Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study the authors describe a novel approach aimed at activating NK cells against tumor cells and support tumor clearance. The approach is based on engineering NK cells with a receptor made up of a surface PD-L1 targeting domain coupled with Syn-Notch intracellular signalling leading to IL-12 expression. IL-12 is a cellular factor able to induce and support activation of immune cells against tumor cellular targets. The article is clearly written and overall the data support the study conclusions. We recommend publication after minor revisions addressing the following points:

-          The figures are not always clear and sometimes seem distorted (e.g.: Fig 3). Can the author re-size the figures and submit higher resolution versions when appropriate

-          Figure 1C: bright panel does not show any cells, just a saturated green background. Normally a nuclear stain is preferred to show cell presence. In any case, the bright field image is not readable. The authors should provide a better image or a nuclear stain (e.g. DAPI).

-          Figure 1 B: a mention of Control and tranfected condition should be put in the figure for better clarity, at the moment is only inferred indirectly form the results.

-          Figure 2: panels C-D are mentioned in the legend but missing in the Figure.

-          Paragraph 3.2: The mentioned softwares and algorithms should be given with relative references (bibliography or web address). Also ANOLEA method is mentioned only in the result sentence line 214) but not mentioned in the previous list, lines 212-3.

-           Figure 3: the y-axis should be renamed (e.g. % PDL-1+ cells) for better clarity.

-          Paragraph 3.4: The authors mentioned only one set of parameters for NK cell transduction and report an efficiency of 40-70%. Did the authors test other conditions to improve this efficiency? Was the transduction method already developed and optimized and here simply applied? Would be interesting to specify....

-          Figure 6: a control with un-transduced cells should be shown to really estimate the contribute of the transduction over background NK cell activity, as done in Figure 5. Here the increase due to the transduction is only inferred indirectly based on the PDL-1 differential expression of the different cell lines.

-          Results in in vivo would be obsviously a “nice to have” to further support this approach and rule out also any potential side-effects, for example. It would be interested to mention at least in the discussion.    

Comments on the Quality of English Language

 

-          Lines 102-3: need rephrasing (main verb missing).

-          Lines 107-9: needs rephrasing for better syntaxis and clarity

-          Lines 117.9: “This allows …” this sentence is redundant and not needed in a method section. I suggest the authors to remove it.

-          Lines 139: CO2, the 2 should be as a lower index

-          Line 299: “of to” remove “to”

-           

Author Response

Dear Reviewer,

Thank you for your valuable feedback and constructive comments on our manuscript titled " Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion" We appreciate the time and effort you have dedicated to reviewing our work. We have carefully considered each of your suggestions and made the necessary revisions to improve the quality and clarity of our study. All the corrections are highlighted in the revised manuscript.

 

1- All figures were rechecked and reconsidered, as requested

2- Thank you for bringing this to our attention. We have removed the fluorescent microscopy bright panel (Fig1C) as the high transfection rate which was already confirmed via flow cytometry (Fig1B).

3- We have revised Fig 1B as requested.

4- We apologize for the oversight regarding the legends for Fig2c and 2d mistake. The legend for Figure 2 has been corrected.

5- Bibliographies and web addresses have been inserted accordingly.

6- Figures have been revised as per your request.

7- We acknowledge the challenges in NK cell culture and transduction. Various conditions were explored to optimize efficiency, and the best-case scenario is highlighted in our method.

8- Corrections have been made to Fig 6 as per your instructions.

9- We have included a statement in the discussion acknowledging the potential benefits of an in vivo study while addressing the limitations preventing its execution.

- All grammatical issues have been addressed.

 

Once again, we sincerely appreciate your feedback and guidance throughout this revision process. We believe that these revisions have significantly strengthened the manuscript, and we remain committed to addressing any further concerns or suggestions you may have.

Reviewer 2 Report

Comments and Suggestions for Authors

*The authors investigated the efficiency of SynNotch Receptor Engineering to release IL-12 from natural killer against tumor cells. The manuscript is well written with minor language issues. 

* Lines 130, and 165: Please, include all catalog numbers of all used kits. 

* I suggest proceeding with the in vivo validation.

 

Comments on the Quality of English Language

* Needed to be revised by an English editing service. 

* Line 72 (., remarkable success).

Author Response

Dear Reviewer,

Thank you for your valuable feedback and constructive comments on our manuscript titled " Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion" We appreciate the time and effort you have dedicated to reviewing our work. We have carefully considered each of your suggestions and made the necessary revisions to improve the quality and clarity of our study. All the corrections are highlighted in the revised manuscript.

1- Catalog numbers have been included within the text.

2- Although we were not capable to investigate, we acknowledge the potential benefits of in vivo evaluation and have already suggested it in the discussion section.

- Grammatical issues have been revised.

Once again, we sincerely appreciate your feedback and guidance throughout this revision process. We believe that these revisions have significantly strengthened the manuscript, and we remain committed to addressing any further concerns or suggestions you may have.

Reviewer 3 Report

Comments and Suggestions for Authors

This study introduces a strategy to enhance the anti-tumor efficacy of natural killer (NK) cells by utilizing PD1-synthetic Notch (synNotch) receptors. The successful integration of PD1-Syn receptors on NK cells was demonstrated, which led to the secretion of IL-12 and target-specific production of IFNγ when interacting with PDL1-positive cells. This resulted in an improvement in the cytotoxic potential of these CAR-NK cells in a target-specific manner. While the study aligns with the journal's focus, a comprehensive revision is recommended prior to acceptance. Key points for revision include:

1.     The rationale behind using sequences from the UniProt database and 3D modeling for chimeric receptor design needs clarification, including its advantages and comparison with existing methods.

2.     More detailed discussion on the challenges and efficiency of expressing PD1-Syn receptors on NK cells through lentiviral transduction is required, alongside a comparative analysis with other studies.

3.     The evaluation of engineered NK cells' functionality, especially their cytotoxicity and cytokine production when co-cultured with PDL1-positive breast cancer cells, needs to be elaborated.

4.     Improvements in figure quality are necessary for clarity, particularly the legibility of text and scale bars in figures such as 1a and 1c.

5.     The omission of Figures 2c and 2d should be addressed, and the rationale behind the chosen models for protein structure modeling needs comparison with other methods like AlphaFold, including a clear representation and differentiation within the figures.

6.     Figure 3 requires the inclusion of original data and details on the number of repetitions for the experiments to support the findings.

7.     Consistency in the scale of the y-axis across Figures 4, 5, and 6 would aid in accurately reflecting the differences observed.

8.     Clarification is needed in Figure 6 regarding the significance of differences observed, specifying the groups involved and ensuring the statistical analysis and labeling are accurate across all figures.

 

Author Response

Dear Reviewer,

Thank you for your valuable feedback and constructive comments on our manuscript titled " Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion" We appreciate the time and effort you have dedicated to reviewing our work. We have carefully considered each of your suggestions and made the necessary revisions to improve the quality and clarity of our study. All the corrections are highlighted in the revised manuscript.

1- We have clarified the aim of our study to design a suitable chimeric receptor targeting PDL1 for NK cell activation and immune response enhancement within the tumor microenvironment. This aspect is now elaborated upon more clearly in the manuscript.

2- The challenges and efficiency of PD1-SynNotch receptors on NK cells have been discussed in comparison to previous studies, emphasizing the use of SynNotch as a receptor.

3- Functional assessments, including cytotoxicity and cytokine production of co-cultured CAR-NK cells, have been explained in greater detail as requested.

4- All figures have been rechecked and reconsidered.

5- We apologize for the oversight regarding the legends for Fig2c and 2d mistake. The legend for Figure 2 has been corrected. We also have provided an explanation of the computational tools used for protein structure prediction, including the I-TASSER server, in the revised manuscript.  It should be noticed that among several different protein modeling methods, we employed the I-TASSER server, as an advanced and one of the best computational tool for protein structure prediction, to generate 3D structure models of the transmembrane and extracellular domains of PD1 and the extracellular region of synNotch receptor. The server goal is to provide the most accurate protein structure and function predictions using state-of-the-art algorithms. In contrast, the Alphafold server is very suitable for known structures and cannot predict novel ones. Since we had some parts of different proteins in our modeling and considering the advantages, we used I-TASSER server and also two other servers for confirmation.

6- We specify that the assessment of PDL1 expression was performed in triplicate in the Figure 3 legend and unfortunately the flow cytometry images are not available due to file damage in the main computer.

7- Since the results from each part in Fig 4, 5, and 6 are compared with its own, we think that it’s not necessary and suited to present “different numbers with similar scales” and also the y-axis are appropriately fitted by the software according to the variable data, enhancing the clarity and accuracy of the presentation.

8- The significance of differences observed in Figure 6 has been elucidated in the results and figure legend to provide a clear understanding of the results.

Once again, we sincerely appreciate your feedback and guidance throughout this revision process. We believe that these revisions have significantly strengthened the manuscript, and we remain committed to addressing any further concerns or suggestions you may have.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all of my concerns. Thanks.