A phase I clinical trial to evaluate the tolerability and safety of an allogeneic iPSC-derived iNKT cell and α-GalCer-pulsed autologous DC combination therapy for patients with recurrent and advanced head and neck cancer: A study protocol

Tomoya Kurokawa; Tomohisa Iinuma; Haruna Ebisu; Tomoha Yanagidaira; Yosuke Inaba; Tadami Fujiwara; Yoko Hattori; Tominaga Fukazawa; Takahiro Aoki; Haruhiko Koseki; Hideki Hanaoka; Toyoyuki Hanazawa; Shinichiro Motohashi

doi:10.1371/journal.pone.0342387

Peer Review History

Original SubmissionSeptember 19, 2025
27 Oct 2025 Decision Letter - Maria de Fátima Macedo, Editor Dear Dr. Motohashi, 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 11 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 . 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Maria de Fátima Matos Almeida Henriques de Macedo, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Competing Interests section: H. Koseki and S. Motohashi received grants from BrightPath Biotherapeutics Co., Ltd. All other authors declare that they have no relevant conflicts of interest. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 3. We note that you have indicated that there are restrictions to data sharing for this study. For studies involving human research participant data or other sensitive data, we encourage authors to share de-identified or anonymized data. However, when data cannot be publicly shared for ethical reasons, we allow authors to make their data sets available upon request. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Before we proceed with your manuscript, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible. Please update your Data Availability statement in the submission form accordingly. 4. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Additional Editor Comments: Please revise according to the reviewers comments [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. Reviewer #1: Recommedation Accept From a statistical perspective this is very well written protocol. The protocol indicates (Page 8, lines 135-139) the use of (3 + 3) dose escalating design with two dose levels (Dose 1 and Dose 2. Thus provided the first three patients experience acceptable levels of dose limiting toxicity (DLT) on Dose 1, the next 3 patients will receive Dose 2. This is carefully set out in Figure 3. The protocol describes carefully (Page 13, lines 244-247) the sensible use of a Confidence Interval at the interpretation stage. I am not sure the P-value will be of much use, but this is a very trivial remark. Reviewer #2: The submitted protocol adheres to international standards for first-in-human studies and provides insight into testing a promising modern cell therapy. I have only a few minor recommendations. The introduction should explain how iPSC-iNKTs are an improvement over standard peripheral blood iNKTs and how donor variability is reduced. iPSCs come from a healthy donor, so the text should reference published data showing that standardized preparation consistently produces iPSC-iNKTs with a similar phenotype and function. It is unnecessary to list the names of commercial cell lines. Mentioning the broad effect and referencing the publication is sufficient. Within the protocol, I would appreciate a mention that cytokine release syndrome and tumor lysis syndrome are monitored. Although these reactions are not expected, they should be mentioned, along with the markers and how treatment is set up in the case of CRS. Feel free to mention that it is set up according to CAR-T therapy guidelines. The protocol contains references to tables that do not exist (e.g., Table 20). I believe these are typos in the protocol revisions, but I recommend checking them in future revisions. Overall, the protocol and the study are of high quality. Reviewer #3: Kurokawa et al. present a study investigating off-the-shelf iPSC-derived invariant NKT (iPSC- iNKT) cells in combination with α-galactosylceramide-pulsed autologous dendritic cells (DC/Gal). The study’s design is both innovative and timely, given the growing interest in off-the- shelf cellular therapies and the clinical application of iPSC-derived products. Its findings are expected to make a significant contribution to the field of cancer immunotherapy, with broad implications for the development of allogeneic or off-the-shelf cellular therapies, while also advancing the translation of iNKT cell–based therapies. At the same time, several aspects of the manuscript could be improved to enhance clarity and readability. Attached, I provide detailed suggestions to strengthen the presentation and ensure that the study’s rationale, methodology, and outcomes are communicated clearly and effectively. ****** 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: No Reviewer #3: 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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: PONE-D-25-33703_Comments_to_the_authors.pdf https://doi.org/10.1371/journal.pone.0342387.r001
Revision 1
9 Dec 2025 Author Response Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf <Response> We have again confirmed that our manuscript meets PLOS ONE’s style requirements, as described in the templates. 2. Thank you for stating the following in the Competing Interests section: H. Koseki and S. Motohashi received grants from BrightPath Biotherapeutics Co., Ltd. All other authors declare that they have no relevant conflicts of interest. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. <Response> We revised the Competing Interests statement in the cover letter as follows: H. Koseki and S. Motohashi received grants from BrightPath Biotherapeutics Co., Ltd. All other authors declare that they have no relevant conflicts of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials. 3. We note that you have indicated that there are restrictions to data sharing for this study. For studies involving human research participant data or other sensitive data, we encourage authors to share de-identified or anonymized data. However, when data cannot be publicly shared for ethical reasons, we allow authors to make their data sets available upon request. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Before we proceed with your manuscript, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible. Please update your Data Availability statement in the submission form accordingly. <Response> We will adhere to the data sharing policy on your journal, and already stated as follows in the “Data Availability” section: No datasets were generated or analyzed during the current study. De-identified research data will be made publicly available upon completion and publication of the study. 4. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. <Response> We added one citation as “[21]” on the previous phase I study using iPSC-iNKT cells which has been published just recently. Reviewer #1: From a statistical perspective this is very well written protocol. The protocol indicates (Page 8, lines 135-139) the use of (3 + 3) dose escalating design with two dose levels (Dose 1 and Dose 2. Thus provided the first three patients experience acceptable levels of dose limiting toxicity (DLT) on Dose 1, the next 3 patients will receive Dose 2. This is carefully set out in Figure 3. The protocol describes carefully (Page 13, lines 244-247) the sensible use of a Confidence Interval at the interpretation stage. I am not sure the P-value will be of much use, but this is a very trivial remark. <Response> We thank the reviewer for the valuable comment regarding the P-value. We agree that the confidence interval provides the primary information for interpreting study results. However, we have included P-values as supplementary information to express the degree of incompatibility between the null hypothesis and the data obtained through our study. We believe that presenting both confidence intervals and P-values will make our results accessible to a broader readership while maintaining statistical rigor. Reviewer #2: The submitted protocol adheres to international standards for first-in-human studies and provides insight into testing a promising modern cell therapy. I have only a few minor recommendations. The introduction should explain how iPSC-iNKTs are an improvement over standard peripheral blood iNKTs and how donor variability is reduced. iPSCs come from a healthy donor, so the text should reference published data showing that standardized preparation consistently produces iPSC-iNKTs with a similar phenotype and function. <Response> We recently published an article on the previous phase I study using iPSC-iNKTs. In the paper, the specifications of the iPSC-iNKTs were described. So, we will add some description on the specification of iPSC-iNKTs with reference in the introduction part. At this time, the data has not been published in a paper. However, the relevant data have been compiled as part of the study, including product quality and nonclinical data, in the protocol (Supporting Information, S4). Therefore, we put the S4 as a reference here. It is unnecessary to list the names of commercial cell lines. Mentioning the broad effect and referencing the publication is sufficient. <Response> As mentioned above, the data have not been published in a paper. We will also revise the main text to eliminate redundant descriptions of cell lines and refer readers to the supporting information describing the in vitro tumor suppressive effects. Within the protocol, I would appreciate a mention that cytokine release syndrome and tumor lysis syndrome are monitored. Although these reactions are not expected, they should be mentioned, along with the markers and how treatment is set up in the case of CRS. Feel free to mention that it is set up according to CAR-T therapy guidelines. <Response> As you mentioned, our monitoring plan includes care for CRS and TLS by vigilant monitoring in the hospital during the first 14 days after administration of study treatment. We will consider including a mention on CRS and TLS in the next protocol amendment. The protocol contains references to tables that do not exist (e.g., Table 20). I believe these are typos in the protocol revisions, but I recommend checking them in future revisions. <Response> Thank you for pointing out the typos. We’ll revise the tables in the next protocol amendment. Overall, the protocol and the study are of high quality. <Response> We appreciate your review and mindful advice. Reviewer#3 Major Revisions Lines 129-132: During the treatment period, we evaluated the dose-limiting toxicity (DLT) from the DC/Gal injection to 14 days after iPSC-iNKT cell administration under hospitalization. The 28-day observation period starts 14 days after administration of the iPSC-iNKT cells, including three visits for the safety evaluation (Fig. 1/2). As patients are monitored throughout both treatment and peri-treatment phases, it would be helpful to clarify at the outset that the monitoring period consists of two phases: a treatment period, during which patients are hospitalized and closely monitored for dose-limiting toxicities (DLTs), and an observation period, during which patients are followed as outpatients with three visits over 28 days. <Response> I agree with your suggestion. It can be more straight forward. Lines 213-215: The outline of this study plan may be revised in the statistical analysis plan if the primary endpoints or the analysis methods are changed. This sentence may be perceived as not methodologically nor regulatory-compliant as it stands. I would suggest revising as follows: “Details of the statistical methods may be further specified in the Statistical Analysis Plan (SAP). If any changes to the primary endpoints or analysis methods are required, they will be implemented through a protocol amendment, and the SAP will be updated accordingly” <Response> I agree with your suggestion. It can be more accurate. Lines 223-225: The purpose of this study is to evaluate the tolerability, safety, and efficacy of the DC/Gal and iPSC-iNKT cell combination therapy. Therefore, a significance test for validation purposes will not be performed. Efficacy: in the abstract, the authors state that the study aims to “explore the immunological changes that occur following this combination treatment”. Therefore, the term “efficacy” could be reconsidered to better reflect the exploratory objective focused on immunological or immune correlates, rather than efficacy assessment per se. Suitable alternatives may include “immune effects” and “tumor response.” Significance test: the protocol is meant to describe a non-comparative, exploratory, early-phase study. It is indeed acceptable not to perform formal statistical significance testing. However, I would suggest stating it more clearly and scientifically, for example: “This study is designed as an exploratory, early-phase evaluation of the safety, tolerability, and exploratory immune changes following treatment with DC/Gal and iPSCiNKT cell combination therapy. Therefore, no formal hypothesis testing will be performed, and analyses will be descriptive and exploratory in nature.” <Response> I agree and replace the language as you suggested. Lines 244-247: This paragraph includes language about CIs and hypothesis testing that may sounds in contradiction with the stated non-comparative design of the study. To improve clarity and use neutral language, I would suggest "This is an exploratory, early-phase study primarily designed to evaluate safety and tolerability. Therefore, no formal hypothesis testing for efficacy is planned and the study is not powered to demonstrate efficacy. Any P values or confidence intervals derived from these analyses will be reported as nominal and exploratory and will not be used to draw definitive conclusions about efficacy." <Response> I agree and replace the language as you suggested. Lines 250-255: Please clarify the manufacturing process. The phrase “was previously manufactured” implies that no further steps are required beyond product release. In contrast, the phrases “The PI will order the manufacturing” and “incubated for purification” suggest that additional procedures are needed and that manufacturing prior to the trial start may not have been completed. <Response> As you mentioned, the word “manufactured” here is inappropriate. We will delete the word. Lines 296-298: These lines would benefit from clarification. Do the authors mean the following: “DLTs are defined as dose-limiting adverse events possibly related to the DC/Gal or iPSC-iNKT cell product that occurs during the period referred to as the treatment period. Other adverse events possibly related to the DC/Gal or iPSC-iNKT cell product, occurring during or outside this period, will be summarized separately.” <Response> Adverse events other than DLTs are not separately summarized, but will be included as “all AEs” regardless of causality, which also includes DLTs. Therefore, we would like to use the first sentence of your proposal. The description on “all AEs” is described in the “Safety measurement” section (Lines 322-325). Line 382: There are few or no examples Please clarify whether no or only a few examples have been reported, and provide references as appropriate. <Response> Thank you for pointing out the misleading part. The intention of this part was to highlight the challenges of clinical trials using regenerative medicines, specifically regarding the combination of autologous cells and allogeneic regenerative therapy. It’s also challenging to determine the prevalence of such a type of clinical trial. Therefore, we’ll replace the sentence as follows: The iPSC-iNKT cells were established from a healthy volunteer and are stocked as a master cell bank at the RIKEN Institute, while αGalCer/DCs are prepared from participants’ peripheral blood in the study site. The combination use of autologous and allogeneic cells requires careful consideration for a feasible study flow. Lines 389-391: Additionally, this trial protocol can inform future clinical trials using immunological agents in combination with patient-derived adjuvant agents. This sentence appears to repeat the information stated in lines 386–387. Immunological (non-cell) agents are in fact under investigation in trials combined with patient-derived adjuvant agents (e.g., NCT03294954). <Response> We intended to highlight that this study protocol would provide insights into organizing challenging clinical trials using separately prepared immune cells. Therefore, we’d like to clarify our intention as follow (underlined: amended language): Additionally, this trial protocol can contribute to future clinical trials using separately prepared immune cell combinations, such as iPS cell-derived immune cell products and patient-derived adjuvant agents. Lines 411-414: because iPSC-iNKT cells exhibited cytotoxic activity against six tumor cell lines, we did not restrict the tumor pathology type in this trial. Although only head and neck cancer cases were included, iPSC-iNKT cells could be applied to other malignant tumors with accessible tumor-feeding arteries. The current phrasing is a bit contradictory: it says the trial did not restrict tumor pathology type, but then notes that only head and neck cancers were included. Consider rewording this passage, for example: “Furthermore, as iPSC-iNKT cells demonstrated cytotoxic activity against six distinct tumor cell lines, their mechanism of action is expected to be broadly applicable across malignancies. Nonetheless, for this first-inhuman trial, enrollment is restricted to patients with head and neck cancer, but in future studies this approach could be applied to other tumors with readily accessible tumorfeeding arteries for targeted administration.” <Response> I agree and replace the language as you suggested. Lines 418-419: Regenerative medicine products are challenging to store long-term, so study materials must be expanded from the working cell bank after each participant’s enrollment. For clarity, it may be helpful to explain what is meant by the bank. For example: “[from the working cell bank] consisting of intermediate cell products [after each participant’s enrollment]”. <Response> I agree and add the language as you suggested. Lines 434-484: The section begins by stating that there are “several limitations,” but the text that follows largely reads as explanations or justifications rather than clearly stated limitations. To frame the limitations explicitly, they should be presented as constraints of the study design or interpretability, followed by brief rea Attachments Attachment Submitted filename: Response to Reviewers_the journal requirements.docx https://doi.org/10.1371/journal.pone.0342387.r002
9 Jan 2026 Decision Letter - Maria de Fátima Macedo, Editor Dear Dr. Shinichiro Motohashi, 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 by reviewer 3. Please submit your revised manuscript by Feb 22 2026 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. A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Maria de Fátima Matos Almeida Henriques de Macedo, Ph.D. Academic Editor PLOS One Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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. Additional Editor Comments: Please address comments done by reviewer 3. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors described where all data underlying the findings will be made available when the study is complete??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics. You may also provide optional suggestions and comments to authors that they might find helpful in planning their study. Reviewer #1: The authors have addressed my very trivial remark about the utility of the P-value in this context. I wish the authors well with this study. Reviewer #2: As part of the revision process, any shortcomings in the already high-quality work were addressed. I have no further comments regarding the work, and I am looking forward to see the results from clinical trial. Reviewer #3: COMMENTS TO THE AUTHORS (PONE-D-25-33703R1) Kurokawa et al. have addressed all the comments and revised their study protocol accordingly. We acknowledge and appreciate the authors’ efforts to incorporate the suggested revisions. The revised manuscript has improved notably in both clarity for readers and accuracy. We would suggest three additional final edits to ensure that all sections are sufficiently clear and detailed for readers to appreciate the quality and impact of this important study. 1) Lines 79-81. Please insert “that” after “responses”. The complete sentence will read as: "They can also promote broader antitumor immune responses that indirectly harm cancer cells by activating NK cells and cytotoxic T lymphocytes (CTLs) through release of interferon (IFN)-γ and other cytokines [12-14]" 2) Tables 1-2. We thank the authors for providing the rationale for the different methods and thresholds used to assess Mycoplasma and Endotoxin. This information may help readers appreciate the implications of generating and applying two distinct products produced under different conditions and in different facilities. We would suggest including this information in the manuscript as brief table footnotes. For example: [Table 2 footnote] "Mycoplasma testing methods are equivalent across products." [Table 1 and Table 2 footnote] "Endotoxin thresholds reflect the validated limit of detection at the manufacturing site." 3) Line 441-443. Please complete the sentence with a main clause. One suitable option is provided below: "Because the DC/Gal and iPSC-iNKT 442 cells are administered at one time, this trial will not assess the tolerability, safety, or efficacy of repeated dosing, restricting evaluation of long-term or repeated 443 combination therapy." With these final improvements, we recommend the manuscript for publication. ****** 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: No Reviewer #3: 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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: PONE-D-25-33703R1_Comments_to_the_authors.pdf https://doi.org/10.1371/journal.pone.0342387.r003
Revision 2
17 Jan 2026 Author Response Reviewer#3 Dear reviewer, We sincerely appreciate your additional comment on our protocol paper. We’ve addressed your comments as below. We would suggest three additional final edits to ensure that all sections are sufficiently clear and detailed for readers to appreciate the quality and impact of this important study. 1) Lines 79-81. Please insert “that” after “responses”. The complete sentence will read as: "They can also promote broader antitumor immune responses that indirectly harm cancer cells by activating NK cells and cytotoxic T lymphocytes (CTLs) through release of interferon (IFN)-γ and other cytokines [12-14]" <Response> The clarity of this sentence has been enhanced by the contribution of your commentary. We revised it as your advice. 2) Tables 1-2. We thank the authors for providing the rationale for the different methods and thresholds used to assess Mycoplasma and Endotoxin. This information may help readers appreciate the implications of generating and applying two distinct products produced under different conditions and in different facilities. We would suggest including this information in the manuscript as brief table footnotes. For example: [Table 2 footnote] "Mycoplasma testing methods are equivalent across products." [Table 1 and Table 2 footnote] "Endotoxin thresholds reflect the validated limit of detection at the manufacturing site." <Response> As you kindly advised, these footnotes would be helpful for readers. We revised it to add the footnotes as your advice. 3) Line 441-443. Please complete the sentence with a main clause. One suitable option is provided below: "Because the DC/Gal and iPSC-iNKT 442 cells are administered at one time, this trial will not assess the tolerability, safety, or efficacy of repeated dosing, restricting evaluation of long-term or repeated 443 combination therapy." <Response> The clarity of this sentence has been enhanced by the contribution of your commentary. We revised it as your advice. Attachments Attachment Submitted filename: Response to Reviewers_260118.docx https://doi.org/10.1371/journal.pone.0342387.r004
21 Jan 2026 Decision Letter - Maria de Fátima Macedo, Editor A Phase I clinical trial to evaluate the tolerability and safety of an allogeneic iPSC-derived iNKT cell and αGalCer-pulsed autologous DC combination therapy for patients with recurrent and advanced Head and Neck Cancer: A study protocol PONE-D-25-33703R2 Dear Dr. Shinichiro Motohashi, 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. For questions related to billing, please contact billing support . 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, Maria de Fátima Matos Almeida Henriques de Macedo, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0342387.r005
Formally Accepted
Acceptance Letter - Maria de Fátima Macedo, Editor PONE-D-25-33703R2 PLOS One Dear Dr. Motohashi, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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 Prof. Maria de Fátima Matos Almeida Henriques de Macedo Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342387.r006

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