora Novakova, We have now received the reviews from the peer reviewers. One of the reviewers has provided feedback indicating that the paper is suitable for publication in its current form. However, the second reviewer has made some constructive comments and suggestions for improvement that we believe would enhance the quality and impact of your manuscript. I recommend that you carefully consider and incorporate the revisions suggested by the second reviewer. These revisions are intended to strengthen your arguments, clarify certain points, and improve the overall presentation of your research. Please find the detailed comments and suggestions of the second reviewer attached to this letter. We ask that you submit a revised version of your manuscript addressing these points, along with a response letter outlining the changes made and how you have addressed the reviewer's concerns. We believe that following the second reviewer's recommendations will significantly enhance the quality of your work Thank you for your attention to this matter. We look forward to receiving your revised manuscript and are confident that the revisions will contribute to the excellence of your publication. Kind Regards, Carlo Ricci [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: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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 aims to develop and functional characterize a bispecific monocyte engager capable of simultaneously targeting PSMA-positive cancer cells and the Fc gamma receptor I (FcγRI/CD64 ) receptor present on the monocyte/macrophage surface. As far as this paper is in my field of expertise (except technical aspects), the manuscript is well written, clear and of interest to readers, thanks to the increasing interest that PSMA has widely spread in recent years. Due to my limited experience in chemistry and radiochemistry, I cannot judge the technical parts. Reviewer #2: The paper needs importante amendments Demonstrate engagement of human macrophage U937 alone Is not convincing Perform functional experiments at different E/T ratios A construct control lacking PSMA Binding is needed ****** 6. PLOS authors have the option to publish the peer review history of their article (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: Yes: Pierosandro Tagliaferri ******** [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. https://doi.org/10.1371/journal.pone.0307353.r002
Revision 1
24 Jan 2025 Author Response Vestec, January 24, 2025 Editorial board of PLOS ONE Dear editors, Thank you very much for the decision letter concerning our manuscript entitled “Targeting prostate cancer by new bispecific monocyte engager directed to prostate-specific membrane antigen”. We appreciate assessment of our work by reviewers, and in the revised version of the manuscript, we addressed all major concerns. According reviewers’ recommendations we added new experimental data related to human macrophages that further evaluate specificity end effect of 5D3-CP33 engager. We discussed all points given by reviewer 2 and proposed detailed explanation of controls used in study. We enhanced the information about effector:target ratio used in cell-based experiments. Changes to the manuscript and detailed explanation are provided in a point-by-point response to reviewers’ comments at the end of this letter. Corrections of grammar and stylistics are shown in track-changes version. Authors kindly notify that the surname of one co-author Jana Nedvedova has been changed to „Campbell“ due to her wedding that happened during year 2024. All gel image data are shown in the body of manuscript (Fig 1D), all original raw gel data are available in Supporting information file „S1_raw_images“ submitted together with revised manuscript. Be so kind to update information in manuscript according amended Funding Statement: "This work was supported by the Czech Academy of Sciences (RVO: 86652036), the Grant Agency of Charles University (GA UK project Number 358321 awarded G.D.), the Ministry of Education, Youth and Sports (LUAUS23254 awarded C.B.), and National Institutes of Health (NIH project R01CA249248). We acknowledge Imaging Methods Core Facility at BIOCEV, institution supported by the MEYS CR (LM2023050 Czech-BioImaging) and BIOCEV Biophysical Techniques CF of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study." We feel that we have provided a thorough response to reviewers’ and editor’s comments and, with their assistance, have strengthened the manuscript by addressing key concerns. Many thanks for seeing our manuscript through the review process. Sincerely, Zora Novakova, PhD Corresponding author Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences Průmyslová 595 252 50 Vestec Czech Republic phone: +420-325 873 736 email: zora.novakova@ibt.cas.cz Review Comments to the Author Reviewer #1: The paper aims to develop and functional characterize a bispecific monocyte engager capable of simultaneously targeting PSMA-positive cancer cells and the Fc gamma receptor I (FcγRI/CD64 ) receptor present on the monocyte/macrophage surface. As far as this paper is in my field of expertise (except technical aspects), the manuscript is well written, clear and of interest to readers, thanks to the increasing interest that PSMA has widely spread in recent years. Due to my limited experience in chemistry and radiochemistry, I cannot judge the technical parts. Reviewer #2: The paper needs importante amendments Demonstrate engagement of human macrophage U937 alone Is not convincing Authors‘ answer: Based on reviewer’s suggestion, effect and specificity of 5D3-CP33 bispecific engager was evaluated on human macrophages differentiated from monocytes (isolated from human PBMCs). Data was inserted as panels D,E in Figure 4. The presence of 5D3-CP33 enhances specific phagocytosis of PSMA-positive but not PSMA-negative cells. In this study, the U937 cell line was employed as a well-established and standardized model routinely used to evaluate phagocytosis. We believe that results related to isolated human macrophages that were added to revised manuscript strengthen main findings of the study. Reviewer #2: Perform functional experiments at different E/T ratios Authors‘ answer: We appreciate reviewer’s comment. Numerous previously published studies show specific phagocytosis of tumor cells driven by linkage via recombinant/synthetic molecules (see papers specified by DOI: 10.1002/ange.201510866, 10.1021/acschembio.0c00112, 10.1002/cpch.88, 10.1021/acscentsci.3c01052, 10.1016/j.biomaterials.2020.120601, 10.1080/19420862.2020.1857100, 10.4049/jimmunol.1402891, 10.1016/j.jim.2007.04.009, 10.1091/mbc.E03-09-0668, 10.18632/oncotarget.18492). In these studies, that utilized monocyte cell lines as well as freshly isolated human monocytes, the E/T ratio varied from 4:1 to 1:1 with significant preference of ratio 1:1. Since our data show superior rate of phagocytosis at E/T ratio 1:1 (Figure 4) that represents standard ratio used preferentially in experimental studies, we conclude that increase in the amount of effector cells would not bring any significant benefit to our study. Specific phagocytosis-supporting effect of 5D3-CP33 was observable even when target cells were present in excess over effectors represented by freshly isolated human monocytes differentiated and polarized into M1 type macrophages (E/T ratio was approximately 0.8:1), see Figure 4E. Reviewer #2: A construct control lacking PSMA Binding is needed Authors‘ answer: We agree with reviewer that implementation of proper controls is critical for the study. The specific binding of both parts of the bispecific engager was evaluated in detail and verified on effector and target cells using flow cytometry (see data in Figure 2 of our study). To show specific effect of 5D3-CP33, we implemented in our study two types of negative control: 1) a mixture of cells with the absence of the bispecific molecule, and 2) PSMA-negative target cells highly similar to PSMA-positive target (PSMA-positive cells represent original cell line overexpressing recombinant PSMA). First negative control shows potential non-specific phagocytosis, whereas second negative control simulates the absence of PSMA binding and is equal to the construct lacking PSMA binding part as suggested by reviewer. The results clearly indicate that PSMA-negative cell line is not attracted by the macrophages, even though 5D3-CP33 engager is present in concentration as high as 1 µM in cell mixture. Both negative controls prove specific binding and stimulatory effect of 5D3-CP33 in phagocytosis of PSMA-positive cells. Additionally, our study includes IgG1 molecule with standard Fc part that serves as a positive control (see Figure 4 of our study), therefore readers could assess directly the efficiency of CP33 part of bispecific engager in binding and activation of macrophages. Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0307353.r003
3 Feb 2025 Decision Letter - Pier Carlo Ricci, Editor Targeting prostate cancer by new bispecific monocyte engager directed to prostate-specific membrane antigen PONE-D-24-27288R1 Dear Dr. Novakova, 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, Pier Carlo Ricci Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A ****** 4. 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 #2: Yes ****** 5. 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 #2: Yes ****** 6. 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 #2: The authors correctly addressed all the points and the paper is now ready for pubblication. Data will add novel information in the field ****** 7. PLOS authors have the option to publish the peer review history of their article (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: Yes: Pierosandro Tagliaferri, MD ******** https://doi.org/10.1371/journal.pone.0307353.r004
Formally Accepted
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