Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo

Shiva Izadi; Mokhtar Jalali Javaran; Sajad Rashidi Monfared; Alexandra Castilho

doi:10.1371/journal.pone.0260796

Peer Review History

Original SubmissionSeptember 15, 2021
10 Nov 2021 Decision Letter - Arijit Biswas, Editor PONE-D-21-29875Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivoPLOS ONE Dear Dr. Castilho, 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 25 2021 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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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This work described transient expression of rPA fused to the Fc region of an IgG1 monoclonal antibody (rPA-Fc) either by the hinge 464 region of the IgG1 (rPA-HFc) or by a flexible (Gly4Ser) linker (rPA-LFc) to ensure correct protein folding and function. The authors showed that point mutations in the CH3 domain of rPA-LFc reduced degradation through inhibition of protein dimerization. The rPA-FC produced recombinantly showed accepptable purity and high activity regarding the clot lysis assay. The paper is written in a logical order and is understandable. The images are well designed with acceptable quality and resolution, and the legends of figures are enough informative. There are some major and minor points which can be considered to improve the data evaluations: Major concerns: In general, some of the assays used (SDS-PAGE and immunoblotting, in vitro degradation assay, and Plasma clot assay) are all semi-quantitative although there are various quantitative assays in this regards. If the authors have access to the systems7assay kits, it is highly recommended that some of the assays suggested below will be used as complementary assays to confirm the activity of rPA. 1. Plate-based activity assay using PA-specific chromogenic/fluorogenic substrates might be used to evaluate the activity of recombinantly produced rPA. This assay can be used not only to evaluate the biological activity of rPA, but also to monitor the degradation of rPA. 2. The rotational thromboelastometry (ROTEM) can be used to evaluate clot lysis assay by spiking citrate pool plasma/whole blood with the rPA and the clot formation and lysis can be monitored. In this way the lysis parameters such as lysis onset time (LOT) and maximum lysis (ML) will be helpful to quantify clot lysis. Minor concern: 1. In the introduction section, Nicotiana benthamiana was repeatedly mentioned as Nicotiana benthamiana or N. benthamiana. After the first introduction it might be mentioned constantly as N. benthamian. Reviewer #2: This is a well written comprehensive report on the biochemical aspects in production of rPA-FC fusions constructs. The presented results are of general interest beyond rPA-Fc-specific aspects and may therefore be published in PLOS ONE. However, I have some comments as stated below. Major: The construct finally used for the clot lysis assay (rPA-LFC) is only specified in Fig. 8 but not within the corresponding results section. Why wasn't rPA-mFC also tested using this assay? Which glycosylation variant was used for assessing the biological activities? The paper would benefit from more physiological experiments, e.g. whole blood ROTEM / TEG analysis. It appears that the discussion does not match the level of the described results (the first paragraph reads like an introduction and some parts of rest like a summary). During revision, it may reasonable to transfer some aspects already adressed within the results section. Minor: There are some typos within the manuscript, please review carefully. For example: Lines: 283/284, 291/292, 350/351, 361, 431, 470 Fig. 2. It would be helpful to also include schematic images on the different structures of the constructs (dimeric, monomeric). The resolutions of Fig. 7 and the lower panel of Fig. 5B need to be increased. ****** 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:** Nasim Shahidi Hamedani 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.0260796.r001
Revision 1
15 Nov 2021 Author Response Reviewer #1: This work described transient expression of rPA fused to the Fc region of an IgG1 monoclonal antibody (rPA-Fc) either by the hinge 464 region of the IgG1 (rPA-HFc) or by a flexible (Gly4Ser) linker (rPA-LFc) to ensure correct protein folding and function. The authors showed that point mutations in the CH3 domain of rPA-LFc reduced degradation through inhibition of protein dimerization. The rPA-FC produced recombinantly showed accepptable purity and high activity regarding the clot lysis assay. The paper is written in a logical order and is understandable. The images are well designed with acceptable quality and resolution, and the legends of figures are enough informative. There are some major and minor points which can be considered to improve the data evaluations: Major concerns: In general, some of the assays used (SDS-PAGE and immunoblotting, in vitro degradation assay, and Plasma clot assay) are all semi-quantitative although there are various quantitative assays in this regards. If the authors have access to the systems7assay kits, it is highly recommended that some of the assays suggested below will be used as complementary assays to confirm the activity of rPA. 1. Plate-based activity assay using PA-specific chromogenic/fluorogenic substrates might be used to evaluate the activity of recombinantly produced rPA. This assay can be used not only to evaluate the biological activity of rPA, but also to monitor the degradation of rPA. 2. The rotational thromboelastometry (ROTEM) can be used to evaluate clot lysis assay by spiking citrate pool plasma/whole blood with the rPA and the clot formation and lysis can be monitored. In this way the lysis parameters such as lysis onset time (LOT) and maximum lysis (ML) will be helpful to quantify clot lysis. Reply: We agree with the reviewer that additional biological assays would improve the manuscript. The aim of our investigation was to set up the basic studies and lay the groundwork for the production of rPA-Fc proteins with added value. We took advantage of the short stay of the PhD student (Ms Izadi) in Iran to carry out simple experiments showing that the plant-derived rPA-Fc is active in vitro and ex vivo. She did indeed try to perform a rotational thromboelastometry (ROTEM) assay but encounter technical problems (even the positive control did not work) and run out of time to optimized the method. At BOKU we are a plant-oriented lab and do not have the facilities or permission to perform experiences involving blood/plasma. We however envision the possibility of making available our proteins to other researchers for further studies in future collaborations. Minor concern: 1. In the introduction section, Nicotiana benthamiana was repeatedly mentioned as Nicotiana benthamiana or N. benthamiana. After the first introduction it might be mentioned constantly as N. benthamiana. Reply: We have modified the manuscript accordingly Reviewer #2: This is a well written comprehensive report on the biochemical aspects in production of rPA-FC fusions constructs. The presented results are of general interest beyond rPA-Fc-specific aspects and may therefore be published in PLOS ONE. However, I have some comments as stated below. Major: The construct finally used for the clot lysis assay (rPA-LFC) is only specified in Fig. 8 but not within the corresponding results section. Why wasn't rPA-mFC also tested using this assay? Which glycosylation variant was used for assessing the biological activities? The paper would benefit from more physiological experiments, e.g. whole blood ROTEM / TEG analysis. Reply: The clot lysis assays were performed at Tarbiat Modares University during a short period visit from Mss Izadi (PhD student). At the moment we were still trying to overcome the issue of instability and the rPA-mFc construct was not yet available for testing. The rPA-mFc construct was used in the zymogram and performed similarly to rPA-LFc. The biological activity of rPA-LFc and rPA-mFc was assessed with variants produced in ∆XF plants. This information is now included in the manuscript. We agree with the reviewer that additional biological assays would improve the manuscript. However, the main aim of our investigation was to set up the basic studies and lay the groundwork for the production of rPA-Fc proteins with added value. We have performed basic experiments to show that our protein is active in vitro and ex vivo. During a short time visit to Tarbiat Modares University, Ms Izadi also tried to perform a rotational thromboelastometry (ROTEM) assay but encounter technical problems (even the positive control did not work) and run out of time to optimized the method. At BOKU, we are a plant-oriented lab and do not have the facilities or permission to perform experiences involving blood/plasma. We however envision the possibility of making available our proteins to other researchers for further studies in future collaborations. It appears that the discussion does not match the level of the described results (the first paragraph reads like an introduction and some parts of rest like a summary). During revision, it may reasonable to transfer some aspects already addressed within the results section. Reply: We agree with the reviewer and modified the discussion section accordingly Minor: There are some typos within the manuscript, please review carefully. For example: Lines: 283/284, 291/292, 350/351, 361, 431, 470 Reply: We have modified the manuscript to correct typos Fig. 2. It would be helpful to also include schematic images on the different structures of the constructs (dimeric, monomeric). Reply: We have included schematic representation of the oligomerization of the constructs in Figure 2. The resolutions of Fig. 7 and the lower panel of Fig. 5B need to be increased. Reply: We have improved resolution of figures Attachments Attachment Submitted filename: Reply to reviewer comments.pdf https://doi.org/10.1371/journal.pone.0260796.r002
17 Nov 2021 Decision Letter - Arijit Biswas, Editor Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo PONE-D-21-29875R1 Dear Dr. Castilho, 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. If you have any billing related questions, 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, Arijit Biswas Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260796.r003
Formally Accepted
18 Nov 2021 Acceptance Letter - Arijit Biswas, Editor PONE-D-21-29875R1 Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo Dear Dr. Castilho: 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. Arijit Biswas Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260796.r004

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