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

. 2026 Feb 26;21(2):e0342387. doi: 10.1371/journal.pone.0342387

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 ^1,², Tomohisa Iinuma ², Haruna Ebisu ¹, Tomoha Yanagidaira ¹, Yosuke Inaba ¹, Tadami Fujiwara ¹, Yoko Hattori ¹, Tominaga Fukazawa ³, Takahiro Aoki ^3,⁴, Haruhiko Koseki ³, Hideki Hanaoka ¹, Toyoyuki Hanazawa ², Shinichiro Motohashi ^4,^*

Editor: Maria de Fátima Matos Almeida Henriques de Macedo⁵

PMCID: PMC12944769 PMID: 41746943

Abstract

Natural killer T (NKT) cells show intense tumor-killing activity through direct and indirect pathways. However, humans have less than 0.01% of NKT cells in the peripheral blood, making it difficult to apply NKT cells for cancer treatment. We have successfully produced invariant NKT cells derived from induced pluripotent stem cells (iPSC-iNKT cells) and demonstrated the tolerability of this product in a previous phase 1 clinical trial. Although the iPSC-iNKT cells showed substantial anti-tumor activity against various tumor cell types when combined with α-Galactosylceramide-pulsed autologous dendritic cells (DC/Gal) in non-clinical experiments, the tolerability of this combination therapy for humans has not demonstrated yet. Thus, we planned this first-in-human phase 1 open-label clinical trial to demonstrate data on tolerability and safety, as well as explore the immunological changes that occur following this combination treatment. We hypothesize that the sequential induction of nasal submucosal DC/Gal injection and intra-arterial administration of the iPSC-iNKT cells is tolerable and has a favorable safety profile for cancer patients. This trial provides vital and fundamental information for next-phase clinical trials and future applications of iPSC-iNKT cells for various cancer patients (jRCTa030220741; URL: https://jrct.mhlw.go.jp/en-latest-detail/jRCTa030220741).

Introduction

Head and neck cancer is the sixth most common cancer worldwide. Although more than 90% of head and neck cancer cases are squamous cell carcinomas, other related pathologies can occur [1]. For non-advanced head and neck cancer, treatment methods such as oral surgery and intensity-modulated radiotherapy are associated with a high success rate while maintaining quality of life for the patient. The primary approaches for advanced head and neck cancer are concurrent chemoradiotherapy and salvage surgery, as needed, or radical resection surgery followed by postoperative chemoradiotherapy [1]. However, despite these highly invasive treatments, the 5-year survival rate for many advanced head and neck squamous cell carcinoma cases remains around 40% to 50% [2–5]. Furthermore, new therapeutic strategies using targeted agents and immune therapy, such as cetuximab, nivolumab, and pembrolizumab, have been developed in recent years [6,7]. Although they demonstrated a response rate of 15% to 35% in head and neck cancer patients [4,8,9], they remain limited. Therefore, additional therapeutic methods are needed for this disease.

Invariant natural killer T (iNKT) cells are a unique type of lymphocyte that express T cell antigen receptors (TCRs) and natural killer receptors (NKRs) on their cell surface [10]. Unlike other T cell types, iNKTs recognize glycolipids, such as alpha-galactosylceramide (αGalCer) [11]. When presented by antigen-presenting cells via CD1d, a monomorphic MHC-like molecule, the glycolipid induces rapid iNKT cell activation. Activated iNKT cells can then recognize CD1d-presented lipid antigens on cancer cells, exerting direct cytotoxicity against CD1d⁺ tumor cells regardless of their major histocompatibility complex (MHC) expression, including through release of perforin and granzymes. 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]. In clinical trials targeting non-small cell lung cancer, intravenously administering autologous activated iNKT cells or αGalCer-pulsed autologous dendritic cells (DC/Gal) showed safety and effectiveness, with no serious adverse events (AEs) observed, confirming the induction of anti-tumor immune responses derived from NKT cells [15,16]. Although the number of peripheral blood iNKT cells is generally decreased in cancer patients, a clinical trial revealed that head and neck cancer patients can retain the peripheral blood iNKT cells. Additionally, unlike regular T cells, iNKT cells also maintain durable IFN-γ production and proliferative capacity following conventional radiation therapy [17]. These promising data suggest that iNKT cell immunotherapy may be useful for head and neck cancer. In clinical studies conducted at Chiba University Hospital, we found that DC/Gal injection into the nasal concha submucosa could induce their migration to cervical lymph nodes and activate iNKT cells [18,19]. From this evidence, we conducted a phase 1 trial of DC/Gal submucosal administration for recurrent and advanced head and neck cancer patients who were not eligible for curative surgery or irradiation. No serious AEs occurred, with eight out of nine cases showing increased anti-tumor immune activity and one case showing tumor shrinkage [20].

However, iNKT cells naturally exist at a sparse frequency of 0.01% to 0.1% among white blood cells, with considerable variation among patients, suggesting limitations in their therapeutic utility. Therefore, we developed a novel therapeutic strategy involving the administration of a sufficient number of iNKT cells, which were differentiated, matured, and proliferated from induced pluripotent stem cells (iPSCs) to head and neck cancer patients via tumor feeding artery including the peripheral branch of the maxillary artery. The iPSC-derived iNKT (iPSC-iNKT) cells are validated by the expression of Va24 iNKT cell-specific rearranged TCR and IFN-γ production. In non-clinical trials, iPSC-derived iNKT (iPSC-iNKT) cells showed an in vitro killing effect on a variety types of cancer cell lines (S3 and S4 Files). Furthermore, the iPSC-iNKT cells showed tumor growth inhibition was observed in human head and neck cancer cells transplanted into mice, with no safety concerns found [21].

From these findings, we conducted a phase 1 trial to evaluate the tolerability and safety of super-selective arterial infusion of the iPSC-iNKT cells for patients with recurrent and advanced head and neck cancer (jRCT2033200116). The results indicated that the iPSC-iNKT cells showed tolerability at a 1.0×10⁸ cells/m² dose without the combined use of DC/Gal [21]. Hence, in the current phase 1 trial, we aimed to demonstrate the tolerability of combining the iPSC-iNKT cells and DC/Gal as the next step towards implementing this therapeutic approach for head and neck cancer patients.

Materials and methods

Trial designs, aim, and settings

This clinical trial is a non-blinded, single arm phase 1 trial targeting patients with recurrent or advanced head and neck cancer who are resistant or intolerant to standard of care. This trial aims to investigate the tolerability, safety, pharmacokinetics, pharmacodynamics, and anti-tumor effects of DC/Gal treatment followed by the administration of iPSC-iNKT cells. This trial starts with a screening period for eligibility after the informed consent process, then a seven-day DC/Gal preparation period for culturing the DC/Gal from each participant. This is followed by a 19-day treatment period, with a single DC/Gal injection and administration of the iPSC-iNKT cell product five days after the DC/Gal injection. We avoid same-day administration of the investigational product to more than two participants, with a minimum interval of at least seven days before administering the investigational product to the following participant. This provision is to prevent multiple individuals from experiencing acute fatal adverse events on the same day. 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 (Figs 1 and 2).

Fig 1 — Abbreviations: ECG: electric cardiogram; CBC: complete blood count; ECOG PS: Eastern Cooperative Oncology Group Performance Status; mMRC: modified Medical Research Council; PK/PD: pharmacokinetic/pharmacodynamic; AE: adverse event; DLT: dose limiting toxicity; HLA: human leukocyte antigen.

Fig 2 — Abbreviations: DC/Gal: dendritic cells pulsed with a-Galactosylceramide; DLT: dose limiting toxicity; iPSC-iNKT: induced pluripotent stem cell-derived invariant natural killer T.

The dosing regimen involves both a submucosal injection of 1.0 × 10⁸ DC/Gal (fixed dose) and tumor arterial infusion of iPSC-iNKT cells at one of two dose levels: initial dose (first dose) cohort (3.0 × 10⁷ cells/m²) or second dose cohort (1.0 × 10⁸ cells/m²) administered at once. We employ the three-by-three (3 + 3) design for dose escalation in this trial. Ultimately, the maximum tolerated dose (MTD) will be determined based on a total of six participants with no or one DLT occurrences (Fig 3).

Fig 3 — The descriptions in the rectangles represent the actions needed. The descriptions in the diamonds represent the assessment for DLT. The numbers above the arrays represent the number of patients who have DLT. Abbreviations: DLT: dose limiting toxicity; MTD: maximum tolerable dose.

Participants

Inclusion criteria. Patients who meet all the following criteria are eligible.

1) Patients with recurrent or advanced head and neck cancer, refractory or intolerant to standard of care, and who have evaluable lesions that can be treated with an intra-arterial infusion to the tumor.
2) Patients who have not been given a previous therapy within 1 month. Any type of previous treatment is acceptable.
3) Patients must be at least 20 years old, but younger than 80 years old.
4) Patients must have an Eastern Cooperative Oncology Group (ECOG) Performance Status of 2 or less.
5) Patients who meet the following laboratory data: hemoglobin ≥ 10 g/dL, white blood cell count ≥ 3000/µL, platelet count ≥ 75,000/µL, serum creatine ≤ 1.5 mg/dL, total bilirubin ≤ 1.5 mg/dL, AST (GOT) and ALT (GPT) < 2.5 times the upper limit of facility reference values, and SpO₂ (under room air) ≥ 93%.
6) Patients who are expected to have a prognosis of 3 months or more.
7) Patients for whom written informed consent has been obtained from the individual.

Exclusion criteria. Patients who meet any of the following conditions are not eligible.

1) Patients who are Hepatitis B Surface (HBs), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), or Human T-lymphotropic virus type 1 (HTLV-1) antibody-positive or HBs antibody-negative, but have Hepatitis B Virus (HBV)-DNA detected by HBV-DNA quantitative testing.
2) Patients who have been taking or injecting corticosteroids (methylprednisolone 10 mg/day or higher or equivalent) or immunosuppressive drugs within at least 2 weeks prior to the start of the study product.
3) Women who are pregnant, lactating, or planning to become pregnant during the study period and men who do not agree with using any of the effective contraceptive methods under the guidance of a physician during the study period or up to 14 days after the last dose of the study product.
4) Patients with active autoimmune disease requiring systemic or immunosuppressive therapy with corticosteroids or biologic agents.
5) Patients who have experienced immune-related AEs with immune checkpoint inhibitor treatment.
6) Patients with poorly controlled diabetes mellitus.
7) Patients with severe lung disease (modified Medical Research Council (mMRC) Breathlessness Scale Grade 2 or higher) or with a history of non-infectious interstitial lung disease requiring steroid treatment.
8) Patients with significant cardiac disease (New York Heart Association (NYHA) class III or greater).
9) Patients with concurrent multiple cancers.
10) Patients who are unable to use contrast agents in radiography, such as from an allergy or kidney disfunction.
11) Patients with a history of hypersensitivity to human serum albumin products or proteins of foreign origin.
12) Patients who, at the time of informed consent, are participating in other clinical trials or clinical studies and are receiving other investigational products or are judged by the principal investigator (PI) or sub-investigator (SI) to have residual effects of AEs caused by such products.
13) Patients with completely identical genotypes of HLA-A, HLA-B, and HLA-C to the investigational product to distinguish the study product from participants’ iNKT cells.
14) Patients who are prohibited from undergoing blood apheresis due to comorbidities, such as unstable angina, A-V block class 2 or greater, Wolff-Parkinson-White (WPW) syndrome, complete left bundle branch block, systolic blood pressure of 90 mmHg or lower, or systolic blood pressure of 170 mmHg or higher.
15) Patients who are judged to be unsuitable to participate in the study.

Statistical analysis and sample size

The statistical analysis plan for this study is summarized below, with the specific details separately described in the statistical analysis plan. 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. Patients enrolled in the study and treated with DC/Gal are the target population for safety analysis. However, cases of non-compliance with the Act on the Safety of Regenerative Medicine are excluded from the safety analysis population. Among the safety analysis population, all participants who could not be appropriately evaluated during the DLT evaluation period for reasons other than DC/Gal- or iPSC-iNKT cell-related toxicity are excluded from the DLT evaluation population. If a participant is excluded from the analysis, then a new participant will be added to the cohort.

This study is designed as an exploratory, early-phase evaluation of the safety, tolerability, and exploratory immune changes following treatment with DC/Gal and iPSC-iNKT cell combination therapy. Therefore, no formal hypothesis testing will be performed, and analyses will be descriptive and exploratory in nature.

We employ a 3 + 3 design for this study. The maximum number of patients to be analyzed is set at 12, with six patients in each of the two dose cohorts. The minimum number of cases is set at two, as the study would be terminated because of the occurrence of two consecutive cases of DLT in the first dose cohort.

For each cohort, the number and proportion of patients experiencing a DLT, with corresponding 95% confidence intervals, are calculated. The following secondary endpoints are analyzed for the purpose of providing supplementary discussion to the primary analysis results. For the safety secondary endpoints, frequency tables are created for all AEs occurring after the DC/Gal or iPSC-iNKT cell administration for each system organ class (SOC) and preferred term (PT) in the Medical Dictionary for Regulatory Activities (MedDRA) Japanese version. Similar frequency tables are created by dose, severity, causal relationship to the DC/Gal or iPSC-iNKT cells, and the common terminology criteria for adverse events (CTCAE) grade. Summary statistics are calculated for the laboratory test results and any changes occurring between baseline and each visit. A trend chart of laboratory values are also created. For the efficacy secondary endpoints, the response rate and disease control rate using response evaluation criteria in solid tumors (RECIST) v.1.1 evaluated by the PI or SI are calculated, with the 95% CI for these rates also calculated. 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. Other items and details are described in the statistical analysis plan.

iPSC-iNKT cells (investigational product)

The iPSC-iNKT cell product was previously cultured, and stored as a frozen master cell bank using a healthy volunteer who provided written informed consent at the RIKEN institute (Yokohama, Kanagawa, Japan). The PI orders the manufacturing of the investigational product at RIKEN following the eligibility confirmation of participant. The iPSC-iNKT cells are incubated for purification for 10–14 days at RIKEN after the order from the PI, then sent to the study site (Chiba University Hospital) in accordance with the standard operating procedure stipulated separately. The product that has passed the product specification test established in advance and agreed upon with the Pharmaceuticals and Medical Devices Agency (PMDA) will be used for this trial (Table 1). The iPSC-iNKT cells are administered into a tumor feeding artery via super-selective arterial infusion. The dose is 3.0 × 10⁷ cells/m²/injection for the initial dose cohort or 1.0 × 10⁸ cells/m²/injection for the second dose cohort.

Table 1. List of specification tests for the induced pluripotent stem cell-derived invariant natural killer T (iPSC-iNKT) cells.

Specification test
Test category	Detail	Method	Tentative Evaluation Standard Values
General characteristic	Appearance	Seeing	No adhesion of foreign matter, peeling of labels, etc. No liquid leakage
	Appearance	Seeing	No abnormality in color tone and no foreign matter
	Cell morphology	Microscopic observation	T cell-like morphology (floating cell)
	Viable cell count (product)	Cell counting	More than 1 × 10⁷ cells/50mL tube
	Viability	Flow cytometry	More than 90% of viability (7AAD-) in the lymphocyte gate
	Cell surface marker expression	Flow cytometry	At least 80% of the CD45 + cells express CD3, and CD3 + cells express NKT-specific TCR (Va24, Vb11)
Safety	Cell purity	PCR	Undifferentiated marker (LIN28)-expressing cells negative
	Sterility	Culture method	Fungi and common bacteria negative
	Sterility	Rapid method	Fungi and common bacteria negative (except anaerobic bacteria)
	Mycoplasma denial	PCR	Negative
	Endotoxin	Colorimetric and turbidimetric methods	Less than the standard value (0.3 EU/mL)^†
Cell function	in vitro IFNγ productivity	ELISA	IFNγ level (Target value: 1.7 times the average of negative samples)
Cell function	in vitro anti-tumor effect	K562 cell killing potential	Confirmation of anti-tumor effect (Target value: 7%)
Process-Related Impurity Testing
Safety (impurity)	BSA	ELISA	<50 ng/dose (25 mL) in final product
Safety (impurity)	Mouse genome	PCR	Below detection limit in final product

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^†: Endotoxin thresholds reflect the validated limit of detection at the manufacturing site.

Study procedures

After receiving written informed consent from the potential participant, the screening tests are conducted. Participant eligibility is ultimately confirmed by the PI and study coordinating physicians. According to the decision from the coordinating physician, we register the eligible participant to this trial. The study schedule details are described in Fig 1.

DC/Gal preparation period. The DC/Gal are manufactured by purifying each eligible patient’s peripheral monocytes after apheresis and incubating them with αGalCer for seven days at Chiba University Hospital. The DC/Gal is validated following the product specification tests (Table 2) before injection into the participant.

Table 2. List of specification tests for the a-galactosyl ceramide-pulsed autologous dendritic cells (DC/Gal).

Test category	Method	Specified value/judgement criteria
Living cell count	Measurement using a blood cell counting plate	More than 1 × 10⁸
Cell viability	Measured using a trypan blue stained blood cell calculator	More than 60%
Rapid viable bacterial test	Membrane filter method/rapid fluorescent staining	Negative
Sterility test	Membrane filter method	Negative
Endotoxin test	Gelation method/limit test method	< 0.25 EU/mL^†
Mycoplasma denial test	NAT Method	Negative^‡
Endotoxin test (simplified test)	kinetic colorimetry (Simplified method)	< 1 EU/mL
Cell surface antigen assay	Flow cytometry	Of CD45 + cells, at least 15% of CD86 + cells
Impurity test	Macro- and Microscopic observation	No foreign matter
Appearance test on product container	Seeing	No abnormalities on the container and package

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^†: Endotoxin thresholds reflect the validated limit of detection at the manufacturing site;

^‡: Mycoplasma testing methods are equivalent across products.

Treatment period. The treatment period begins with the injection of DC/Gal (1.0 × 10⁸ cells/injection) into the submucosa of the inferior nasal concha of each participant. The iPSC-iNKT cell product is administered to the tumor feeding artery five days after the DC/Gal injection. Both the DC/Gal and iPSC-iNKT cells are administered only once during this trial. We carefully monitor the participant until two weeks after iPSC-iNKT cell product administration. All the participants must be hospitalized throughout the treatment period.

Observation period. All the participants must visit the hospital three times by two weeks (days 1, 15, and 28 of the observation period), with potential AEs carefully monitored during the treatment period. If there are any AEs related to this trial at the final visit or time of discontinuation of this study, we will continue to follow the participant until the AEs relieve.

Outcome measurements

DLTs. 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. The grade is evaluated using the CTCAE ver. 5.0 Japanese version JCOG edition. The following AEs are included:

1) Grade 4 or higher hematological toxicities;
2) Any non-disease-related blood toxicity requiring any transfusion or granulocyte-colony stimulating factor (G-CSF) administration;
3) Grade 3 or higher non-hematological toxicities (excluding transient clinical laboratory abnormalities, diarrhea, nausea, vomiting, or other manageable systemic symptoms that recovered to grade 2 or below under appropriate treatments); and
4) AEs leading to blood transfusion.

If DLT occurrence is suspected, then the PI can consult the Independent Data Monitoring Committee (IDMC). The IDMC determines if the patient should continue or terminate the treatment.

DLT assessment is evaluated at each step, with the DLTs of the first three participant assessed in the first dose cohort. If any of these three patients experienced the DLT, then we will add an additional three patients. If two or more patients experience a DLT at the first dose cohort, the dose will not be escalated, and the study will be stopped. If one or none of the six patients have DLT, then the dose can be considered to be tolerable. The study can then be continued to assess the second dose cohort in the same manner. Finally, if one or none of the six patients have DLT in the second dose cohort, then the IDMC will define the dose as the MTD. DLT assessment is conducted by evaluating all the safety information, including DLT. The IDMC then decides whether to proceed with the next dose level cohort or the MTD.

Safety measurement. We assess the DC/Gal and iPSC-iNKT cell safety information throughout the study. We record all AEs that occurred after DC/Gal or iPSC-iNKT cell administration, regardless of the causality. All the AEs are specified in its terminology using the MedDRA, frequency, and severity.

Efficacy measurement. As a preliminarily efficacy measurement of the study treatments, we evaluate the tumors using contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) before the DC/Gal injection and at day 15 of the observation period or the time of discontinuation.

Exploratory outcomes. We assess the pharmacokinetics or pharmacodynamics of the iPSC-iNKT cells, as well as the HLA type of the peripheral blood cells to detect the iPSC-iNKT cells in the peripheral blood of the participants. In addition, we conduct an immune cell repertoire analysis and omics analysis of the peripheral blood and tumor specimens to evaluate the effects of the study treatments on the immune system of the participants and tumor microenvironment, respectively. Tumor specimens are optionally collected before the study treatment and/or within two days after iPSC-iNKT cell administration, only if the participant consents to this procedure.

Ethics consideration and data management plans

This study is conducted in accordance with the Declaration of Helsinki, the Pharmaceutical and Medical Device Act, the Act on the Safety of Regenerative Medicine, and the Japanese GCP for regenerative medicine. This study has been approved by the Certified Review Board for Regenerative Medicine of Chiba University and the regenerative medicine review board of Japanese Ministry of Health, Labour, and Welfare (MHLW), and registered with the Japan Registry of Clinical Trials (jRCT) (jRCT number: jRCTa030220741; URL: https://jrct.mhlw.go.jp/en-latest-detail/jRCTa030220741). Additionally, all the study procedures follow the study protocol and associated standard operating procedures stipulated separately. All participants are well informed and provide written consent to participate in this trial using the informed consent form (S1 File) by the PI before the eligibility screening procedures.

All the study data are collected using the electronic case report forms (eCRFs) and stored through the electronic data capturing system “the Fountayn.” Documents or records pertaining to the study are preserved at a storage location deemed appropriate until 30 years have elapsed after the study is discontinued or terminated. These data managements are conducted according to the standard operating procedure of this study stipulated separately.

Safety considerations

We assess the body weight, vital signs, ECOG PS, tumor status, blood tests, urine test, and radiography for all participants throughout this trial. In addition, the participants need to be hospitalized during the treatment period for intensive observation and prompt identification of DLTs. When the participants are discharged, we ensure that the patient can contact our hospital and nearby medical facilities promptly in case of AEs. We monitor AEs from the time the patient provides consent until the end of the observation period (discontinuation). If AEs with a plausible relationship to the investigational product remain unresolved at the end of the observation period, then the investigations will be continued until the recovery, stabilization, or death from such AEs, whichever occurs earliest. Additionally, the IDMC monitors the safety of the clinical trial and make recommendations regarding the dosage and administration intervals. The IDMC provides recommendations for further safety evaluation at a specified dose level and the determination of the next dose evaluation.

Trial status

At the time of this submission, patient recruitment has commenced. We started recruitment from March 30, 2023, and will end in December 2027 (six participants recruited at the time of submission), on estimation. All data will be collected by the end of February 2027, and this study will be completed by April 2027. The findings of this trial will be disseminated through peer-reviewed publications and scientific conferences.

Discussion

This is the first-in-human clinical trial using the combination of DC/Gal and iPSC-iNKT cells for head and neck cancer patients. 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. This study will provide not only the tolerability, safety, exploratory efficacy, and biological background of the iPSC-iNKT cells for head and neck cancer, but also important evidence guiding the feasible study design and procedures for the combination use of autologous and allogenic cells. This clinical study is essential for developing the iPSC-iNKT cell therapy toward the next phase 2 study to evaluate its efficacy against malignant tumors. Additionally, this trial protocol can contribute to future clinical trials using separately prepared immune cells products, such as an iPS cell-derived immune cell product and patient-derived adjuvant agents.

As a first-in-human study, we schedule frequent blood draws for the PK/PD evaluation during the first week after the first and second administration of the iPSC-iNKT cells. We use HLA genotyping to detect residual iPSC-iNKT cells in participants’ peripheral blood. When doing this, we exclude patients with identical HLA genotypes as the iPSC-iNKT cell product from this study.

We set the administration schedule of the DC/Gal and iPSC-iNKT cells on days 1 and 6 of the treatment period, respectively. This treatment sequence was determined from the previous study using DC/Gal [19]. We showed that the DC/Gal are widely distributed toward the neck lymph area 2–7 days after injection into the inferior nasal concha submucosa [18]. We therefore adopt sequential administration of the DC/Gal and iPSC-iNKT cells to maximize the immunological interaction between them. The starting iPSC-iNKT cell dose (3.0 × 10⁷ cells/m²) was determined from a pre-clinical study using severely immunodeficient NOG (NOD/Shi-scid/IL-2Rgamma null) mice (unpublished data). The pre-clinical safety study indicated that the no adverse event level (NOAEL) of the combination use of iPSC-iNKT cells with DC/Gal was 2.0 × 10⁶ cells/body. This NOAEL is equivalent to 3.0 × 10⁷ cells/m² for humans following the FDA “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” [22]. From this, we consider this starting dose to be ethically and scientifically acceptable. 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-in-human 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 tumor-feeding arteries for targeted administration.

Another notable aspect of the scheduling of this study is the preparation period for iPSC-iNKT cell product and the delays allowed for the treatment initiation. With regenerative medicine, which is associated with long-term storage difficulties, the study products need to be expanded from working cell bank consisting of intermediate cell products after the enrollment of each participant. The iPSC-iNKT cell and DC/Gal preparations take 10–14 days and five days, respectively. We determined the screening duration and treatment initiation time after considering the preparation period for study treatment. We also set the allowance to seven days for the treatment initiation time to account for the delay of cell preparation.

This trial began with only single dose cohort (3.0 × 10⁷ cells/m² of the iPSC-iNKT cells) because the tolerability assessment of the second dose (1.0 × 10⁸ cells/m²) of the iPSC-iNKT cells was simultaneously in progress in the previous phase 1 trial at the time this trial was initiated (S5 and S6 Files). After confirmation of the tolerability of the second iPSC-iNKT cell dose, we amended the study design to add the second dose cohort. In addition to the amendment, we added the optional tumor biopsy from the participants to examine the anti-tumor effects and immune landscape of the tumor microenvironment. This tumor biopsy is conducted before and after administration of the combination therapy only for participants with an accessible bulky tumor and who agree with this extra biopsy given the ethical considerations.

This phase 1 study has some limitations. Because the DC/Gal and iPSC-iNKT 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 combination therapy. The repetitive use of DC/Gal is not feasible considering the burden of blood apheresis on the patient for preparation. However, the allogenic iPSC-iNKT cell product may be able to be administered repeatedly in future trials. Because more than 99.6% of the iPSC-iNKT product was found to be excreted within 48 hours in the non-clinical study, we believe that the bi-weekly use of this product would not accumulate and increase the risk of use. Another limitation of this study is the participants. We enrolled head and neck cancer patients who were unamenable to standard of care in this study in accordance with “the guideline for Evaluation of anti-cancer agent [23]” and because of its approach for the tumor feeding artery. The study design limits the generalizability of the findings to other malignancies. Nevertheless, the iPSC-iNKT cells showed a killing effect not only in head and neck cancer cells, but also in other cell types, suggesting that the results of this trial would be vital and fundamental for the future application of iPSC-iNKT cells for various cancer patients.

Supporting information

S1 File. Example of the informed consent form (translated into English).

(PDF)

pone.0342387.s001.pdf^{(343KB, pdf)}

S2 File. SPIRIT checklist.

Recommended items to address in a clinical trial protocol and related documents.

(PDF)

pone.0342387.s002.pdf^{(127.3KB, pdf)}

S3 File. The latest version (v.3.0) of the full protocol (Japanese original version).

(PDF)

pone.0342387.s003.pdf^{(2.9MB, pdf)}

S4 File. The latest version (v.3.0) of the full protocol (English version).

(PDF)

pone.0342387.s004.pdf^{(2.2MB, pdf)}

S5 File. The previous version (v.1.8) of the full protocol (Japanese original version).

(PDF)

pone.0342387.s005.pdf^{(2.8MB, pdf)}

S6 File. The previous version (v.1.8) of the full protocol (English version).

(PDF)

pone.0342387.s006.pdf^{(2.8MB, pdf)}

Acknowledgments

We thank Kazue Namiki, Chikako Inamata, and Natsumi Kondo for trial coordinating support. We thank J. Iacona, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Abbreviations

ALT: alanine aminotransferase
AST: aspartic aminotransferase
CTCAE: common terminology criteria for adverse events
CTL: Cytotoxic T lymphocyte
DC: dendritic cells
DLT: dose limiting toxicity
EDC: electronic data capture
ECOG: Eastern Cooperative Oncology Group
GCP: good clinical practice
HLA: human leukocyte antigen
IDMC: Independent Data Monitoring Committee
mMRC: modified Medical Research Council
MTD: maximum tolerated dose
NYHA: New York Heart Association
PS: performance status
RECIST: response evaluation criteria in solid tumors
α-GalCer: α-Galactosylceramide

Data Availability

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.

Funding Statement

HK: 21bk0104119h0001, 24bk0104173h0001 Japan Agency for Medical Research and Development https://www.amed.go.jp/en/index.html The funder didn’t play any role in the study design, data collection and analysis, decision to publish, nor preparation of the manuscript.

References

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PLoS One. doi: 10.1371/journal.pone.0342387.r001

Decision Letter 0

Maria de Fátima Macedo

27 Oct 2025

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.

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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

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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

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Reviewer #2: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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.

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Reviewer #1: Recommedation

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.

**********

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Reviewer #2: No

Reviewer #3: No

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Attachment

Submitted filename: PONE-D-25-33703_Comments_to_the_authors.pdf

pone.0342387.s007.pdf^{(61.3KB, pdf)}

PLoS One. 2026 Feb 26;21(2):e0342387. doi: 10.1371/journal.pone.0342387.r002

Author response to Decision Letter 1

9 Dec 2025

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

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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 include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

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.

Before we proceed with your manuscript, please address the following prompts:

Please update your Data Availability statement in the submission form accordingly.

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.

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.

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.

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.

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.

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.

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.

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.

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”

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.”

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."

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.

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.”

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.

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).

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.”

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]”.

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

Attachment

Submitted filename: Response to Reviewers_the journal requirements.docx

pone.0342387.s008.docx^{(31KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0342387.r003

Decision Letter 1

Maria de Fátima Macedo

9 Jan 2026

Dear Dr. Shinichiro Motohashi,

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'.

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.

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Additional Editor Comments:

Please address comments done by reviewer 3.

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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

**********

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.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Attachment

Submitted filename: PONE-D-25-33703R1_Comments_to_the_authors.pdf

pone.0342387.s009.pdf^{(40KB, pdf)}

PLoS One. 2026 Feb 26;21(2):e0342387. doi: 10.1371/journal.pone.0342387.r004

Author response to Decision Letter 2

17 Jan 2026

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.

The clarity of this sentence has been enhanced by the contribution of your commentary. We revised it as your advice.

2) Tables 1-2.

[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."

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:

The clarity of this sentence has been enhanced by the contribution of your commentary. We revised it as your advice.

Attachment

Submitted filename: Response to Reviewers_260118.docx

pone.0342387.s010.docx^{(25.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0342387.r005

Decision Letter 2

Maria de Fátima Macedo

21 Jan 2026

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.

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Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0342387.r006

Acceptance letter

Maria de Fátima Macedo

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Example of the informed consent form (translated into English).

(PDF)

pone.0342387.s001.pdf^{(343KB, pdf)}

S2 File. SPIRIT checklist.

Recommended items to address in a clinical trial protocol and related documents.

(PDF)

pone.0342387.s002.pdf^{(127.3KB, pdf)}

S3 File. The latest version (v.3.0) of the full protocol (Japanese original version).

(PDF)

pone.0342387.s003.pdf^{(2.9MB, pdf)}

S4 File. The latest version (v.3.0) of the full protocol (English version).

(PDF)

pone.0342387.s004.pdf^{(2.2MB, pdf)}

S5 File. The previous version (v.1.8) of the full protocol (Japanese original version).

(PDF)

pone.0342387.s005.pdf^{(2.8MB, pdf)}

S6 File. The previous version (v.1.8) of the full protocol (English version).

(PDF)

pone.0342387.s006.pdf^{(2.8MB, pdf)}

Attachment

Submitted filename: PONE-D-25-33703_Comments_to_the_authors.pdf

pone.0342387.s007.pdf^{(61.3KB, pdf)}

Attachment

Submitted filename: Response to Reviewers_the journal requirements.docx

pone.0342387.s008.docx^{(31KB, docx)}

Attachment

Submitted filename: PONE-D-25-33703R1_Comments_to_the_authors.pdf

pone.0342387.s009.pdf^{(40KB, pdf)}

Attachment

Submitted filename: Response to Reviewers_260118.docx

pone.0342387.s010.docx^{(25.4KB, docx)}

Data Availability Statement

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.

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PERMALINK

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

Roles

Abstract

Introduction

Materials and methods

Trial designs, aim, and settings

Fig 1. Study schedule of enrollment, interventions, and assessments.

Fig 2. The study outline, including all study visits and timeline.

Fig 3. The 3 + 3 design for dose escalation.

Participants

Statistical analysis and sample size

iPSC-iNKT cells (investigational product)

Table 1. List of specification tests for the induced pluripotent stem cell-derived invariant natural killer T (iPSC-iNKT) cells.

Study procedures

Table 2. List of specification tests for the a-galactosyl ceramide-pulsed autologous dendritic cells (DC/Gal).

Outcome measurements

Ethics consideration and data management plans

Safety considerations

Trial status

Discussion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Maria de Fátima Macedo

Roles

Author response to Decision Letter 1

Decision Letter 1

Maria de Fátima Macedo

Roles

Author response to Decision Letter 2

Decision Letter 2

Maria de Fátima Macedo

Roles

Acceptance letter

Maria de Fátima Macedo

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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