Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models

Yanxia Li; Nelusha Amaladas; Marguerita O’Mahony; Jason R. Manro; Ivan Inigo; Qi Li; Erik R. Rasmussen; Manisha Brahmachary; Thompson N. Doman; Gerald Hall; Michael Kalos; Ruslan Novosiadly; Oscar Puig; Bronislaw Pytowski; David A. Schaer

doi:10.1371/journal.pone.0268244

Peer Review History

Original SubmissionDecember 17, 2021
8 Feb 2022 Decision Letter - Francesco Bertolini, Editor PONE-D-21-39856Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor modelsPLOS ONE Dear Dr. Puig, 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 by both Reviewers. Please submit your revised manuscript by Mar 10 2022 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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We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Yes ****** 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: This manuscript deals with the effect on tumor growth of a combination of anti-VEGFR2 and anti-PDL1 antibodies in murine models. It is evident that the combination therapy is more effective compared to monotherapy. This effect is accompanied by a stronger T cell infiltration, activation of macrophages and immunological memory to further rechallenge with tumor cells. Overall, the manuscript is well organized and written and the flow of experiments are straightforward. I suggest some improvements to further reinforce the main message of this work. a- the analysis of infiltrating lymphocytes is not well described. Indeed, it is not clear how large are the tumors analyzed, whether a whole scan of tumor sections have been performed, and whether a software of analysis has been used to get the level of infiltration. Also, no analysis of markers involved in lymphocyte migration and localization has been considered. It is conceivable that T cells and myeloid cells can home to the tumor, expressing some receptors involved in endothelial migration. b- it is not stated that the cell lines used in the tumor murine models are indeed PDL-1 positive, this is important to support the treatment with anti PDL-1 antibodies. c- all the data are coming from the in vivo murine models. It would be fine to demonstrate that TIL, splenic cells, lymph node T cells, PB lymphocytes can recognize the tumor cells in vitro. In other words, are CTLs present in the mice-treated specific for the tumor cells used to induce tumors? Do NK cells play a role in killing tumor cells, and can the antibodies used for therapy trigger ADCC? Reviewer #2: Li et al. uncovered the treatment with a VEGFR-2 antibody to be potentially associated with intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models. Points to be considered: 1. When discussing the methodology applied for number selection it is not clear for my understanding, how the author selected the sufficient experimental setting. Indeed, in the in vivo experiments, sample size has been should be calculated in a rigorous way, i.e. by using GPower software (power of for example 80% and 0.05 statistical level, etc.). Assuming an effect-size of for example, 0.4 with statistical significance of α <;0.05 and a power of 80%. A given 9 mice for each group for a total of 18 mice were extimated. This number should be increased to 20 considering an expected drop-out rate of 10% for the treatment. Can the authors comment on this? 2. Did the authors normalize for time exposure immunofluorescence in figure 1 and suppl. 1? 3. Did the authors employ unstained/isotype control compensation in fig. 4? 4. Did the author checked for normally distributed (Gaussian) data before performing parametric tests? 5. Available research, patent analysis and existing platform for the cross-talk between angiogenetic addicted and potentially immunosensitive cancers are already available can be ameliorated: describe how the authors' manuscript goes beyond the state-of-the-art, and the extent to the proposed work is ambitious. This reviewer personally misses some state-of-the-art standpoint regarding angiogenesis and immune-infiltration in cancer. As is now well known, tumors grow and evolve through a constant crosstalk with the surrounding microenvironment, and emerging evidence indicates that angiogenesis and immunosuppression frequently occur simultaneously in response to this crosstalk. Accordingly, strategies combining anti-angiogenic therapy and immunotherapy seem to have the potential to tip the balance of the tumor microenvironment and improve treatment response (refer to PMID: 32456352 and expand introduction/discussion). 6.Innovation: describe where the proposed work is positioned in terms of research and innovation R&I maturity (from the idea of application to lab to market/clinic), providing an indication of the Technology Readiness Level, possibly distinguishing the start and by the end of the manuscript. 7. Methodology: the authors should be able to describe and explain the overall methodology, including CONCEPT, MODELS and ASSUMPTIONS that underpin this project. It should be explained how this will enable to deliver the project’s objectives. Any important challenges of the chosen methodology should be identified and methods to overcome them should be clearly stated. 8. Few bullets regarding a computationally specific approach are also needed, referring to the EFFECTS of the projects, and R&I in general in this field technological outcomes: the manuscript should bring new products, services, processes to the clinician, increasing efficiency (quantify), decreasing costs, increasing profits, decreasing mortality, and improving decision making in the oncology field. Remarkably, the manuscript should give an indication of the SCALE and significance of the project’s contribution to the expected outcomes and impacts, should the project be successful (High risk/high gain balance). SCALE refers to how the outcomes and impacts are likely to be, i.e. in terms of the size of the target group or the proportion of that group, that should benefit over time; SIGNIFICANCE refers to the importance or value of those benefits (number of additional healthy life years, etc.) It should be always explained at the baseline, benchmarks and assumptions used for those estimates. Wherever possible, quantify the estimation of the effects expected from the project. Only one methodology should be used for calculating the estimates for each region and country. ******* 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: Yes:** Alessandro Poggi MSc, MD 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. https://doi.org/10.1371/journal.pone.0268244.r001
Revision 1
3 Apr 2022 Author Response Francesco Bertolini, MD, PhD Academic Editor PLOS ONE Dear Dr. Bertolini, On behalf of myself and my co-authors, I would like to thank the reviewers for their valuable feedback and for the time they devoted to reviewing the article. We have addressed all feedback with a point-to-point response below (the reviewers’ comments are reproduced in italic and authors responses are in blue). Tracked and clean versions of our resubmission addressing the points raised have been provided. We hope that our letter and corresponding changes in the manuscript in response to the reviewers' comments are satisfactory. Reviewers' comments: Reviewer #1: This manuscript deals with the effect on tumor growth of a combination of anti-VEGFR2 and anti-PDL1 antibodies in murine models. It is evident that the combination therapy is more effective compared to monotherapy. This effect is accompanied by a stronger T cell infiltration, activation of macrophages and immunological memory to further rechallenge with tumor cells. Overall, the manuscript is well organized and written and the flow of experiments are straightforward. I suggest some improvements to further reinforce the main message of this work. a- the analysis of infiltrating lymphocytes is not well described. Indeed, it is not clear how large are the tumors analyzed, whether a whole scan of tumor sections have been performed, and whether a software of analysis has been used to get the level of infiltration. Also, no analysis of markers involved in lymphocyte migration and localization has been considered. It is conceivable that T cells and myeloid cells can home to the tumor, expressing some receptors involved in endothelial migration. We thank the reviewer and apologize for the omission of important details on how IF and IHC were analyzed. We have now added them to the methods section (lines #219-239). We were limited in the number of markers explored, and unfortunately, we did not use lymphocyte migration markers in our analysis. The reviewer is correct, specific migration markers would have been very informative in determining accurately the amount of T cell infiltration. b- it is not stated that the cell lines used in the tumor murine models are indeed PDL-1 positive, this is important to support the treatment with anti PDL-1 antibodies. Thank you for this request. We have now added the appropriate references showing that the cell lines used in the mouse models are indeed PD-L1 positive (line #205). c- all the data are coming from the in vivo murine models. It would be fine to demonstrate that TIL, splenic cells, lymph node T cells, PB lymphocytes can recognize the tumor cells in vitro. In other words, are CTLs present in the mice-treated specific for the tumor cells used to induce tumors? Do NK cells play a role in killing tumor cells, and can the antibodies used for therapy trigger ADCC? It’s a great question, and unfortunately, we don’t know the answer. Due to limitation in tumor size, it would have been challenging to determine whether tumor TILs in treated animals responded specifically to tumors. We could have used peripheral blood or splenic T cells, but unfortunately, we didn’t do it, and there is no guarantee these cells represent the same T cells present in tumors. We included NK markers in flow analysis, but we did not detect a change of percentages or absolute numbers of NK cells in the TME after treatment, suggesting NK cells are not critical in the responses observed. Finally, we know that DC101 can’t trigger ADCC, but PD-L1 Ab 178G7 has not been tested for ADCC activity, so we cannot fully answer the reviewer’s question. We have added all these caveats to the discussion (lines #438-444). “This suggests that the increased T cell infiltration observed after DC101 therapy resulted not only from inhibition of tumor angiogenesis but also through activation of the vasculature causing T cell recruitment. It is also possible that tumor infiltrated T cells are further activated by the treatment to recognize tumor antigens, but our experiments were not designed to address this question and further research will be needed. Finally, we did not observe any changes in NK cell percentages or counts, suggesting NK cells are not critical driving the observed response.” Reviewer #2: Li et al. uncovered the treatment with a VEGFR-2 antibody to be potentially associated with intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models. Points to be considered: 1. When discussing the methodology applied for number selection it is not clear for my understanding, how the author selected the sufficient experimental setting. Indeed, in the in vivo experiments, sample size has been should be calculated in a rigorous way, i.e. by using GPower software (power of for example 80% and 0.05 statistical level, etc.). Assuming an effect-size of for example, 0.4 with statistical significance of α <;0.05 and a power of 80%. A given 9 mice for each group for a total of 18 mice were extimated. This number should be increased to 20 considering an expected drop-out rate of 10% for the treatment. Can the authors comment on this? The reviewer is correct: power calculations indicate that a larger number of animals is generally needed to unequivocally determine small effect sizes like the ones observed in this study. However, there are also practical reasons that are critical when planning animal studies, and it’s not always straightforward to design studies that account for both, statistical and practical factors: animal welfare, cost, animal housing space available, and the ability to take down animals and to handle samples sequentially in a given period of time, while preserving tumor factors that easily degrade if not processed in a timely fashion. 5-6 animals per treatment arm is a standard number used in animal studies that allows a balance between detecting signals, cost and effectively handling tumor samples. Further, 5-6 animals per treatment arm is widely used in the literature. 2. Did the authors normalize for time exposure immunofluorescence in figure 1 and suppl. 1? Thank you for this question. We failed to indicate that isotype controls were used in all experiments to ensure immunofluorescence signals were properly normalized. This has been now clarified in the methods section (line #230). “with appropriate isotype control IgG antibodies” 3. Did the authors employ unstained/isotype control compensation in fig. 4? Thank you for the request. We did not use isotype controls, but we used unstained and “Fluorescence Minus One” controls. This has now been clarified in the methods section (line #248) “Appropriate unstained and “Fluorescence Minus One” controls were used.” 4. Did the author checked for normally distributed (Gaussian) data before performing parametric tests? Yes, this was confirmed in each analysis. Model diagnostics were reviewed to confirm that parametric statistical analysis assumptions (i.e. normally distributed error with constant variance) were sufficiently met for all reported parametric analysis results. 5. Available research, patent analysis and existing platform for the cross-talk between angiogenetic addicted and potentially immunosensitive cancers are already available can be ameliorated: describe how the authors' manuscript goes beyond the state-of-the-art, and the extent to the proposed work is ambitious. This reviewer personally misses some state-of-the-art standpoint regarding angiogenesis and immune-infiltration in cancer. As is now well known, tumors grow and evolve through a constant crosstalk with the surrounding microenvironment, and emerging evidence indicates that angiogenesis and immunosuppression frequently occur simultaneously in response to this crosstalk. Accordingly, strategies combining anti-angiogenic therapy and immunotherapy seem to have the potential to tip the balance of the tumor microenvironment and improve treatment response (refer to PMID: 32456352 and expand introduction/discussion). Thank you very much for this suggestion. We agree with the reviewer and have now added a paragraph in the introduction to expand the crosstalk between angiogenesis and aberrant tumor responses (lines #120-127) “Angiogenesis, the formation of new blood vessels from pre-existing ones, is an important step in cancer progression [1]. Tumor-induced angiogenesis is highly dysregulated compared to normal angiogenesis, and results in structurally aberrant vessels with insufficient pericyte coverage and leaky nascent vessels [2]. The interplay between aberrant tumor vessels and protumoral immune cells generates a vicious cycle that severely disturbs anti-cancer responses and promotes tumor progression; abnormal tumor vessels promote immune cell evasion, which in turn enhances tumor angiogenesis. This aberrant crosstalk hinders T-cell infiltration into the tumor and impairs T-cell effector functions [3-6].” 6.Innovation: describe where the proposed work is positioned in terms of research and innovation R&I maturity (from the idea of application to lab to market/clinic), providing an indication of the Technology Readiness Level, possibly distinguishing the start and by the end of the manuscript. Prior work referenced in our manuscript showed that combination of checkpoint inhibitors and anti-angiogenic treatments leads to enhanced antitumor activity, but there was limited evidence of immunological memory. Our results shows that in addition to enhanced antitumor activity, DC101+anti-PD-L1 treatment also leads to immunological memory. We have highlighted this in the discussion (lines #464 and #485) “Notably, the combined treatment led to immunological memory, since animals that responded to treatment were resistant to tumor rechallenge”. “resulting in enhanced antitumor activity and immunological memory.” 7. Methodology: the authors should be able to describe and explain the overall methodology, including CONCEPT, MODELS and ASSUMPTIONS that underpin this project. It should be explained how this will enable to deliver the project’s objectives. Any important challenges of the chosen methodology should be identified and methods to overcome them should be clearly stated. We apologize, but we do not understand what exactly the reviewer is asking us to do. We believe that with the new modifications, the methods section is now clear and concise. We are happy to incorporate any further suggestions the reviewer may have. 8. Few bullets regarding a computationally specific approach are also needed, referring to the EFFECTS of the projects, and R&I in general in this field technological outcomes: the manuscript should bring new products, services, processes to the clinician, increasing efficiency (quantify), decreasing costs, increasing profits, decreasing mortality, and improving decision making in the oncology field. We agree with the reviewer that it is important to highlight how this work impacts the field. We believe that the current discussion (lines #478-482) highlights the importance of this preclinical work in the field of angiogenesis and checkpoint inhibition, and the impact in clinical development of ramucirumab. Perhaps the reviewer can suggest how we could do this better? “Several clinical trials combining the anti-VEGFR-2 antibody, ramucirumab, and PD1 or PD-L1 antibodies are ongoing in several indications, including non-small-cell lung cancer, recurrent/metastatic head and neck squamous cell carcinoma, and mesothelioma (NCT03904108, NCT03650764, NCT03502746), and early indications suggest the results of this combination will be promising. Remarkably, the manuscript should give an indication of the SCALE and significance of the project’s contribution to the expected outcomes and impacts, should the project be successful (High risk/high gain balance). SCALE refers to how the outcomes and impacts are likely to be, i.e. in terms of the size of the target group or the proportion of that group, that should benefit over time; SIGNIFICANCE refers to the importance or value of those benefits (number of additional healthy life years, etc.) It should be always explained at the baseline, benchmarks and assumptions used for those estimates. Wherever possible, quantify the estimation of the effects expected from the project. Only one methodology should be used for calculating the estimates for each region and country. Please see our prior responses. We believe that the current discussion section reflects well the reviewer’s concerns. It highlights the importance of this preclinical work in the field of angiogenesis and checkpoint inhibition, and the impact in clinical development of ramucirumab. Again, we are happy to incorporate any further suggestions the reviewer may have. ________________________________________ 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. We thanks the journal for providing us with this option. We think it’s an excellent practice making the entire process transparent to the reader. We thank again both reviewers for helping us improve this manuscript. Their time and contributions have been critical to improve the final manuscript. Regards The authors Attachments Attachment Submitted filename: Response to Reviewers v2.docx* https://doi.org/10.1371/journal.pone.0268244.r002
26 Apr 2022 Decision Letter - Francesco Bertolini, Editor Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models PONE-D-21-39856R1 Dear Dr. Puig, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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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 #1: All comments have been addressed 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 #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 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 ****** 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 #1: Yes 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 #1: The authors have replied to all my queries. So, I endorse the manuscript for publication in PlosOne. Reviewer #2: The authors have clarified several of the questions I raised in my previous review. Most of the major problems have been addressed by this revision. ****** 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 #1: Yes:** Alessandro Poggi MSc, MD Reviewer #2: No https://doi.org/10.1371/journal.pone.0268244.r003
Formally Accepted
8 Jul 2022 Acceptance Letter - Francesco Bertolini, Editor PONE-D-21-39856R1 Treatment with a VEGFR-2 antibody results in intra-tumor immune modulation and enhances anti-tumor efficacy of PD-L1 blockade in syngeneic murine tumor models Dear Dr. Puig: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Francesco Bertolini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0268244.r004

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