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. 2025 Feb 6;116(4):884–896. doi: 10.1111/cas.70008

Antibody‐Dependent Cellular Cytotoxicity of iPS Cell‐Derived Natural Killer T Cells by Anti‐GD2 mAb for Neuroblastoma

Katsuhiro Nishimura 1,2, Takahiro Aoki 1,3,4, Midori Kobayashi 1, Mariko Takami 1, Ko Ozaki 1, Keita Ogawa 1, Wang Hongxuan 1, Daiki Shimizu 1, Daisuke Katsumi 1,2, Hiroko Yoshizawa 1,2, Shugo Komatsu 2, Tomozumi Takatani 3, Kiyoshi Hirahara 5, Haruhiko Koseki 4, Tomoro Hishiki 2, Shinichiro Motohashi 1,✉
PMCID: PMC11967243  PMID: 39916425

ABSTRACT

While antibody‐dependent cellular cytotoxicity (ADCC) by anti‐disialoganglioside GD2 monoclonal antibody (mAb) has succeeded in increasing the survival rate of high‐risk patients with neuroblastoma, approximately 40%–50% of patients die from the disease. Recently, we developed induced pluripotent stem cell‐derived natural killer T (iPS‐NKT) cells, which exhibit NK‐like cytotoxicity. However, whether iPS‐NKT cells can induce ADCC function is unclear. Here, we investigated the ADCC of iPS‐NKT cells and the efficacy of the combination treatment of anti‐GD2 mAb and iPS‐NKT cells against neuroblastoma. Anti‐GD2 mAb enhanced the cytotoxicity and secretion of cytokines and cytotoxic granules of iPS‐NKT cells, which expressed CD16 to GD2‐expressing neuroblastoma cell lines. We also examined which Fcγ receptors contribute to ADCC of iPS‐NKT cells. CD16 stimulation against iPS‐NKT cells caused cytotoxicity and secretion of interferon‐gamma, tumor necrosis factor, and granzyme B. In contrast, CD32 and CD64 stimulation did not. In vivo, the intratumor administration of anti‐GD2 mAb and iPS‐NKT cells significantly inhibited tumor growth compared with the other treatment groups: no treatment, anti‐GD2 mAb alone, and iPS‐NKT cells alone. In conclusion, iPS‐NKT cells exhibit CD16‐mediated ADCC, and the addition of iPS‐NKT cells to anti‐GD2 mAb therapy may be a potential approach for immunotherapy against neuroblastoma.

Keywords: antibody‐dependent cellular cytotoxicity, anti‐GD2 monoclonal antibody, induced pluripotent stem cell, invariant natural killer T cell, neuroblastoma

Unlike invariant natural killer T (iNKT) cells, induced pluripotent stem cell‐derived NKT (iPS‐NKT) cells exert anti‐GD2 monoclonal antibody(mAb)‐mediated antibody‐dependent cell cytotoxicity(ADCC) and exhibit CD16‐dependent cytotoxic activity and produce cytokines. Combination therapy with iPS‐NKT cells and anti‐GD2 mAb inhibited tumor growth in a mouse model of neuroblastoma subcutaneous tumor.

graphic file with name CAS-116-884-g001.jpg

Abbreviations

ADCC

antibody‐dependent cellular cytotoxicity

GD2

disialoganglioside

iNKT cell

invariant natural killer T cell

iPS‐NKT cell

induced pluripotent stem cell–derived natural killer T cell

1. Introduction

Neuroblastoma is a tumor of the sympathetic nervous system and the most common extracranial solid malignant tumor in children. Approximately, 50% of neuroblastoma patients have metastases at diagnosis, and despite surgery, chemotherapy, and radiation therapy, neuroblastoma patients have poor long‐term survival [1, 2]. The disialoganglioside GD2 is a tumor‐associated antigen expressed in neuroblastoma, melanomas, and other malignant tumors [3]. Because GD2 expression in normal tissues is restricted to some tissues such as nerve fibers and melanocytes, GD2 is considered a suitable target for immunotherapy and has been investigated as a therapeutic target over the past 50 years [4, 5, 6]. In 2010, Yu et al. reported the efficacy of anti‐GD2 monoclonal antibody (mAb) in combination with interleukin (IL)‐2 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) against neuroblastoma [7]. Anti‐GD2 mAb therapy improved the outcome of patients with high‐risk neuroblastoma compared with prior standard therapy [7, 8] and is the standard immunotherapy for high‐risk neuroblastoma. However, the 5‐year event‐free survival rate is still approximately 50%–60% [8], and therefore, the identification of new treatments is critical to improve patient outcomes.

Invariant natural killer T (iNKT) cells play an important role in tumor immunity. iNKT cells are activated by a specific glycolipid ligand, α‐galactosylceramide, presented on CD1d molecules. Activated iNKT cells enhance both innate and type I acquired immunity [9, 10]. Studies have shown that activated iNKT cells have an adjuvant effect on anti‐GD2 mAb–mediated antibody‐dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells, [11] and iNKT cell infiltration into neuroblastoma is correlated with a favorable prognosis [12].

While iNKT cells are competent effector cells in cancer immunity, their use in immunotherapy is limited by the low number of cells in human peripheral blood. To overcome this problem, a method for reprograming induced pluripotent stem cells (iPSCs) from human peripheral blood iNKT cells was established. iPSC‐derived NKT (iPS‐NKT) cells exhibited anti‐tumor activity in various cancer cell lines and adjuvant activity for human NK cells [13]. A phase I clinical trial of iPS‐NKT cell therapy for head and neck cancer was conducted (jRCT2033200116). However, whether iPS‐NKT cells show anti‐tumor activity in neuroblastoma and whether iPS‐NKT cells possess ADCC have not been determined.

In this study, we examined the anti‐tumor effects of iPS‐NKT cells against neuroblastoma. We also investigated the efficacy of the combination treatment of anti‐GD2 mAb and iPS‐NKT cells.

2. Material and Methods

2.1. Flow Cytometry

Flow cytometric data were acquired with a FACSVerse or LSRFortessa‐X20 instrument (BD Biosciences, Franklin Lakes, NJ, USA) running BD FACSuite or FACSDiva, respectively, and analyzed with FlowJo software (FlowJo LLC). iNKT cells and iPS‐NKT cells were identified using FITC‐labeled anti‐Vα24 (clone C15, Beckman Coulter, Brea, CA, USA), PE‐labeled anti‐Vβ11 (clone C21, Beckman Coulter), and Pacific Blue–labeled anti‐CD3 (clone UCHT1, BioLegend, San Diego, California, USA) antibodies and APC‐labeled anti‐CD45 (clone 2D1, BioLegend). Pacific Blue–labeled anti‐CD16 (clone 3G8, BioLegend), PE‐labeled anti‐CD32 (clone FUN‐2, BioLegend), and APC‐labeled anti‐CD64 (clone S18012C, BioLegend) antibodies were used to detect the expression of Fcγ receptors on the surface of iNKT cells and iPS‐NKT cells. PE‐labeled anti‐CD69 (clone FN50, BD Bioscioences) were used to evaluate the activation of iNKT and iPS‐NKT cells. Propidium iodide (Sigma‐Aldrich, St. Louis, MO, USA) and 7‐amino‐actinomycin D (7‐AAD, BD Biosciences) were used to identify dead cells.

2.2. Generation of iNKT Cells

We generated iNKT cells as described in a previous report [11]. Briefly, venous blood was obtained from three healthy adult volunteer donors with informed consent, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll‐Paque (GE Healthcare, Chicago, IL, USA). PBMCs were cultured in complete RPMI 1640 medium for 9–14 days in the presence of 100 U/mL of recombinant human IL‐2 (Imunace; Shionogi, Osaka, Japan) and 200 ng/mL of αGalCer (KRN7000; REGiMMUNE, Tokyo, Japan). Next, iNKT cells were isolated with MiniMACS Separator or MidiMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) using fluorescein isothiocyanate (FITC)‐labeled anti‐Vα24 antibody and anti‐FITC microbeads (Miltenyi Biotec). The purified iNKT cells were cultured in complete medium containing IL‐2 until experiments (within 3 days after isolation). Even if the donor was the same, NKT cells purified from PBMCs collected at different times were assigned to different lots. The institutional review board of Chiba University approved all experiments.

2.3. Generation of Vα24 iNKT Cells From NKT‐iPSCs

We used previously developed NKT‐iPSCs induced from two donors [13]. Vα24 iNKT cells were generated from NKT‐iPSCs, which we named iPS‐NKT cells, using the OP9/OP9DLL1 stromal cell co‐culture system as previously described with slight modifications [13]. In brief, confluent NKT‐iPSC colonies were mechanically removed from dishes and plated on mitomycin‐C (Sigma‐Aldrich)–treated OP9 overconfluent cells in 10 cm dishes containing 10 mL of OP9 medium: α‐MEM (Thermo Fisher Technologies, Waltham, MA, USA) with 20% FCS and penicillin–streptomycin solution (Wako, Osaka, Japan). On day 13, colonies were treated for 20 min with 5 mL of Accumax (Innovative Cell Technologies, San Diego, CA, USA) at 37°C. To remove stromal cells and aggregated cells, cells were passed through an EASYstrainer (100 μm, Greiner Bio‐One, Kremsmünster, Austria). Cells were plated on mitomycin‐C–treated OP9/DLL1 semi‐confluent cells in dishes containing OP9 medium with 5 ng/mL of hIL‐7, 5 ng/mL of hFlt‐3 L (PeproTech, Cranbury, NJ, USA) and 10 ng/mL of hSCF (PeproTech). On day 16 and day 23, semiadherent cells were collected and passaged into a new dish plated with mitomycin C‐treated OP9/DLL1 cells. On day 30, cells were collected and cultured with 5 ng/mL of hIL‐7 and 10 ng/mL of hIL‐15 for 9–14 days. Even if the donor is the same, iPS‐NKT cells purified and cultured from NKT‐iPSc induced at different times were assigned to different lots.

2.4. Cell Lines

The human neuroblastoma cell lines IMR‐32 and SK‐N‐SH were obtained from the RIKEN BRC through the National Bio‐Resource Project of the MEXT/AMED, Japan. SH‐SY5Y cells were kindly provided by Dr. A. Nakagawara (Chiba, Japan). All cell lines were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat‐inactivated FCS. The PE‐labeled anti‐human ganglioside GD2 antibody (clone 14.G2a, BioLegend) was used to detect GD2 expression in human neuroblastoma cell lines with flow cytometry.

2.5. CD107a Degranulation Assay

iPS‐NKT cells (2×105 cells) were incubated for 5 h at 37°C in the complete medium in the presence of 2.5 μg anti‐CD107a antibody (clone H4A3, BioLegend) and 2 μmol/L monensin (BioLegend) with 2×105 target cells reacted anti‐GD2 mAb or with antibodies cross‐linking Fcγ receptors. iPS‐NKT cells were then collected and stained with anti‐Vα24 and anti‐CD3 antibodies for 20 min. After staining, the expression of CD107a in iPS‐NKT cells was measured by flow cytometry. CD107a expression indicates the percentage of CD107a‐positive fraction induced in iPS‐NKT cells in which target cells or cross‐linking receptors were added compared with that in unstimulated iPS‐NKT cells.

2.6. In Vitro Cytotoxicity Assay

For the Annexin V assay, tumor cells were labeled with Cell Trace Violet (CTV, Invitrogen, Carlsbad, CA, USA) for identification following the manufacturer's protocol. CTV‐labeled tumor cells (4×104 cells) were seeded in wells of a 96‐well plate with or without anti‐GD2 mAbs (1 μg/mL). We used two anti‐GD2 mAbs: 14.G2a (BD Pharmingen, Franklin Lakes, NJ, USA), a purified mouse anti‐human GD2 monoclonal antibody (mAb), and ch14.18 (dinutuximab, United Therapeutics, Silver Spring, MD, USA), a purified chimera anti‐human GD2 monoclonal antibody. Effector cells, purified iNKT cells or iPS‐NKT cells, were added to wells at several effector: target (E: T) ratios and incubated for 4 h at 37°C in 5% CO2. APC‐labeled Annexin V (BioLegend) and 7‐AAD in Annexin V binding buffer (BioLegend) were added to identify apoptotic tumor cells following the manufacturer's protocol. After staining, the frequency of live CTV‐labeled tumor cells was determined by flow cytometry. Annexin V−/7‐AAD− cells were counted as living cells. Cytotoxicity (%) was calculated as 100×(living cells without effector cells—living cells with effector cells). Representative FACS data and the gating strategy for an Annexin V assay are shown in Figure S1.

2.7. Cytokine Measurements

The indicated numbers of purified iNKT cells or iPS‐NKT cells were incubated for 24 h at 37°C in 200 μL complete medium with 4×104 target cells. The supernatants were collected and stored at −80°C until analysis. The levels of interferon‐γ (IFN‐γ), tumor necrosis factor (TNF), and Granzyme B were determined in supernatants using a BD Cytometric Bead Array System (BD Bioscience) following the manufacturer's protocol. Data were acquired on FACSVerse and analyzed with FCAP Array software (BD Biosciences).

2.8. Blocking Assay

To block Fcγ receptor CD16, CD32, and CD64 expressed on iPS‐NKT cells, the cells were pre‐incubated for 30 min with 10 μg/mL blocking antibodies at 37°C. We used purified anti‐human CD16 antibody (clone 3G8, BioLegend), CD32 antibody (clone FUN‐2, BioLegend), and CD64 antibody (clone S18012C, BioLegend) for blocking Fcγ receptors. We then conducted the in vitro cytotoxicity assay and cytokine measurement experiment as described above.

2.9. Mice

Recipient hIL‐7 and hIL‐15 knock‐in (IL‐7×15) NSG mice (NOD.Cg‐PrkdcscidIL2rgtm1Wjl/SzJ) were provided by Dr. Koseki and Dr. Ishikawa (RIKEN) [14]. Mice were maintained under specific pathogen‐free conditions and studied in compliance with institutional guidelines for the care and use of laboratory animals. The Chiba University Institutional Animal Care and Use Committee approved all animal procedures.

2.10. Generation of Luciferase‐Transduced Cell Lines

For in vivo bioluminescence imaging experiments, we generated the firefly luciferase (Luc) transgenic neuroblastoma cell lines using a lentiviral vector. Lentiviral V5‐Luciferase expression vector, pLenti PGK V5‐LUC Puro (w543‐1), was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 19360; Watertown, MA, USA). pLenti PGK V5‐LUC Puro was co‐transfected with a lentiviral packaging plasmid psPAX2 (Addgene) and a VSV‐G envelope expressing plasmid pMD2.G (Addgene) into HEK‐293 T cells using polyethylenimine (PEI 25K; Polysciences, Warrington, PA, USA). The supernatant medium containing virus was collected after 48 h. Virus‐containing medium and polybrene were added to cultured IMR‐32 cells for transduction. After infection, the virus‐containing medium was replaced with fresh medium for 48–72 h. Transduced IMR‐32 cells were collected and subjected to single‐cell cloning by the limiting dilution method, and the clone with the strongest luminescence was expanded to purify IMR‐32‐Luc.

2.11. In Vivo Tumor Models and Treatments

Female 8–10‐week‐old mice were anesthetized with isoflurane (Viatris Pharmaceuticals Japan, Tokyo, Japan) and injected subcutaneously with IMR‐32‐Luc cells (5×106) suspended in 100 μL of sterile PBS with Matrigel (CORNING, Corning, NY, USA). At 6 days after tumor cell inoculation, we confirmed tumor cell implantation in mice. We randomly assigned animals to the following treatment groups: (i) control, (ii) ch14.18 (United Therapeutics) alone, (iii) iPS‐NKT cells alone, or (iv) the combination of iPS‐NKT cells and ch14.18 (n = 5 mice per group; total n = 20 mice). Mice received intratumor administration of treatments on days 6 and 7 after tumor cell inoculation: (i) 50 μL PBS/day i.t., (ii) 5 μg of ch14.18/day i.t., (iii) 1×107 iPS‐NKT cells/day i.t., or (iv) 5 μg of ch14.18 and 1×107 iPS‐NKT cells/day i.t. Ch14.18 and iPS‐NKT cells were administered in the same volume of PBS as the control group. The amounts of tumor cells were quantified every 5 days by luciferase bioluminescence imaging. All mice used in the experiments were included in the analysis. Mice were euthanized when they lost more than 20% of their body weight or when the tumor diameter reached more than 20 mm.

2.11.1. Comparison of ADCC for iNKT and iPS‐NKT Cells In Vivo

In accordance with the above, we prepared mice with subcutaneous tumors of IMR‐32‐Luc and randomly assigned them to the following three treatment groups: ch14.18 alone, iNKT cells with ch14.18, and iPS‐NKT cells with ch14.18 (n = 6 mice per group; total n = 18 mice). Mice received intratumoral administration of treatments on days 5 and 6 after tumor cell inoculation: 5 μg of ch14.18/day i.t., 5×106 iNKT cells, or iPS‐NKT cells with 5 μg of ch14.18/day i.t. The amount of tumor cells was quantified every 6 days by luciferase bioluminescence imaging. Once the subcutaneous tumor reached a measurable size, the tumor volume of at least 5 mm on one side was measured once a week. Tumor volume was calculated as length [mm] × width [mm] × height [mm] × 0.52. The time to tumor diameter of 20 mm was measured, and the mice were euthanized at that time.

2.11.2. Analysis of iNKT and iPS‐NKT Cells in the Tumor Microenvironment (TME)

We subcutaneously inoculated mice with IMR‐32‐Luc cells (5×106) and allowed them to grow for 40 days until the tumors became palpable. On day 41 after tumor inoculation, 1×107 iNKT or iPS‐NKT cells with 10 μg of ch14.18 were administered intratumorally, and the tumor was resected the following day by sacrificing the mice. Resected tumor specimens were cut into small fragments and dissociated into single cells using a gentle MACS octo dissociator with heaters and the tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol. Tumor‐derived cell suspensions were separated into tumor‐infiltrating lymphocyte (TIL), that is, tumor‐injected iNKT cells and iPS‐NKT cells, and the other cells using the MidiMACS separator with CD45 MicroBeads, human (Miltenyi Biotec). Isolated human‐CD45 positive cells were stained with anti‐Vα24, CD3, and CD69 to identify and evaluate the activation of iNKT and iPS‐NKT cells and analyzed by flow cytometry.

2.12. Bioluminescence Imaging

Mice received 150 mg/kg D‐luciferin (beetle luciferin, E1605; Promega, Madison, WI, USA) intraperitoneally and were anesthetized with 2% isoflurane. After 10 min, which is the peak of maximum bioluminescence, imaging was performed with an IVIS Lumina2 system (Perkin Elmer, Courtaboeuf, France). The whole body was photographed, and the emission intensity was measured by setting the region of interest at the subcutaneous tumor. Bioluminescence intensity was expressed as total flux (photons/s). The researchers responsible for IVIS imaging were blinded as to which mice were assigned to which treatment group.

2.13. Statistical Analysis

The sample size for this study was determined with reference to previous studies of similar experiments [11, 13]. We used unpaired two‐sided Student's t‐test and one‐way analysis of variance followed by Tukey's multiple comparison test to compare data from different experimental conditions. The association between the frequency of CD16 expression on iPS‐NKT cells and cytotoxicity was evaluated using Pearson's correlation coefficient and expressed as the corresponding correlation coefficient R2. All tests were two‐sided, and p < 0.05 indicated statistical significance. All statistical analyses were performed using GraphPad Prism 10 (version 10.1.0 for Mac OS, GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

3. Results

3.1. iPS‐NKT Cells Express CD16 and Exert ADCC via the Anti‐GD2 mAb 14.G2a

To determine whether iPS‐NKT cells have the potential to show ADCC by anti‐GD2 mAb, we analyzed the expression of CD16 (FcγRIII), one of the Fcγ receptors which mediate ADCC, on the surface of iPS‐NKT cells by flow cytometry. Consistent with previous findings [11], CD16 expression was low in iNKT cells and moderately expressed in iPS‐NKT cells. While expression variations were observed, iPS‐NKT cells expressed CD16 at an average frequency of 35% (Figure 1A,B).

FIGURE 1.

FIGURE 1

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iPS‐NKT cells express CD16 and exert ADCC via 14.G2a. (A) CD16 expression on the surface of iNKT cells and iPS‐NKT cells. Shaded histograms represent isotype controls. Data are from a representative experiment of independent experiments. (B) Percentage of iNKT cells and iPS‐NKT cells expressing CD16. Each symbol represents a difference in a donor, and each dot represents a difference in a lot. The data represents 8 lots of iNKT cells from 3 donors and 13 lots of iPS‐NKT cells from 2 donors used in this study. Data are represented as mean ± standard deviation (SD) of each lot. (C) Cytotoxicity of iNKT cells or iPS‐NKT cells against IMR‐32, a neuroblastoma cell line. iNKT cells and iPS‐NKT cells were cultured with IMR‐32 (4×104 cells) at an E: T ratio of 10:1 in the absence or presence of 14.G2a for 4 h. (D) Amounts of cytokines secreted from iPS‐NKT cells (1×105 cells) cultured with IMR‐32 (1×104 cells) in the absence or presence of 14.G2a for 4 h. (E) Amounts of cytokines secreted from iNKT cells (4×105 cells) cultured with IMR‐32 (4×104 cells) in the absence or presence of 14.G2a for 4 h. Cytokine levels were measured by cytometric bead array. The data on cytotoxicity and cytokine levels are represented as mean ± SD of triplicate in one representative experiment (each experiment was repeated two or three times and at least one experiment with a different lot). The symbols used in this figure indicate the following donors: ◆, iNKT donor 1; ▲, iNKT donor 2; ▼, iNKT donor 3; ●, iPS‐NKT donor 1; and ■, iPS‐NKT donor 2. Statistical analysis was performed using Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non‐significant.

We next performed a cytotoxicity assay to determine whether iPS‐NKT cells exhibit anti‐GD2 mAb–mediated ADCC. iNKT cells or iPS‐NKT cells were co‐cultured with the IMR‐32 neuroblastoma cell line and cytotoxicity with and without anti‐GD2 mAb (14.G2a) were analyzed. As reported [11], anti‐GD2 mAb did not enhance the cytotoxicity of iNKT cells against IMR‐32 cells. However, the cytotoxicity of iPS‐NKT cells was significantly enhanced by anti‐GD2 mAb (p = 0.02) (Figure 1C). Anti‐GD2 mAb also enhanced the production of cytokines such as IFN‐γ and TNF or granzyme B from iPS‐NKT cells (Figure 1D). In iNKT cells, while no significant increase in cytotoxicity was detected, the secretion of cytokines and cytotoxic factors was slightly increased in the presence of 14.G2a (Figure 1E). These results indicate that iPS‐NKT cells exhibit anti‐GD2 mAb–mediated ADCC against a neuroblastoma cell line in vitro.

3.2. Ch14.18 (Dinutuximab) Induces Degranulation and Cytotoxicity of iPS‐NKT Cells More Potently Than 14.G2a

Ch14.18 (dinutuximab) is a chimeric human‐murine anti‐GD2 mAb that has been shown to induce higher ADCC of NK cells against melanoma and neuroblastoma cells than 14.G2a, a murine anti‐GD2 mAb [15]. To examine differences in ADCC of iPS‐NKT cells mediated by 14.G2a and ch14.18, the degranulation and cytotoxic activities of iPS‐NKT cells induced by the anti‐GD2 mAbs were evaluated.

To examine the degranulation activities of iPS‐NKT cells with or without anti‐GD2 mAbs, iPS‐NKT cells were co‐cultured with IMR‐32 cells in the absence or presence of 14.G2a or ch14.18, and CD107a (LAMP1) expression was determined by flow cytometry. CD107a is a degranulation marker on lymphocytes including NKT cells. We also performed experiments on iNKT cells at the same time as the control. The expression of CD107a in iPS‐NKT cells was increased in the presence of the anti‐GD2 mAbs, and CD107a expression was significantly higher in iPS‐NKT cells cultured with ch14.18 compared with that in those cultured with 14.G2a (p < 0.0001). In contrast, CD107a expression on iNKT cells was not increased by any type of anti‐GD2 mAbs (Figure 2A,B). Similarly, iPS‐NKT cells co‐cultured with IMR‐32 cells in the presence of ch14.18 showed significantly more potent cytotoxicity against IMR‐32 cells than iPS‐NKT cells co‐cultured with 14.G2a (p = 0.003) (Figure 2C). Enhancement of CD107a expression and ADCC via ch14.18 was also demonstrated in iPS‐NKT cells induced from different donors (Figure 2A,B,D).

FIGURE 2.

FIGURE 2

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ch14.18 (dinutuximab) induced degranulation and cytotoxicity of iPS‐NKT cells more potently than 14.G2a. (A) CD107a expression on the surface of iNKT and iPS‐NKT cells. These cells were cultured with the same number of IMR‐32 cells in the absence or presence of 14.G2a or ch14.18 for 5 h and collected. CD107a expression was determined by flow cytometry. Data are from a representative experiment of independent experiments. (B) Percentage of CD107a‐positive fraction induced in iNKT cells and iPS‐NKT cells. (C) Cytotoxicity of iPS‐NKT cells against IMR‐32 cells. iPS‐NKT cells were cultured with IMR‐32 cells (4×104 cells) at various E: T ratios in the absence or presence of 14.G2a or ch14.18 for 4 h. (D) Cytotoxicity of iPS‐NKT cells with different donors against IMR‐32. iPS‐NKT cells were cultured with IMR‐32 (4×104 cells) at an E: T ratio of 10:1 in the absence or presence of ch14.18 for 4 h. (E) The expression of GD2 and CD1d on neuroblastoma cell lines. Shaded histograms represent isotype controls. (F) Cytotoxicity of iPS‐NKT cells against neuroblastoma cell lines with high GD2 expression (IMR‐32 and SH‐SY5Y) and SK‐N‐SH with low GD2 expression. iPS‐NKT cells were cultured with neuroblastoma cell lines at E: T ratio of 10:1 in the absence or presence of ch14.18 for 4 h. The data on frequency and cytotoxicity are represented as mean ± SD of triplicate in one representative experiment (each experiment was repeated two to three times and at least one experiment with a different lot). The symbols used in this figure indicate the following donors: ▲, iNKT donor 2; ●, iPS‐NKT donor 1; and ■, iPS‐NKT donor 2. Statistical analysis was performed using Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non‐significant.

We next examined the ch14.18‐mediated ADCC of iPS‐NKT cells against other neuroblastoma cell lines. Three neuroblastoma cell lines (IMR‐32, SH‐SY5Y, and SK‐N‐SH) with different levels of GD2 expression were used to evaluate ch14.18‐mediated ADCC of iPS‐NKT cells. IMR‐32 and SH‐SY5Y cells have high GD2 expression, while SK‐N‐SH cells exhibit low GD2 expression. These three cell lines do not express CD1d, which is required for the interaction of invariant TCR with iNKT cells (Figure 2E). While iPS‐NKT cells did not show ADCC against SK‐N‐SH cells with low GD2 expression, they exhibited ADCC against IMR‐32 and SH‐SY5Y cells with high GD2 expression (Figure 2F). Cytotoxicity by iPS‐NKT cells against SH‐SY5Y cells was demonstrated even in the absence of ch14.18, indicating that iPS‐NKT cells exhibit direct cytotoxicity against SH‐SY5Y cells without ADCC. It has been reported that iPS‐NKT cells express the NK cell markers TRAIL, FasL, and NKG2D [16]. These markers and their corresponding receptors and ligands are considered to potential candidates for inducing antibody‐independent cytotoxicity.

3.3. CD16 Stimulation Promotes iPS‐NKT Cell Degranulation and Cytokine Production

CD16 is an important Fcγ receptor for the exertion of ADCC of NK cells against malignant tumors, including neuroblastoma [17, 18]. Other Fcγ receptors and effector cells are involved in ADCC [16, 19, 20, 21]. With regard to anti‐GD2 mAb therapy for neuroblastoma, not only cytotoxicity by NK cells via CD16 but also anti‐tumor activity by neutrophils and macrophages via CD32 (FcγRII) is known [19, 21, 22]. Therefore, we examined the expression of Fcγ receptors on iNKT and iPS‐NKT cells and determined which Fcγ receptors contribute to cytotoxic activity against neuroblastoma cells. Unlike iNKT cells, iPS‐NKT cells express CD32 and CD64 (FcγRI) in addition to CD16 (Figure 3A,B). Fcγ receptor‐stimulated degranulation and cytokine production of iPS‐NKT cells were evaluated by CD107a assay and cytometric bead array. Degranulation of iPS‐NKT cells was significantly induced only when cells were stimulated with CD16 antibodies (p < 0.0001) (Figure 3C). Similarly, the production of IFN‐γ and cytotoxic molecules such as TNF and granzyme B significantly increased only by stimulation with CD16 antibodies (Figure 3D). To clarify the role of each Fcγ receptor on ADCC for neuroblastoma cells, we performed a cytotoxicity assay by blocking Fcγ receptors on iPS‐NKT cells. Only the inhibition of CD16 on iPS‐NKT cells significantly suppressed ch14.18‐mediated ADCC against IMR‐32 cells (p = 0.006); inhibition of CD32 or CD64 did not influence cytotoxicity (Figure 3E,F). While inhibition of CD16 suppressed the ADCC of iPS‐NKT cells, there was no correlation between the frequency of CD16 expression and the cytotoxicity of iPS‐NKT cells (Figure S2). These results suggest that ch14.18‐mediated ADCC of iPS‐NKT cells is mainly regulated by CD16 as in NK cells, but it may also be regulated by other factors in addition to CD16.

FIGURE 3.

FIGURE 3

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CD16 stimulation promotes iPS‐NKT cell degranulation and cytokine production. (A) CD32 and CD64 expression on the surface of iNKT and iPS‐NKT cells. Shaded histograms represent isotype controls. Data are from a representative experiment of independent experiments. (B) The data represents 5 lots of iNKT cells from 3 donors and 8 lots of iPS‐NKT cells from 2 donors used in this study. Data are represented as mean ± SD of each lot. (C) CD107a expression on iPS‐NKT cells after stimulation of Fcγ receptors. iPS‐NKT cells were cultured on 96‐well plates coated with CD16, CD32, or CD64 antibodies to stimulate the Fcγ receptors for 5 h, and CD107a expression was detected by flow cytometry. (D) Amounts of cytokines secreted from iPS‐NKT cells (1×105 cells) stimulated by Fcγ receptors were measured by cytometric bead array. (E and F) Cytotoxicity of iPS‐NKT cells with Fcγ receptor inhibition against IMR‐32 cells. iPS‐NKT cells were pretreated with blocking antibodies against CD16, CD32, or CD64 and cultured with IMR‐32 (4×104 cells) at various E: T ratios with or without ch14.18 for 4 h. The bar graph in F is part of the experimental data in E and compares the cytotoxicity of each group at an E: T ratio of 20: 1. The data on frequency, cytokine levels, and cytotoxicity are represented as mean ± SD of triplicate in one representative experiment (each experiment was repeated two to three times and at least one experiment with a different lot). The symbols used in this figure indicate the following donors: ◆, iNKT donor 1; ▲, iNKT donor 2; ▼, iNKT donor 3; ●, iPS‐NKT donor 1; ■, iPS‐NKT donor 2. Statistical analysis was performed using Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.4. Combination Therapy of ch14.18 and iPS‐NKT Cells Inhibits Tumor Growth In Vivo

To examine ch14.18‐mediated ADCC of iPS‐NKT cells against neuroblastoma in vivo, we evaluated the effects of the combination therapy of ch14.18 and iPS‐NKT cells using a subcutaneous neuroblastoma model in IL‐7×15 NSG mice. The experimental protocol is shown in Figure 4A. Luciferase‐transduced IMR‐32 (IMR‐32‐Luc) cells were subcutaneously injected into the right side back of mice. After subcutaneous tumors were confirmed by IVIS (day 6), mice were divided into four treatment groups: (i) control, (ii) ch14.18 monotherapy, (iii) iPS‐NKT cell monotherapy, or (iv) ch14.18 + iPS‐NKT cell combination therapy. Mice received the indicated treatment, and tumors were monitored every five days by IVIS. The results are shown in Figure 4B and Figure S3. On day 11, five days after the start of treatment, mice treated with ch14.18 + iPS‐NKT cell combination therapy showed a reduction in tumor volume compared with the other three groups of mice. On day 21, two weeks after treatment, the tumor volume in the combination therapy group was significantly smaller compared with the control group (p = 0.0005) and ch14.18 monotherapy group (p = 0.0017). Although not statistically significant, there was a trend toward decreased tumor volume in the combination therapy group compared with the iPS‐NKT cell monotherapy group (p = 0.0896). Mice treated with ch14.18 alone or iPS‐NKT cells alone also showed a reduction in tumor volume compared with mice in the control group, but the difference was not statistically significant. These results indicate that iPS‐NKT cells show ch14.18‐mediated ADCC against neuroblastoma in vivo.

FIGURE 4.

FIGURE 4

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Combination therapy of ch14.18 and iPS‐NKT cells inhibits tumor growth in vivo. (A) Schematic of the experimental protocol. IL7 × 15 NSG mice were inoculated with IMR‐32‐Luc (5×106 cells) on the right side of the back. Five days after tumor cell inoculation, we confirmed tumor growth. Mice were divided into four groups: (i) control, (ii) ch14.18 monotherapy, (iii) iPS‐NKT cell monotherapy, or (iv) ch14.18 + iPS‐NKT cell combination therapy. Mice received the indicated treatments on days 6 and 7 after tumor cell inoculation as described in Methods. The amounts of tumor cells were quantified every five days by IVIS. (B) Total flux of subcutaneous tumors in each treatment group are shown. Values of total flux were log10 transformed. Data are from a representative experiment of two independent experiments. Data are shown as mean + SD. Statistical analysis was performed using Student's t‐test and one‐way analysis of variance followed by Turkey's multiple comparison test. ***p < 0.001, i.t., intratumoral injection; IVIS, in vivo imaging system; ns, non‐significant; s.c., subcutaneous injection. Figure 4A was created with BioRender.

3.5. The Difference in the Effects of iNKT Cells and iPS‐NKT Cells In Vivo

To compare the therapeutic effects and side effects of ch14.18 with iNKT cells or iPS‐NKT cells, we conducted an experiment using the protocol shown in Figure 5A. Two out of six mice in the group treated with iNKT cells and ch14.18 showed weight loss and poor grooming within four weeks of treatment initiation. These two mice were euthanized when their body weight decreased to less than 20% of their pre‐treatment weight. Flow cytometry analysis showed that the splenocytes of these two mice had been almost completely replaced by human T cells, not iNKT cells. These observations suggested the development of graft‐versus‐host disease (GVHD) and were excluded from the analysis. None of the mice in the groups treated with ch14.18 alone or iPS‐NKT cells with ch14.18 developed GVHD. IVIS imaging and tumor volume measurements showed that tumor growth was significantly suppressed in mice treated with iPS‐NKT cells with ch14.18 compared to mice treated with iNKT cells with ch14.18 (Figure 5B,C and Figure S4). One of the six mice in the group treated with iNKT cells and ch14.18 did not develop GVHD but died of unknown causes before the tumor reached 20 mm in diameter. In three out of six mice treated with iPS‐NKT cells and ch14.18, tumor growth was suppressed even in the long term (Figure 5D). To analyze iNKT cells and iPS‐NKT cells in the TME, iNKT cells or iPS‐NKT cells were injected together with ch14.18 into palpable subcutaneous tumors, and after one day, the tumors were resected and iNKT cells or iPS‐NKT cells were isolated (Figure 5E). In the TME, iPS‐NKT cells showed increased expression of CD69, an activation marker, compared to before injection. In contrast, iNKT cells showed decreased expression of CD69 in the TME (Figure 5F,G).

FIGURE 5.

FIGURE 5

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The differences in the effects of iNKT cells and iPS‐NKT cells in vivo. (A) Schematic of the experimental protocol. IL7 × 15 NSG mice were inoculated with IMR‐32‐Luc (5×106 cells) on the right side of the back. Four days after tumor cell inoculation, tumor growth was confirmed. Mice were divided into four groups: (i) ch14.18 monotherapy, (ii) ch14.18 + iNKT cell combination therapy, and (iii) ch14.18 + iPS‐NKT cell combination therapy. Mice received the indicated treatments on days 5 and 6 after tumor cell inoculation as described in Methods. The amounts of tumor cells were quantified every six days by IVIS. (B) Total flux of subcutaneous tumors in each treatment group are shown. Total flux values were log10 transformed. Data are shown as mean + SD. (C) Growth curve of subcutaneous tumor volume up to 77 days after tumor inoculation. Tumor diameter measurements were taken once a week after the tumor became palpable. The tumor volume on day 77, the day when the first mouse with a tumor diameter greater than 20 mm appeared, was compared between the groups. (D) Kaplan–Meier curve showing the time until the tumor diameter reached 20 mm. A mouse in the group treated with iNKT cells and ch14.18 that died before the tumor reached 20 mm in diameter, is plotted as censored on day 56. (E) Schematic of the experimental protocol to analyze iNKT cells and iPS‐NKT cells in the TME. (F) CD69 expression on the surface of iNKT and iPS‐NKT cells. Representative data from one of three mice are shown. (G) Expression levels of CD69 on iNKT cells and iPS‐NKT cells before tumor injection and after isolation from the TME were compared using MFI. The delta (△) MFI was calculated by subtracting the MFI of each cell before injection from the MFI of corresponding cells in the TME. Data are shown as mean ± SD. Statistical analysis was performed using Student's t‐test and one‐way analysis of variance followed by Tukey's multiple comparison test. **p < 0.01, ***p < 0.001, i.t., intratumoral injection; IVIS, in vivo imaging system; MFI, mean fluorescence intensity; ns, non‐significant; s.c., subcutaneous injection; TME, tumor microenvironment. Figure 5A,E was created with BioRender.

4. Discussion

In this study, we found that iPS‐NKT cells exhibit anti‐GD2 mAb‐mediated ADCC against neuroblastoma in vitro and in vivo. Anti‐GD2 mAb binds the GD2 antigen on the neuroblastoma cell surface to the CD16 on the iPS‐NKT cell, inducing iPS‐NKT cell degranulation, cytokine production, and cytotoxicity against neuroblastoma. In our experiments in a mouse model of neuroblastoma, the injection of iPS‐NKT cells and ch14.18 into the tumor site resulted in a reduction in tumor size and inhibition of tumor growth.

Anti‐GD2 antibodies originating from 14.18 [3] and 3F8 [4] have been developed and studied as therapeutic agents against neuroblastoma. The murine IgG3 monoclonal antibody 14.18 was class switched to mouse IgG2a (14.G2a) and chimerized with human IgG1 (ch14.18, dinutuximab) to enhance ADCC activity [22]. Ch14.18 has been shown to have 50‐ to 100‐fold ADCC activity against melanoma compared with 14.G2a [15]. Similarly, we found that ch14.18 potently induced ADCC from iPS‐NKT cells compared with 14.G2a. Humanized 3F8 (naxitamab) is another anti‐GD2 mAb that is a modified murine 3F8 [23], and the combination with GM‐CSF exhibited efficacy in refractory or relapsed neuroblastoma with bone or bone marrow involvement in a phase 1/2 trial [24, 25]. Another humanized anti‐GD2 mAb with K322A point mutation, hu14.18K322A, was developed to increase ADCC by lowering fucosylation and removing complement‐mediated cytotoxicity to reduce the adverse effect of pain [26]. While only 14.G2a and ch14.18 were examined in this study, other new anti‐GD2 mAbs also exert ADCC mainly through CD16 [22], so iPS‐NKT cells may also exhibit ADCC via new anti‐GD2 mAbs. The reason for the variability in the frequency of CD16 expression in iPS‐NKT cells remains unclear. Preliminary data suggest that CD16 expression in iPS‐NKT cells begins to emerge around day 30 of the generation process. Since CD16 expression tends to be consistent within the same lot, it is plausible that variations in the intrinsic conditions of the cells during the differentiation induction process may influence CD16 expression.

We speculated that iPS‐NKT cells not only exhibit ADCC themselves but may also enhance the ADCC of NK cells. Yamada et al. reported that iPS‐NKT cells have an adjuvant effect on NK cells [13]. Mise et al. reported that activated iNKT cells enhance the anti‐GD2 mAb–mediated ADCC of NK cells against neuroblastoma in vitro [11]. In our experiments, we observed a trend toward increased secretion of IFN‐γ, TNF, and granzyme B by iNKT cells in the presence of anti‐GD2 mAb, although no enhancement of cytotoxic activity was observed (Figure 1C,E). The reason for the increased secretion of cytokines and cytotoxic factors in iNKT cells, which do not express CD16, remains unclear. It is possible that this is attributable to an unidentified function of iNKT cells or it could result from the adjuvant effect of iNKT cells on NK cells that were mixed in during the purification process of iNKT cells.

As reported in other malignancies, clinical trials of chimeric antigen receptor (CAR) engineering‐based immunotherapy as a novel treatment for neuroblastoma have been conducted. Several trials of CAR‐T cells with CARs directed at GD2 have been reported, but none have demonstrated objective efficacy [27, 28, 29]. Recently, GD2‐CART01, autologous, third‐generation GD2‐CAR T cells expressing the inducible caspase 9 suicide gene, showed safety and efficacy against high‐risk neuroblastoma in a phase 1/2 clinical trial (NCT03373097) [30]. In the trial, 27 children with heavily pretreated neuroblastoma were enrolled and received GD2‐CART01. Cytokine release syndrome occurred in 20 of the 27 patients and was mild in 19 of 20. Among the 27 patients, 17 patients had a response to the treatment: 9 patients had a complete response (CR) and 8 had a partial response (PR). Heczey et al. demonstrated the anti‐tumor activity and safety of autologous NKTs co‐expressing a GD2‐specific CAR with IL‐15 (GD2‐CAR.15 NKTs). In a phase 1 trial (NCT03294954), 12 children with neuroblastoma received GD2‐CAR.15 NKTs; no patients had dose‐limiting toxicities and three patients experienced a therapeutic effect, including two PR and one CR [31]. While CAR‐T and CAR‐NKT cells were generated without problems in most patients in the above clinical trials, there is a risk that a sufficient number of cells may not be obtained from patients for treatment because cells are generated from patients' blood. iPS‐NKT cells can be abundantly and uniformly generated by iPSC technology, thus resolving the challenge of patient dependence. Notably, iPS‐NKT cells exert anti‐tumor effects via mechanisms distinct from CAR‐T cell therapy. CAR‐T cells are exclusively targeted to cancers that express particular antigens. Different from CAR‐T cells, iPS‐NKT cells can exert indirect cytotoxicity against a wide range of cancers by inducing antigen‐specific cytotoxic T cells for unknown antigens and also by activating NK cells for cancers lacking the expression of HLA [32]. The first‐in‐human clinical trial using the GMP‐grade iPS‐NKT cells for patients with advanced head and neck cancer is currently ongoing. If the safety and efficacy of iPS‐NKT cells are demonstrated in this trial, we speculate that iPS‐NKT cell therapy combined with anti‐GD2 mAb may be a potential therapeutic option for neuroblastoma in the future.

In conclusion, we found that iPS‐NKT cells exert ADCC via anti‐GD2 mAbs against neuroblastoma in vitro and in vivo. Similar to NK cells, iPS‐NKT cells exhibit cytotoxic activity through CD16‐mediated degranulation and cytokine production. These findings suggest that iPS‐NKT cells can be applied to anti‐GD2 mAb therapy against neuroblastoma as effector cells in the future.

Author Contributions

Katsuhiro Nishimura: conceptualization, investigation, writing – original draft. Takahiro Aoki: conceptualization, investigation, methodology, writing – review and editing. Midori Kobayashi: investigation. Mariko Takami: methodology. Ko Ozaki: investigation. Keita Ogawa: investigation. Wang Hongxuan: investigation. Daiki Shimizu: investigation. Daisuke Katsumi: investigation. Hiroko Yoshizawa: investigation. Shugo Komatsu: investigation. Tomozumi Takatani: resources. Kiyoshi Hirahara: supervision. Haruhiko Koseki: resources. Tomoro Hishiki: supervision. Shinichiro Motohashi: methodology, supervision, writing – review and editing.

Ethics Statement

Approval of the research protocol by an Institutional Reviewer Board: The study design was approved by the Research Ethics Committee of the Graduate School of Medicine, Chiba University.

Consent

Written informed consent was obtained from all human participants in this study.

Conflicts of Interest

Haruhiko Koseki received research funding support from BrightPath Biotherapeutics Co. Ltd. Other authors declare no conflicts of interest.

Animal Studies

The Chiba University Institutional Animal Care and Use Committee approved all animal procedures.

Supporting information

Figure S1. Representative FACS data showing the gating strategy for an Annexin V assay.

Figure S2. Correlation between the frequency of CD16 expression on iPS‐NKT cells and cytotoxicity.

Figure S3. IVIS imaging in therapeutic experiments using ch14.18 and iPS‐NKT cells.

Figure S4. IVIS imaging in therapeutic experiments using the ch14.18 antibody in combination with iNKT cells or iPS‐NKT cells.

CAS-116-884-s001.pdf (1.2MB, pdf)

Acknowledgments

We thank Gabrielle White Wolf, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. Some figures were created using BioRender (https://www.biorender.com/) with written permission from the copyright owners to reproduce the material.

Funding: This work was supported by Kashiwado Memorial Foundation for Medical Research. Japan Society for the Promotion of Science, 23K07306.

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Associated Data

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

Supplementary Materials

Figure S1. Representative FACS data showing the gating strategy for an Annexin V assay.

Figure S2. Correlation between the frequency of CD16 expression on iPS‐NKT cells and cytotoxicity.

Figure S3. IVIS imaging in therapeutic experiments using ch14.18 and iPS‐NKT cells.

Figure S4. IVIS imaging in therapeutic experiments using the ch14.18 antibody in combination with iNKT cells or iPS‐NKT cells.

CAS-116-884-s001.pdf (1.2MB, pdf)

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