An innovative treatment for lung cancer using gene-engineered human-induced pluripotent stem cell- derived natural killer cells

An innovative treatment for lung cancer using gene-engineered human-induced pluripotent stem cell- derived natural killer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An innovative treatment for lung cancer using gene-engineered human-induced pluripotent stem cell- derived natural killer cells Yuka Sato, Kumiko Goto, Shigehiro Yagishita, Kotoko Miyata, Noriko Uesugi, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8246785/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 10 You are reading this latest preprint version Abstract Various therapeutic approaches have been developed for lung cancer, including chemotherapy, radiation therapy, and immune checkpoint inhibitors. However, none of these approaches, including chimeric antigen receptor (CAR)-T cell therapy, have been effective against solid tumors. To enhance the therapeutic effect, we focused on the multiple effects of a new modality of cell therapy and created engineered natural killer (eNK) cells, which are gene-engineered induced pluripotential stem cell (iPSC)-derived NK cells armed with CC motif ligand 19 (CCL19), CC chemokine receptor type 2B (CCR2B), high-affinity cluster of differentiation 16 (CD16), interleukin (IL)-15, and natural killer group 2, member D (NKG2D)-DNAX-activating protein 10 (DAP10) complex. In vitro studies showed that eNK cells showed significant long-lasting cytotoxicity and antibody dependent cell-mediated cytotoxicity (ADCC) against human lung cancer cell lines. Intravenous and intra-tumoral treatment with eNK cells almost completely inhibited tumor growth in both orthotopically and subcutaneously transplanted cell line-derived xenograft (CDX) models, respectively. Moreover, intra-tumoral eNK cells administered as a single dose or in combination with cetuximab in the patient-derived xenograft (PDX) model of lung cancer showed clear tumor growth inhibition. This study demonstrates that eNK cells exhibit significant antitumor effects in both CDX and PDX models, and that these effects are further enhanced via ADCC. We successfully demonstrated that eNK cells having the ability to migrate to tumor cells and become activated in the tumor microenvironment and having the ability to induce tumor-specific killing activity without targeting CAR, are an innovative mode of treating lung cancer in pre-clinical studies. iPSC (induced pluripotent stem cell) NK (natural killer) cells Genetic engineering Cell therapy Lung cancer PDX (Patient-derived xenograft) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction There has been remarkable progress in the research and development of innovative therapeutic methods such as immune checkpoint inhibitors, cell therapy, gene therapy, and nucleic acid medicine therapy. However, none of these therapies address the challenges of treating refractory solid cancers, and more promising therapeutic methods are needed. Regarding innovative cell therapies, various CAR-T therapies have been investigated in non-clinical and clinical trials targeting solid tumors 1,2) ; however, no breakthrough effects have been achieved and their efficacy against solid tumors is still limited. On the other hand, NK cells are attracting attention because they attack targets in a non-HLA-restricted manner, have the advantage of not causing graft-versus-host disease (GVHD), and are safer than T cells. 3) Our concept is to create groundbreaking cell therapies that could universally recognize cancer cells and kill them directly, rather than targeting limited numbers of targets with CARs for highly heterogeneous solid tumors. Engineered natural killer (eNK) cells have the potential to be highly effective in treating various refractory solid cancers. In this report, we present our research into lung cancer treatment, which is particularly useful. Lung cancer is the leading cause of cancer-related deaths worldwide and consistently ranks high when it comes to annual mortality rates for both men and women. Lung cancer is often diagnosed when it is in an advanced stage. Although the number of treatment options, including chemotherapy drugs, immune checkpoint inhibitors, and antibodies is increasing, almost no effective therapy options are available for patients with advanced disease. On the other hand, it has been reported that intratumoral injection into solid tumors is a promising strategy for enhancing the efficacy of cancer treatment and avoiding systemic adverse effects. 4, 5) First, because there have been few successful cases of systemic administration of cell therapy, we investigated whether eNK cells can exert an anti-solid tumor effect by injecting them directly into the tumor mass. We evaluated this strategy using cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) and obtained results that are expected to be highly accurate for clinical application. We hypothesized that cell therapy-specific organ-dependent distribution would be the same whether the cells are administered intravenously or directly. As hypothesized, eNK cells were remarkably effective in lung cancer treatment, and we succeeded in inducing an innovative antitumor effect. The in vivo persistence and antitumor effects of eNK cells can be enhanced by gene transfer and further enhanced by combining gene transfer with antibodies that exert an ADCC mediated by transfected CD16. The interaction of cancer cells with immune cells in the tumor microenvironment (TME) is a crucial factor in cancer immunotherapy. To enhance the homing and infiltration of immune cells into the TME, we transfected CC motif ligand 19 (CCL19) 6,7) to recruit patient immune cells to tumor tissue, and CC chemokine receptor type 2B (CCR2B) 8, 9) to enable eNK cells to migrate toward CCL2-expressing cancer cells. For immunological cells to be effective, they needed to have strong cytotoxic activity to cancer cells and persist in the tumor and surrounding tissues. Because long-term persistence of CAR-T cells and in vivo expansion of CAR-T cell number are associated with a better clinical response, 10) we transfected interleukin ( IL ) 15 gene to achieve suitable lymphocyte persistence and activation levels, 11) and natural killer group 2, member D ( NKG2D ) and DNAX-activating protein 10 ( DAP10 ) genes to improve cancer cell detectability. Moreover, in the case of NK cells, antibody-dependent cellular cytotoxicity (ADCC) activity can be effectively utilized, so we attempted to enhance the expression of cluster of differentiation (CD)16 and increase the value of our therapy by combining it with antibodies. 12) As a result of these creative hypotheses, we developed eNK cells, which are gene-engineered human induced pluripotent stem cell (hiPSC)-derived NK cells armed with NKG2D, IL-15, CD16, CCL19, and CCR2B molecules (Fig. 1 ). 13) Materials and Methods Cell lines and cell culture The lung cancer cell lines used were A549, NCI-H1975-Luc (luciferase expressing NCI-H1975), NCI-H460-Luc (luciferase expressing NCI-H460), Lu99, NCI-H520, and SBC-3. A549-GFP and NCI-H1975-Luc-GFP (H1975-GFP) were established by transfection of EF1α-GFP-IRES-Puro or Hygro-hGHpA vector using the piggyBac transposon system into each cell type and subsequent cloning. A549 and NCI-H520 were purchased from American Type Culture Collection. NCI-H1975-Luc, NCI-H460-Luc, Lu99, and SBC-3 were purchased from National Institute of Biomedical Innovation, Health, and Nutrition (Osaka, Japan). These cells were grown in monolayer culture in Ham’s F-12K (Kaighn’s) medium (A549 and A549-GFP), RPMI 1640 (NCI-H1975-Luc, NCI-H1975-GFP, NCI-H460-Luc, Lu99, NCI-H520), MEM (SBC-3) supplemented with 10% fetal bovine serum (FBS) (Biosera, Cholet, France), in a humidified incubator with at 37°C with 5% CO 2 . J-PDX_E0050 tumor was provided by the National Cancer Center J-PDX Library, Japan (Tokyo, Japan) and maintained by subcutaneous transplantation in NOD/Shi-scid IL-2R gamma (null) (NOG) mice. Animals NOG mice 14) were purchased from In-Vivo Science Inc. (Kawasaki, Japan) and maintained under specific pathogen-free conditions with ad libitum access to water and food. CDX model experiments were performed in accordance with the relevant institutional and national guidelines and regulations, and were approved by the Institutional Animal Care and Use Committee of HEALIOS K.K. PDX model experiments were conducted at Mediford Corporation (Tokyo, Japan)(formerly LSI Medience Corporation, Tokyo, Japan). The experiments were approved by the Ethics Committee in accordance with the "Ethics Review Regulations for Research, etc. Involving the Handling of Human Samples and Human Information", by the Safety Committee in accordance with the "Regulations of the Genetically Modified Organisms Safety Committee (Kumamoto)", and by the Animal Experimentation Committee and the head of the institution in accordance with the "Guidelines for Animal Experiments". The use of human-derived PDX was carried out in accordance with the "Ethical Guidelines for Medical Research Involving Human Subjects." Lactate dehydrogenase release assay Lung cancer cells were seeded at 3.75 × 10 4 cells/well in a 96-well plate and incubated overnight. eNK cells were co-cultured with tumor cells at an effector/target (E/T) ratio ranging from 0.3 to 10 for 4 h at 37℃ and 5% CO 2 . After co-culture with eNK cells, supernatants were collected, and lactate dehydrogenase (LDH) release was measured using an LDH cytotoxicity assay kit (Dojindo Molecular Technology, Kumamoto, Japan). Cytotoxicity (% of lysis) was calculated by using the following formula. For determining maximal LDH release, cell lysis was induced by 1% Triton X-100 solution. $$\:\text{C}\text{y}\text{t}\text{o}\text{t}\text{o}\text{x}\text{i}\text{c}\text{i}\text{t}\text{y}\:\left(\text{%}\:\text{o}\text{f}\:\text{l}\text{y}\text{s}\text{i}\text{s}\right)=\frac{(\text{E}\text{x}\text{p}\text{e}\text{r}\text{i}\text{m}\text{e}\text{n}\text{t}\text{a}\text{l}\:\text{L}\text{D}\text{H}\:\text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\:－\:\text{S}\text{p}\text{o}\text{n}\text{t}\text{a}\text{n}\text{e}\text{o}\text{u}\text{s}\:\text{L}\text{D}\text{H}\:\text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\:\text{o}\text{f}\:\text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\text{o}\text{r}\:\text{c}\text{e}\text{l}\text{l}\text{s})}{(\text{M}\text{a}\text{x}\text{i}\text{m}\text{u}\text{m}\:\text{L}\text{D}\text{H}\:\text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\:－\:\text{S}\text{p}\text{o}\text{n}\text{t}\text{a}\text{n}\text{e}\text{o}\text{u}\text{s}\:\text{L}\text{D}\text{H}\:\text{r}\text{e}\text{l}\text{e}\text{a}\text{s}\text{e}\:\text{o}\text{f}\:\text{t}\text{a}\text{r}\text{g}\text{e}\text{t}\:\text{c}\text{e}\text{l}\text{l}\text{s})}\times\:100$$ Incucyte cytotoxicity assay To generate tumor spheroids, A549 and H1975 cells stably expressing green fluorescent protein (GFP) were seeded at 1.0 × 10 4 cells/well in a 96-well round-bottom plate (Corning, NY, USA) in Ham’s F-12K (Kaighn’s) medium (A549-GFP) or RPMI 1640 (H1975-GFP) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Biosera). After centrifugation for 10 min at 130 × g, plates were cultured in an incubator at 37℃ and 5% CO 2 for 4 days. Thirty thousand eNK cells were added to the wells containing one tumor spheroid in AIM-V medium (Thermo Fisher Scientific) supplemented with 5% FBS (Merck, Darmstadt, Germany), 50 ng/mL stem cell factor (SCF) (PeproTech, Cranbury, NJ, USA), and 50 ng/mL IL-15 (PeproTech) in the absence or presence of 2.5 µg/mL cetuximab (Cmab; Merck) and 3 µg/mL necitumumab (Nmab; Nippon Kayaku Co., Ltd., Tokyo, Japan). Cytotoxicity was monitored at 120 min intervals using the Incucyte SX5 imaging system (Sartorius, Göttingen, Germany). In vivo persistence assay A total of 5.0× 10 6 eNK cells were intravenously administered to NOG mice. Peripheral blood, lung, liver, and spleen samples were collected on 1, 7, 14, and 28 days after administration. The genomic DNA (gDNA) was extracted from the peripheral blood and tissues using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The DNA content of eNK cells per gDNA content in each sample was determined by qPCR using an Alu sequence-specific primer set. 15) On the final day, mice were anesthetized by inhalation of vaporized isoflurane and euthanized by exsanguination from the inferior vena cava. In vivo antitumor activity assay NOG mice were injected intravenously or subcutaneously with H1975-Luc or A549-Luc cells. However, any models in which cancer cells engrafted in organs other than the lungs were excluded from the study. hIL2 (PeproTech) and hIL15 were intraperitoneally injected to encourage or enhance persistence of eNK cells. An orthotopic lung cancer model was established by intravenous injection of tumor cells. Tumor progression was assessed by bioluminescence imaging using the IVIS Spectrum system (PerkinElmer, Shelton, CT, USA). For subcutaneous tumors, the length (L), width (W), and height (H) were measured using calipers. Tumor volume (mm 3 ) was calculated using the following formula: L × W × H × π/6. For histopathological analysis, tumors were collected from tumor-bearing mice in the satellite group that did not receive eNK cells treatment. Tissue collection was performed on the first day of eNK cells administration, following euthanasia under anesthesia and exsanguination. H&E staining and Ki67 immunostaining using anti-Ki-67 antibody (clone: MIB-1, Dako) were performed at Sapporo General Pathology Laboratory Co., Ltd. (Sapporo, Japan). PDX model The experiments were conducted at Mediford Corporation in research facilities that had obtained AAALAC International Certification (Tokyo, Japan). Human lung cancer PDX lines provided by the National Cancer Center Japan (Tokyo, Japan) were subcutaneously implanted into NOG mice. eNK cells and Cmab were administered three times a week for three weeks intratumorally and intraperitoneally respectively. hIL2 was intraperitoneally injected to encourage or enhance persistence of eNK cells. The tumor size was measured using a caliper and the estimated tumor volume was calculated using the following formula. Tumor volume (mm 3 ) = W (mm) × L (mm) × L (mm) × 1/2. Statistical analysis All data are presented as the mean ± standard deviation (SD). Dunnett's multiple comparison test was performed as a statistical significance examination. Results The cytotoxicity of eNK cells and the contribution of the gene-engineering of CD16 in vitro First, we evaluated the cytotoxicity of eNK cells against A549 (wild-type epidermal growth factor receptor [EGFR]), NCI-H1975-Luc (mutant EGFR; L858R, T790M), NCI-H460-Luc (PIK3CA mutant), LU99 (wild-type EGFR), NCI-H520 (wild-type EGFR), and SBC-3 using the LDH release assay. eNK cells showed dose dependent cytotoxicity in all lung cancer cell lines (Fig. 2 ). Next, we evaluated the sustained cytotoxicity of eNK cells using the Incucyte cytotoxicity assay. A549-GFP and NCI-H1975-GFP cell spheroids were respectively incubated with eNK cells in E/T ratios ranging from 0.3 to 10 and monitored for cell death by measuring GFP fluorescence intensity using the Incucyte live-cell imaging system. eNK cells were clearly cytotoxic for A549-GFP cells at an E/T ratio of 3 and inhibited their growth at an E/T ratio of 1. Against NCI-H1975-GFP cells, eNK cells were cytotoxic at an E/T ratio of 0.3 or more. Moreover, eNK cells retained cytotoxic activity until 120 h after the treatment (Fig. 3 a, b). We evaluated the ability of eNK cells to induce ADCC against a wild type-EGFR-positive tumor cell line (A549) via Cmab 16) and Nmab 17) binding to FcgRIII (CD16a). A549-GFP spheroids were incubated with eNK cells and Cmab or Nmab (Fig. 3 c, d). Cmab and Nmab alone were not cytotoxic, however the addition of these antibodies further enhanced the cytotoxicity ofeNK cells. The antitumor effects of intratumoral eNK cell administration on the subcutaneously transplanted lung cancer cell line, H1975-Luc We examined whether the intratumoral route of injection affects the antitumor effect of eNK cells on subcutaneously transplanted H1975-Luc, because it had been reported that intratumoral injection into solid tumors was a promising strategy for enhancing cancer treatment efficacy. 4, 5) To examine whether eNK cells could inhibit tumor growth, H1975-Luc cells were transplanted subcutaneously in NOG mice (day 0), and eNK cells were administered intratumorally three times a week for two weeks starting on day 14 after tumor transplantation. The results clearly showed that eNK cells dose-dependently suppress tumor growth (Fig. 4 ). The effects of eNK cells in the PDX mouse model derived from a lung cancer patient To improve the predictability of tumor growth inhibition by eNK cells in clinical applications, we examined the effects of eNK cells in a high-predictive-value PDX mouse model. The patient who provided the J-PDX_E0050 tumor used in the PDX mouse model (Fig. 5 a, non-small cell lung cancer, adenocarcinoma) was resistant to chemotherapy and radiotherapy. J-PDX_E0050 clearly expressed EGFR in histological examination (Fig. 5 b). eNK cells at doses of 3 × 10 6 and 1 × 10 7 cells/mouse were intratumorally administered three times a week for 3 weeks to NOG mice bearing subcutaneous PDX tumors. eNK cells at a dose of 1 × 10 7 cells/mouse reduced tumor volume by 28% on day 35 after the first administration (Fig. 5 c). Next, the combined effect of eNK cells and Cmab was examined based on ADCC. Injection of eNK cells at a dose of 1 × 10 7 cells/mouse and Cmab at a dose of 0.5 mg/body into mice bearing PDX tumors resulted in 18% and 36% inhibition of tumor growth on day 35, respectively. eNK cells combined with Cmab resulted in 53% inhibition, the enhancement of the inhibitory effect having been attributed to ADCC (Fig. 5 d). These results suggested that eNK cells would be effective both as a single agent or in combination with antibody in clinical practice. Persistence of eNK cells in normal NOG mice eNK cells were administered intravenously to normal NOG mice and its pharmacokinetics were investigated. eNK cells were injected intravenously at a dose of 5 × 10 6 cells in male and female NOG mice. Human-specific Alu sequence analysis using qPCR was conducted in gDNA extracted from blood and organs collected on day 1, 7, 14, and 28 after injection. The blood concentration of human-specific Alu sequences decreased 1 to 7 days after administration and then increased on days 14 and 28 after administration (Fig. 6 a). The time-dependent patterns of changes in concentration in the lungs, liver, and spleen were similar to those in the blood (Fig. 6 b, c, d). In addition, eNK cells were most abundantly distributed in the lungs among the organs evaluated, suggesting that they are efficiently distributed to the sites of lung cancer involvement and exhibit antitumor effects. Anti-tumor effects of eNK cells against the human lung cancer cell line, H1975-Luc, intravenously injected into an orthotopically transplanted mouse model of lung cancer As shown in Figs. 4 and 5 , eNK cells showed clear antitumor effect when injected intratumorally into subcutaneously transplanted tumors. In contrast, systemic administration of eNK cells resulted in substantial accumulation in the lungs. Notably, this pulmonary accumulation resembled the localized concentration observed following intratumoral injection. Based on this observation, eNK cells were systemically administered to a lung orthotopic transplantation model to evaluate their antitumor efficacy. NOG mice were intravenously injected with the H1975-Luc human lung cancer cell line. eNK cells were then intravenously at doses of 1 × 10⁶, 3 × 10⁶, and 1 × 10⁷ cells per mouse, three times per week from day 14 to day 25. Seven days after the final administration, eNK cells significantly inhibited tumor growth. At doses of 1 × 10 7 and 3 × 10 6 cells /mouse, bioluminescence signals returned to baseline and matched those observed in the IVIS control group without tumor transplantation, suggesting near-complete tumor regression (Fig. 7 a). Tumor regression was clearly visualized by IVIS imaging on 32, 7 days after the last eNK cell administration (Fig. 7 b). Histological analysis on day 14 confirmed that H1975-Luc tumor cells were successfully engrafted and distributed within the lung tissue (Fig. 7 c). These results strongly suggest that eNK cells possess robust therapeutic efficacy, even when used as monotherapy. Enhancement of the anti-tumor effect of eNK cells by necitumumab-mediated ADCC in A549-Luc lung tumor orthotopic graft bearing mice We evaluated whether the antitumor effect of eNK cells could be enhanced via ADCC activity in tumor bearing model mice with wild-type EGFR-positive A549-Luc cells. eNK cells exhibited a clear dose-dependent tumor growth inhibition in A549-Luc tumor-bearing mice, similar to the effect observed in H1975-Luc tumor-bearing mice. NOG mice were intravenously injected with A549-Luc tumor cells on day 0. eNK cells were administered intravenously at a dose of 5 × 10 6 cells/mouse, three times per week for 2 weeks starting on day 4. This dose was selected as a partially effective dose based on prior evaluations. Nmab was administered intraperitoneally at a dose of 0.08 mg/body, twice weekly for 2 weeks starting on day 4. Both eNK cells and Nmab inhibited tumor growth, and their combination resulted in more pronounced antitumor effect than that either treatment alone. These results suggest that the potent antitumor effects of the combination of eNK cells and Nmab are likely mediated cytotoxicity (ADCC) (Fig. 8 ). Discussion Administration of antibodies against immune checkpoint molecules such as PD-1, PD-L1, and CTLA4 have been shown to inhibit the growth of, and have a therapeutic effect against, malignant advanced cancers such as advanced non-small cell lung cancer (NSCLC) and extend survival time. 18,19,20) However, only about 20% of lung cancers are sensitive to immune checkpoint inhibitor therapies, for example, PD-1/PD-L1 antibody therapy, 19) so to address this issue, development of new immunotherapies and novel treatments is needed for cancer patients. For immune checkpoint inhibitors to be effective, it is important that patients have tumor-reactive T cells. 20) However, the supply of T cells is often exhausted in cancer patients. 21,22) In addition, the more mutant the antigens in a tumor are, the higher is the probability of inducing tumor-specific T cells, 23) but if there are few mutant antigens, fewer tumor-specific T cells are induced and the effects of immune checkpoint molecules may be reduced. In a comparison of comprehensive genetic analysis of cancer cells and treatment outcomes, the frequency of highly antigenic mutant proteins in tumors was correlated with survival rates after PD-1/PD-L1 treatment. 24,25) The development of CAR-T cell therapy has led to the discovery that the inhibitory activity against tumor cells expressing specific antigens can be therapeutic, but the therapeutic effect on heterogeneous solid tumors is attributable to lack of persistence and exhaustion of CAR-T cells in the patient, as well as the limited ability of CAR-T cells to home in on target tumor sites. We have developed eNK cell therapy for solid tumors by generating gene-engineered iPSC-derived NK cells. 13) We believe that this is a promising concept for the treatment of solid tumors with heterogeneity, rather than an established one relying on the achievement of efficacy by CAR targeting. 26,27,28) We report the remarkable in vivo antitumor effects of eNK cells active against lung cancer in lung cancer models, including the PDX model, which has a high predictive value clinically, in this publication. As previously reported, 13) eNK cells, which are transfected with genes encoding NKG2D , DAP10 , CCL19 , CCR2B , CD16 , and IL-15 , have the following functions: infiltration and migration into cancer tissue and cancer cells, 7, 29) recruitment of the patient's own immune cells for activation of the immune system in the tumor microenvironment and enhancement of cytotoxic activity, 4, 5) , and maintenance of cell activation and persistence. 9, 30) Based on the in vivo distribution characteristics of cell therapy products, it is expected that systemically administered eNK cells will accumulate in the lungs. 31, 32, 33) The present results suggest that eNK cells have similar properties. In addition, it was found that the number of eNK cells decreased after reaching a peak at 24 h following intravenous administration. This reduced level was maintained for approximately one week. Subsequently the number of eNK cells increased, likely due to proliferation, and eventually returned to the level observed at 24 h post administration, where it remained for up to 28 days. The persistence of eNK concentrations is considered to contribute to adequate efficacy. The CDX model, in which intravenously administered cancer cells become engrafted in an animal’s lungs, 34) is an orthotopic lung cancer engraftment model. Intravenously administered eNK cells clearly inhibited tumor growth in this model. The remarkable efficacy of eNK cells administered via the clinical route in an orthotopic lung cancer model indicates their strong potential for clinical application. Since eNK cells were primarily localized in the lungs after intravenous administration, we believe that local intratumoral administration of eNK cells in subcutaneously transplanted PDX tumors can be used to predict the results of systemic administration. Therefore, the fact that eNK cells demonstrated antitumor effects in PDX models, which have high predictive value clinically, 35, 36) suggests their usefulness in clinical applications. Targeting EGFR is an important treatment modality for many solid tumors including NSCLC. Cmab and Nmab are monoclonal antibodies containing the constant region of human IgG1 with high affinity to EGFR and exhibiting ADCC activity, 14, 37, 38) It was reported that the addition of Cmab to chemotherapy in the first-line treatment improves overall survival of patients with EGFR-expressing advanced NSCLC. 39) But no clear combined effects 40) and toxicity concerns were reported despite the improvement in antitumor effects when Cmab and chemotherapy were combined. 41, 42) Moreover, retrospective analyses revealed that mutational status and copy number of EGFR were not predictive for cetuximab benefit. 43) Nmab also was unable to achieve sufficient efficacy when combined with chemotherapy because Nmab showed adverse effects in combination therapy. 38) Therefore, Cmab or Nmab and eNK cells are useful in clinical applications, since eNK cells show anti-tumor effects and the combination of eNK cells with Cmab and Nmab enhances these effects through ADCC activity. Currently, treatment options are limited, and in refractory cases of lung cancer, new treatment modalities, such as gene therapy and cell therapy, are actively developed. Because cell therapy is thought to be very useful for treating lung cancer based on cell dynamics studies showing that most cells are distributed in lung tissue, 44, 45) lung cancer was selected as the first solid tumor target for systemic treatment, and the results of evaluation have demonstrated the significant efficacy of systemic treatment. eNK cell therapy is a new concept. eNK cells are gene-transduced iPS cell-derived NK cells that improve immune function in the tumor microenvironment, immune cell homing and migration to tumor sites, and the distribution of immune cells in the targets of treatment (lung tissue and lung cancer lesions). Leveraging these properties, eNK cells have tumor damaging activity that is different from the antitumor activity engendered by CAR-T cells, and we believe that its antitumor effects on solid tumors and lung cancer are groundbreaking. In conclusion, eNK cell therapy is a novel and promising treatment option for refractory lung cancer. However, the antitumor effect of eNK cells alone is limited in preclinical models, so further evaluation is necessary to optimize therapeutic outcomes. Combination strategies with other antibody-based drugs or refined dosing schedules may be essential to maximize efficacy and ensure the translational potential of this approach. Declarations Competing Interests H.K. is Executive Officer of HEALIOS K.K.; K.T. is a former CSO of HEALIOS K.K.; F.N. is a former employee of HEALIOS K.K.; Y.S., K.G., K.M., N.U., Y.-s.T., Y.N., R.T., R.S., Y.T., K.K., M.Y., and Y.H. are current employees of HEALIOS K.K. Author Contribution Y.S. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing–original draft; K.G. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing – review & editing; S.Y. : Resources, Performed the experiments, Writing – review & editing, Funding acquisition, Supervision; K.M. , N.U. , Y.-s.T. and Y.N. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing – review & editing; R.T. and R.S. : Performed the experiments, Data curation, Data analysis; Y.T. and M.Y. : Methodology, Writing – review & editing, Project administration, Supervision; K. K. : Resources, Writing – review & editing, Project administration, Supervision; Y. H. and F. N. : Writing – review & editing, Project administration; H. K. : Conceptualization, Writing – review & editing, Project administration, Supervision; A. H. : Resources, Writing – review & editing, Funding acquisition, Supervision; K. T. : Conceptualization, Writing – review & editing, Supervision, Funding acquisition.All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank Oriental Bio Service, Kobe BM Laboratory (Kobe, Japan) for animal housing and care. We also thank Dr. Takashi Murakami (Saitama Medical University) for kindly providing the NCI-H1975-Luc and NCI-H460-Luc cell lines. PDX model experiments were conducted at Mediford Corporation (Tokyo, Japan) (formerly LSI Medience Corporation, Tokyo, Japan), and we are grateful for their technical support. Hematoxylin and eosin (H&E) staining and Ki-67 immunostaining were performed at Sapporo General Pathology Laboratory Co., Ltd. (Sapporo, Japan). We thank Sumisho Pharma International Co., Ltd. (Tokyo, Japan) for their support with IVIS imaging analysis. The National Cancer Center J-PDX library used in this study is partly supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number 17pc0101011h0001, and by the National Cancer Center Research and Development Fund, Japan. English proofreading services were provided by ASCA Corporation (Osaka, Japan). The authors would like to thank all HEALIOS lab members for their technical assistance and helpful discussions. References Albelda SM (2024) CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn. Nat Reviews Clin Oncol 21:47–66. doi.org/10.1038/s41571-023-00832-4 Uslu U, June CH (2024) Beyond the blood: expanding CAR T cell therapy to solid tumors. 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Stem Cells Dev 18(5):683–691. 10.1089/scd.2008.0253 Pichardo AH, Wilm B, Liptrott N, Murray P (2023) Intravenous Administration of Human Umbilical Cord Mesenchymal Stromal Cells Leads to an Inflammatory Response in the Lung. Stem Cells Int Article ID 7397819. doi.org/10.1155/2023/7397819 Additional Declarations Competing interest reported. H.K. is Executive Officer of HEALIOS K.K.; K.T. is a former CSO of HEALIOS K.K.; F.N. is a former employee of HEALIOS K.K.; Y.S., K.G., K.M., N.U., Y.-s.T., Y.N., R.T., R.S., Y.T., K.K., M.Y., and Y.H. are current employees of HEALIOS K.K. Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2026 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 15 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviews received at journal 07 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers invited by journal 04 Dec, 2025 Editor assigned by journal 03 Dec, 2025 Submission checks completed at journal 03 Dec, 2025 First submitted to journal 01 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:15:42","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155796,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/0350d89dae88cf3e5c6f47e9.html"},{"id":97766383,"identity":"57e2de7b-901e-4e82-89ea-7246964bf22b","added_by":"auto","created_at":"2025-12-09 07:15:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114398,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of the eNK cell.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/1dd11f5fb43b08baf89b36a7.jpg"},{"id":97896701,"identity":"58043315-ac29-44d4-b615-30dfdb93223f","added_by":"auto","created_at":"2025-12-10 15:36:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124918,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of eNK cells against human lung cancer cells.\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity of eNK cells against human lung cancer cell lines such as A549, NCI-H1975-Luc, NCI-H460-Luc, Lu99, NCI-H520, and SBC-3 were evaluated using an LDH release assay. Human lung cancer cells were plated. On the next day, eNK cells, which were cultured for 3 days after thawing for recovery from the frozen state, were added at various ratios (in triplicate). Media were collected after 4 h of A549 cell/eNK cell co-culture and the level of LDH release was measured. The horizontal axis shows the E/T ratio, that is, the eNK cell/cancer cell ratio.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/6f0e24d4d9eef8c4551c34f2.jpg"},{"id":97897203,"identity":"ec33820b-c91b-42b0-adf8-aa9e73f4bfb1","added_by":"auto","created_at":"2025-12-10 15:37:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":422568,"visible":true,"origin":"","legend":"\u003cp\u003eChange in eNK cell cytotoxicity against A549 and H1975 lung cancer spheroids as determined by live-cell analysis using the Incucyte® system\u003c/p\u003e\n\u003cp\u003eA549 (a, c, d) and H1975 (b) cells labeled with green fluorescent protein (GFP) were plated on 96-well ULA plates and cultured 4 days. eNK cells, which were frozen and cultured for 3 days after thawing, and cetuximab (Cmab, 2.5 μg/mL) (c) were added to the pre-incubated GFP-labeled tumor cells. (All determinations were carried out in triplicate.) The Incucyte® single spheroid assay was conducted for 5 days in the co-cultures containing eNK cells and Cmab. Cytotoxicity was defined as % GFP fluorescence intensity in the co-cultures against total GFP fluorescence intensity of A549 or H1975 single culture. E/T ratio was calculated using the initial number of A549 cells or H1975 cells before the formation of spheroids.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/0bc0506d7c3babbdf4fb493a.jpg"},{"id":97897792,"identity":"66856abb-ae16-4103-9036-90aa63c87063","added_by":"auto","created_at":"2025-12-10 15:38:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164039,"visible":true,"origin":"","legend":"\u003cp\u003eThe anti-tumor effects of eNK intratumoral treatment on H1975-Luc tumors that developed from subcutaneously transplanted H1975-Luc cells in mice\u003c/p\u003e\n\u003cp\u003eeNK cells at a dose of 3 × 10\u003csup\u003e5\u003c/sup\u003e, 1 × 10\u003csup\u003e6\u003c/sup\u003e, 3 × 10\u003csup\u003e6\u003c/sup\u003e, and 1 × 10\u003csup\u003e7\u003c/sup\u003e cells/mouse were intratumorally administered three times a week for 2 weeks from day 14 after NOG mice were subcutaneously injected with 5 × 10\u003csup\u003e5\u003c/sup\u003e H1975-Luc tumor cells (day 0). hIL2 and hIL15 were intraperitoneally injected to enhance the persistence of eNK cells. Mean ± SD, n=5 (a; n=4), Arrow: eNK cell administration, **; p\u0026lt;0.01, ***; p\u0026lt;0.001 vs Control (Vehicle) (Dunnett's\u0026nbsp;multiple comparison test)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/6b05bf2cfda6eac2f2322ed0.jpg"},{"id":97766387,"identity":"98e25c9a-40bc-4d6d-ae17-c6700f5ce39a","added_by":"auto","created_at":"2025-12-09 07:15:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":881189,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of eNK cells on the PDX mouse model derived from lung cancer patients\u003c/p\u003e\n\u003cp\u003e(a) Characterization of J-PDX_E0050. (b) Expression of epidermal growth factor receptor (EGFR) in J-PDX_E0050. (c) The tumor mass from PDX was maintained in mice, then cut into pieces (approximately 2-3 mm in size), and finally implated subcutaneously into NOG mice. When the tumors grew to approximately 200 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomized (day 0). Administration of eNK cells was started on day 0 (c). Administration of eNK cells and Cmab was started on day 0 and day 1, respectively (d). eNK cells and Cmab were administered three times a week for 3 weeks. hIL2 was intraperitoneally injected to enhance the persistence of eNK cells. Mean ± SD, n=5, ( ): number of animals, Red Arrow: eNK cell administration, Black Arrow: Cmab administration\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/b3faf9f9da4e32b376181bde.jpg"},{"id":97766390,"identity":"6d2d95db-f82a-448f-8e4d-c6053fc0033f","added_by":"auto","created_at":"2025-12-09 07:15:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":132934,"visible":true,"origin":"","legend":"\u003cp\u003ePersistence of eNK cells in normal NOG mice\u003c/p\u003e\n\u003cp\u003eeNK cells at a dose of 5 × 10\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;cells were injected intravenously into male and female NOG mice. The DNA content of eNK cells extracted from blood (a) and organs (b, c, d) collected on day 1, 7, 14, and 28 after injection was determined by a human-specific Alu sequence qPCR assay. Lower limit of quantification (LLOQ): 0.12 pg DNA of eNK cells/10 or 100 ng gDNA, n=3\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/edd80a331051f2f6546d80c6.jpg"},{"id":97896772,"identity":"4f4d5232-1bc0-4e45-9d32-8da0e557e293","added_by":"auto","created_at":"2025-12-10 15:37:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":570365,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effects of eNK cells in H1975-Luc lung tumor orthotopic graft bearing mice\u003c/p\u003e\n\u003cp\u003eeNK cells were intravenously administered three times a week for 2 weeks from day 14 after NOG mice intravenously transplanted with H1975-Luc tumor (3 × 10\u003csup\u003e5\u003c/sup\u003e cells) (day 0). H1975-Luc tumor growth was measured by IVIS fluorescence intensity (a,b). At the start of administration 2 weeks after H1975-Luc transplatation, it was confirmed that lung cancer cells had taken root in the lungs, and therapeutic administration was carried out (c). The upper photo shows H\u0026amp;E stained cells, the lower photo shows Ki67 immunostained cells. The scale bar in the photographs indicates 100 µm. hIL2 and hIL15 were intraperitoneally injected to enhance persistence of eNK cells. Mean ± SD, n=6 (IVIS control: n=2), Arrow (a): eNK administration, Arrow (c): Tumor cell mass.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/e307e458e74da6f84b8af59c.jpg"},{"id":97766392,"identity":"07b15ee5-5f24-465d-b529-b69b9fcc1475","added_by":"auto","created_at":"2025-12-09 07:15:42","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":201616,"visible":true,"origin":"","legend":"\u003cp\u003eEnhancement of the effects of necitumumab-mediated ADCC by eNK cells in A549-Luc lung tumor orthotopic graft bearing mice\u003c/p\u003e\n\u003cp\u003eeNK cells at a dose of 5 × 10\u003csup\u003e6\u003c/sup\u003e cells/mouse were intravenously administered three times a week for 2 weeks from day 4 after NOG mice were intravenously injected with A549-Luc tumor (2 × 10\u003csup\u003e5\u003c/sup\u003e cells). Nmab at a dose of 0.08 mg/body was intraperitoneally administered twice a week for 2 weeks from day 4. A549-Luc tumor growth was measured by IVIS luminescence intensity. hIL2 and hIL15 were intraperitoneally injected to enhance persistence of eNK cells. Mean ± SD, n=5, Red arrow: eNK administration, Black arrow: Nmab administration, **; p\u0026lt;0.01 vs Control (Vehicle) (Dunnett's\u0026nbsp;multiple comparison test)\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/5892da062604567c4a7ed4a1.jpg"},{"id":106343886,"identity":"d8099748-306a-46a2-a403-4147ab2a3802","added_by":"auto","created_at":"2026-04-07 16:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3511554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8246785/v1/5b04cdf6-1ba8-48cb-9039-54755d9cfeed.pdf"}],"financialInterests":"Competing interest reported. H.K. is Executive Officer of HEALIOS K.K.; K.T. is a former CSO of HEALIOS K.K.; F.N. is a former employee of HEALIOS K.K.; Y.S., K.G., K.M., N.U., Y.-s.T., Y.N., R.T., R.S., Y.T., K.K., M.Y., and Y.H. are current employees of HEALIOS K.K.","formattedTitle":"An innovative treatment for lung cancer using gene-engineered human-induced pluripotent stem cell- derived natural killer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThere has been remarkable progress in the research and development of innovative therapeutic methods such as immune checkpoint inhibitors, cell therapy, gene therapy, and nucleic acid medicine therapy. However, none of these therapies address the challenges of treating refractory solid cancers, and more promising therapeutic methods are needed.\u003c/p\u003e\u003cp\u003eRegarding innovative cell therapies, various CAR-T therapies have been investigated in non-clinical and clinical trials targeting solid tumors\u003csup\u003e1,2)\u003c/sup\u003e; however, no breakthrough effects have been achieved and their efficacy against solid tumors is still limited. On the other hand, NK cells are attracting attention because they attack targets in a non-HLA-restricted manner, have the advantage of not causing graft-versus-host disease (GVHD), and are safer than T cells.\u003csup\u003e3)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOur concept is to create groundbreaking cell therapies that could universally recognize cancer cells and kill them directly, rather than targeting limited numbers of targets with CARs for highly heterogeneous solid tumors.\u003c/p\u003e\u003cp\u003eEngineered natural killer (eNK) cells have the potential to be highly effective in treating various refractory solid cancers. In this report, we present our research into lung cancer treatment, which is particularly useful. Lung cancer is the leading cause of cancer-related deaths worldwide and consistently ranks high when it comes to annual mortality rates for both men and women. Lung cancer is often diagnosed when it is in an advanced stage. Although the number of treatment options, including chemotherapy drugs, immune checkpoint inhibitors, and antibodies is increasing, almost no effective therapy options are available for patients with advanced disease. On the other hand, it has been reported that intratumoral injection into solid tumors is a promising strategy for enhancing the efficacy of cancer treatment and avoiding systemic adverse effects.\u003csup\u003e4, 5)\u003c/sup\u003e First, because there have been few successful cases of systemic administration of cell therapy, we investigated whether eNK cells can exert an anti-solid tumor effect by injecting them directly into the tumor mass. We evaluated this strategy using cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) and obtained results that are expected to be highly accurate for clinical application. We hypothesized that cell therapy-specific organ-dependent distribution would be the same whether the cells are administered intravenously or directly. As hypothesized, eNK cells were remarkably effective in lung cancer treatment, and we succeeded in inducing an innovative antitumor effect. The \u003cem\u003ein vivo\u003c/em\u003e persistence and antitumor effects of eNK cells can be enhanced by gene transfer and further enhanced by combining gene transfer with antibodies that exert an ADCC mediated by transfected CD16.\u003c/p\u003e\u003cp\u003eThe interaction of cancer cells with immune cells in the tumor microenvironment (TME) is a crucial factor in cancer immunotherapy. To enhance the homing and infiltration of immune cells into the TME, we transfected CC motif ligand 19 (CCL19)\u003csup\u003e6,7)\u003c/sup\u003e to recruit patient immune cells to tumor tissue, and CC chemokine receptor type 2B (CCR2B)\u003csup\u003e8, 9)\u003c/sup\u003e to enable eNK cells to migrate toward CCL2-expressing cancer cells. For immunological cells to be effective, they needed to have strong cytotoxic activity to cancer cells and persist in the tumor and surrounding tissues. Because long-term persistence of CAR-T cells and \u003cem\u003ein vivo\u003c/em\u003e expansion of CAR-T cell number are associated with a better clinical response,\u003csup\u003e10)\u003c/sup\u003e we transfected interleukin (\u003cem\u003eIL\u003c/em\u003e)\u003cem\u003e15\u003c/em\u003e gene to achieve suitable lymphocyte persistence and activation levels,\u003csup\u003e11)\u003c/sup\u003e and natural killer group 2, member D (\u003cem\u003eNKG2D\u003c/em\u003e) and DNAX-activating protein 10 (\u003cem\u003eDAP10\u003c/em\u003e) genes to improve cancer cell detectability. Moreover, in the case of NK cells, antibody-dependent cellular cytotoxicity (ADCC) activity can be effectively utilized, so we attempted to enhance the expression of cluster of differentiation (CD)16 and increase the value of our therapy by combining it with antibodies.\u003csup\u003e12)\u003c/sup\u003e As a result of these creative hypotheses, we developed eNK cells, which are gene-engineered human induced pluripotent stem cell (hiPSC)-derived NK cells armed with NKG2D, IL-15, CD16, CCL19, and CCR2B molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003csup\u003e13)\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and cell culture\u003c/h2\u003e\u003cp\u003eThe lung cancer cell lines used were A549, NCI-H1975-Luc (luciferase expressing NCI-H1975), NCI-H460-Luc (luciferase expressing NCI-H460), Lu99, NCI-H520, and SBC-3. A549-GFP and NCI-H1975-Luc-GFP (H1975-GFP) were established by transfection of EF1α-GFP-IRES-Puro or Hygro-hGHpA vector using the piggyBac transposon system into each cell type and subsequent cloning. A549 and NCI-H520 were purchased from American Type Culture Collection. NCI-H1975-Luc, NCI-H460-Luc, Lu99, and SBC-3 were purchased from National Institute of Biomedical Innovation, Health, and Nutrition (Osaka, Japan). These cells were grown in monolayer culture in Ham\u0026rsquo;s F-12K (Kaighn\u0026rsquo;s) medium (A549 and A549-GFP), RPMI 1640 (NCI-H1975-Luc, NCI-H1975-GFP, NCI-H460-Luc, Lu99, NCI-H520), MEM (SBC-3) supplemented with 10% fetal bovine serum (FBS) (Biosera, Cholet, France), in a humidified incubator with at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eJ-PDX_E0050 tumor was provided by the National Cancer Center J-PDX Library, Japan (Tokyo, Japan) and maintained by subcutaneous transplantation in NOD/Shi-scid IL-2R gamma (null) (NOG) mice.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eNOG mice\u003csup\u003e14)\u003c/sup\u003e were purchased from In-Vivo Science Inc. (Kawasaki, Japan) and maintained under specific pathogen-free conditions with \u003cem\u003ead libitum\u003c/em\u003e access to water and food.\u003c/p\u003e\u003cp\u003e CDX model experiments were performed in accordance with the relevant institutional and national guidelines and regulations, and were approved by the Institutional Animal Care and Use Committee of HEALIOS K.K.\u003c/p\u003e\u003cp\u003ePDX model experiments were conducted at Mediford Corporation (Tokyo, Japan)(formerly LSI Medience Corporation, Tokyo, Japan). The experiments were approved by the Ethics Committee in accordance with the \"Ethics Review Regulations for Research, etc. Involving the Handling of Human Samples and Human Information\", by the Safety Committee in accordance with the \"Regulations of the Genetically Modified Organisms Safety Committee (Kumamoto)\", and by the Animal Experimentation Committee and the head of the institution in accordance with the \"Guidelines for Animal Experiments\". The use of human-derived PDX was carried out in accordance with the \"Ethical Guidelines for Medical Research Involving Human Subjects.\"\u003c/p\u003e\n\u003ch3\u003eLactate dehydrogenase release assay\u003c/h3\u003e\n\u003cp\u003eLung cancer cells were seeded at 3.75 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate and incubated overnight. eNK cells were co-cultured with tumor cells at an effector/target (E/T) ratio ranging from 0.3 to 10 for 4 h at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. After co-culture with eNK cells, supernatants were collected, and lactate dehydrogenase (LDH) release was measured using an LDH cytotoxicity assay kit (Dojindo Molecular Technology, Kumamoto, Japan). Cytotoxicity (% of lysis) was calculated by using the following formula. For determining maximal LDH release, cell lysis was induced by 1% Triton X-100 solution.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{C}\\text{y}\\text{t}\\text{o}\\text{t}\\text{o}\\text{x}\\text{i}\\text{c}\\text{i}\\text{t}\\text{y}\\:\\left(\\text{%}\\:\\text{o}\\text{f}\\:\\text{l}\\text{y}\\text{s}\\text{i}\\text{s}\\right)=\\frac{(\\text{E}\\text{x}\\text{p}\\text{e}\\text{r}\\text{i}\\text{m}\\text{e}\\text{n}\\text{t}\\text{a}\\text{l}\\:\\text{L}\\text{D}\\text{H}\\:\\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\:－\\:\\text{S}\\text{p}\\text{o}\\text{n}\\text{t}\\text{a}\\text{n}\\text{e}\\text{o}\\text{u}\\text{s}\\:\\text{L}\\text{D}\\text{H}\\:\\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\:\\text{o}\\text{f}\\:\\text{e}\\text{f}\\text{f}\\text{e}\\text{c}\\text{t}\\text{o}\\text{r}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s})}{(\\text{M}\\text{a}\\text{x}\\text{i}\\text{m}\\text{u}\\text{m}\\:\\text{L}\\text{D}\\text{H}\\:\\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\:－\\:\\text{S}\\text{p}\\text{o}\\text{n}\\text{t}\\text{a}\\text{n}\\text{e}\\text{o}\\text{u}\\text{s}\\:\\text{L}\\text{D}\\text{H}\\:\\text{r}\\text{e}\\text{l}\\text{e}\\text{a}\\text{s}\\text{e}\\:\\text{o}\\text{f}\\:\\text{t}\\text{a}\\text{r}\\text{g}\\text{e}\\text{t}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s})}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eIncucyte cytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eTo generate tumor spheroids, A549 and H1975 cells stably expressing green fluorescent protein (GFP) were seeded at 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well round-bottom plate (Corning, NY, USA) in Ham\u0026rsquo;s F-12K (Kaighn\u0026rsquo;s) medium (A549-GFP) or RPMI 1640 (H1975-GFP) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Biosera). After centrifugation for 10 min at 130 \u0026times; g, plates were cultured in an incubator at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e for 4 days. Thirty thousand eNK cells were added to the wells containing one tumor spheroid in AIM-V medium (Thermo Fisher Scientific) supplemented with 5% FBS (Merck, Darmstadt, Germany), 50 ng/mL stem cell factor (SCF) (PeproTech, Cranbury, NJ, USA), and 50 ng/mL IL-15 (PeproTech) in the absence or presence of 2.5 \u0026micro;g/mL cetuximab (Cmab; Merck) and 3 \u0026micro;g/mL necitumumab (Nmab; Nippon Kayaku Co., Ltd., Tokyo, Japan). Cytotoxicity was monitored at 120 min intervals using the Incucyte SX5 imaging system (Sartorius, G\u0026ouml;ttingen, Germany).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003epersistence assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 5.0\u0026times; 10\u003csup\u003e6\u003c/sup\u003e eNK cells were intravenously administered to NOG mice. Peripheral blood, lung, liver, and spleen samples were collected on 1, 7, 14, and 28 days after administration. The genomic DNA (gDNA) was extracted from the peripheral blood and tissues using a DNeasy Blood \u0026amp; Tissue Kit (Qiagen, Hilden, Germany). The DNA content of eNK cells per gDNA content in each sample was determined by qPCR using an \u003cem\u003eAlu\u003c/em\u003e sequence-specific primer set.\u003csup\u003e15)\u003c/sup\u003e On the final day, mice were anesthetized by inhalation of vaporized isoflurane and euthanized by exsanguination from the inferior vena cava.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eantitumor activity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNOG mice were injected intravenously or subcutaneously with H1975-Luc or A549-Luc cells. However, any models in which cancer cells engrafted in organs other than the lungs were excluded from the study. hIL2 (PeproTech) and hIL15 were intraperitoneally injected to encourage or enhance persistence of eNK cells.\u003c/p\u003e\u003cp\u003eAn orthotopic lung cancer model was established by intravenous injection of tumor cells. Tumor progression was assessed by bioluminescence imaging using the IVIS Spectrum system (PerkinElmer, Shelton, CT, USA). For subcutaneous tumors, the length (L), width (W), and height (H) were measured using calipers. Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) was calculated using the following formula: L \u0026times; W \u0026times; H\u0026thinsp;\u0026times;\u0026thinsp;π/6.\u003c/p\u003e\u003cp\u003eFor histopathological analysis, tumors were collected from tumor-bearing mice in the satellite group that did not receive eNK cells treatment. Tissue collection was performed on the first day of eNK cells administration, following euthanasia under anesthesia and exsanguination.\u003c/p\u003e\u003cp\u003eH\u0026amp;E staining and Ki67 immunostaining using anti-Ki-67 antibody (clone: MIB-1, Dako) were performed at Sapporo General Pathology Laboratory Co., Ltd. (Sapporo, Japan).\u003c/p\u003e\n\u003ch3\u003ePDX model\u003c/h3\u003e\n\u003cp\u003eThe experiments were conducted at Mediford Corporation in research facilities that had obtained AAALAC International Certification (Tokyo, Japan). Human lung cancer PDX lines provided by the National Cancer Center Japan (Tokyo, Japan) were subcutaneously implanted into NOG mice. eNK cells and Cmab were administered three times a week for three weeks intratumorally and intraperitoneally respectively. hIL2 was intraperitoneally injected to encourage or enhance persistence of eNK cells. The tumor size was measured using a caliper and the estimated tumor volume was calculated using the following formula. Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;W (mm) \u0026times; L (mm) \u0026times; L (mm) \u0026times; 1/2.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Dunnett's multiple comparison test was performed as a statistical significance examination.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe cytotoxicity of eNK cells and the contribution of the gene-engineering of CD16\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFirst, we evaluated the cytotoxicity of eNK cells against A549 (wild-type epidermal growth factor receptor [EGFR]), NCI-H1975-Luc (mutant EGFR; L858R, T790M), NCI-H460-Luc (PIK3CA mutant), LU99 (wild-type EGFR), NCI-H520 (wild-type EGFR), and SBC-3 using the LDH release assay. eNK cells showed dose dependent cytotoxicity in all lung cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we evaluated the sustained cytotoxicity of eNK cells using the Incucyte cytotoxicity assay. A549-GFP and NCI-H1975-GFP cell spheroids were respectively incubated with eNK cells in E/T ratios ranging from 0.3 to 10 and monitored for cell death by measuring GFP fluorescence intensity using the Incucyte live-cell imaging system. eNK cells were clearly cytotoxic for A549-GFP cells at an E/T ratio of 3 and inhibited their growth at an E/T ratio of 1. Against NCI-H1975-GFP cells, eNK cells were cytotoxic at an E/T ratio of 0.3 or more. Moreover, eNK cells retained cytotoxic activity until 120 h after the treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe evaluated the ability of eNK cells to induce ADCC against a wild type-EGFR-positive tumor cell line (A549) via Cmab\u003csup\u003e16)\u003c/sup\u003e and Nmab\u003csup\u003e17)\u003c/sup\u003e binding to FcgRIII (CD16a). A549-GFP spheroids were incubated with eNK cells and Cmab or Nmab (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Cmab and Nmab alone were not cytotoxic, however the addition of these antibodies further enhanced the cytotoxicity ofeNK cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe antitumor effects of intratumoral eNK cell administration on the subcutaneously transplanted lung cancer cell line, H1975-Luc\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe examined whether the intratumoral route of injection affects the antitumor effect of eNK cells on subcutaneously transplanted H1975-Luc, because it had been reported that intratumoral injection into solid tumors was a promising strategy for enhancing cancer treatment efficacy.\u003csup\u003e4, 5)\u003c/sup\u003e To examine whether eNK cells could inhibit tumor growth, H1975-Luc cells were transplanted subcutaneously in NOG mice (day 0), and eNK cells were administered intratumorally three times a week for two weeks starting on day 14 after tumor transplantation. The results clearly showed that eNK cells dose-dependently suppress tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effects of eNK cells in the PDX mouse model derived from a lung cancer patient\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo improve the predictability of tumor growth inhibition by eNK cells in clinical applications, we examined the effects of eNK cells in a high-predictive-value PDX mouse model. The patient who provided the J-PDX_E0050 tumor used in the PDX mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, non-small cell lung cancer, adenocarcinoma) was resistant to chemotherapy and radiotherapy. J-PDX_E0050 clearly expressed EGFR in histological examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eeNK cells at doses of 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mouse were intratumorally administered three times a week for 3 weeks to NOG mice bearing subcutaneous PDX tumors. eNK cells at a dose of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mouse reduced tumor volume by 28% on day 35 after the first administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Next, the combined effect of eNK cells and Cmab was examined based on ADCC. Injection of eNK cells at a dose of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mouse and Cmab at a dose of 0.5 mg/body into mice bearing PDX tumors resulted in 18% and 36% inhibition of tumor growth on day 35, respectively. eNK cells combined with Cmab resulted in 53% inhibition, the enhancement of the inhibitory effect having been attributed to ADCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eThese results suggested that eNK cells would be effective both as a single agent or in combination with antibody in clinical practice.\u003c/p\u003e\n\u003ch3\u003ePersistence of eNK cells in normal NOG mice\u003c/h3\u003e\n\u003cp\u003eeNK cells were administered intravenously to normal NOG mice and its pharmacokinetics were investigated. eNK cells were injected intravenously at a dose of 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in male and female NOG mice. Human-specific Alu sequence analysis using qPCR was conducted in gDNA extracted from blood and organs collected on day 1, 7, 14, and 28 after injection. The blood concentration of human-specific Alu sequences decreased 1 to 7 days after administration and then increased on days 14 and 28 after administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The time-dependent patterns of changes in concentration in the lungs, liver, and spleen were similar to those in the blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c, d). In addition, eNK cells were most abundantly distributed in the lungs among the organs evaluated, suggesting that they are efficiently distributed to the sites of lung cancer involvement and exhibit antitumor effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-tumor effects of eNK cells against the human lung cancer cell line, H1975-Luc, intravenously injected into an orthotopically transplanted mouse model of lung cancer\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, eNK cells showed clear antitumor effect when injected intratumorally into subcutaneously transplanted tumors. In contrast, systemic administration of eNK cells resulted in substantial accumulation in the lungs. Notably, this pulmonary accumulation resembled the localized concentration observed following intratumoral injection. Based on this observation, eNK cells were systemically administered to a lung orthotopic transplantation model to evaluate their antitumor efficacy.\u003c/p\u003e\u003cp\u003eNOG mice were intravenously injected with the H1975-Luc human lung cancer cell line. eNK cells were then intravenously at doses of 1 \u0026times; 10⁶, 3 \u0026times; 10⁶, and 1 \u0026times; 10⁷ cells per mouse, three times per week from day 14 to day 25. Seven days after the final administration, eNK cells significantly inhibited tumor growth. At doses of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e and 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells /mouse, bioluminescence signals returned to baseline and matched those observed in the IVIS control group without tumor transplantation, suggesting near-complete tumor regression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Tumor regression was clearly visualized by IVIS imaging on 32, 7 days after the last eNK cell administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Histological analysis on day 14 confirmed that H1975-Luc tumor cells were successfully engrafted and distributed within the lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). These results strongly suggest that eNK cells possess robust therapeutic efficacy, even when used as monotherapy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnhancement of the anti-tumor effect of eNK cells by necitumumab-mediated ADCC in A549-Luc lung tumor orthotopic graft bearing mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated whether the antitumor effect of eNK cells could be enhanced via ADCC activity in tumor bearing model mice with wild-type EGFR-positive A549-Luc cells. eNK cells exhibited a clear dose-dependent tumor growth inhibition in A549-Luc tumor-bearing mice, similar to the effect observed in H1975-Luc tumor-bearing mice. NOG mice were intravenously injected with A549-Luc tumor cells on day 0. eNK cells were administered intravenously at a dose of 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mouse, three times per week for 2 weeks starting on day 4. This dose was selected as a partially effective dose based on prior evaluations. Nmab was administered intraperitoneally at a dose of 0.08 mg/body, twice weekly for 2 weeks starting on day 4.\u003c/p\u003e\u003cp\u003eBoth eNK cells and Nmab inhibited tumor growth, and their combination resulted in more pronounced antitumor effect than that either treatment alone. These results suggest that the potent antitumor effects of the combination of eNK cells and Nmab are likely mediated cytotoxicity (ADCC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAdministration of antibodies against immune checkpoint molecules such as PD-1, PD-L1, and CTLA4 have been shown to inhibit the growth of, and have a therapeutic effect against, malignant advanced cancers such as advanced non-small cell lung cancer (NSCLC) and extend survival time.\u003csup\u003e18,19,20)\u003c/sup\u003e However, only about 20% of lung cancers are sensitive to immune checkpoint inhibitor therapies, for example, PD-1/PD-L1 antibody therapy,\u003csup\u003e19)\u003c/sup\u003e so to address this issue, development of new immunotherapies and novel treatments is needed for cancer patients. For immune checkpoint inhibitors to be effective, it is important that patients have tumor-reactive T cells.\u003csup\u003e20)\u003c/sup\u003e However, the supply of T cells is often exhausted in cancer patients.\u003csup\u003e21,22)\u003c/sup\u003e In addition, the more mutant the antigens in a tumor are, the higher is the probability of inducing tumor-specific T cells,\u003csup\u003e23)\u003c/sup\u003e but if there are few mutant antigens, fewer tumor-specific T cells are induced and the effects of immune checkpoint molecules may be reduced. In a comparison of comprehensive genetic analysis of cancer cells and treatment outcomes, the frequency of highly antigenic mutant proteins in tumors was correlated with survival rates after PD-1/PD-L1 treatment.\u003csup\u003e24,25)\u003c/sup\u003e The development of CAR-T cell therapy has led to the discovery that the inhibitory activity against tumor cells expressing specific antigens can be therapeutic, but the therapeutic effect on heterogeneous solid tumors is attributable to lack of persistence and exhaustion of CAR-T cells in the patient, as well as the limited ability of CAR-T cells to home in on target tumor sites.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe have developed eNK cell therapy for solid tumors by generating gene-engineered iPSC-derived NK cells.\u003csup\u003e13)\u003c/sup\u003e We believe that this is a promising concept for the treatment of solid tumors with heterogeneity, rather than an established one relying on the achievement of efficacy by CAR targeting.\u003csup\u003e26,27,28)\u003c/sup\u003e We report the remarkable \u003cem\u003ein vivo\u003c/em\u003e antitumor effects of eNK cells active against lung cancer in lung cancer models, including the PDX model, which has a high predictive value clinically, in this publication.\u003c/p\u003e\u003cp\u003eAs previously reported,\u003csup\u003e13)\u003c/sup\u003e eNK cells, which are transfected with genes encoding \u003cem\u003eNKG2D\u003c/em\u003e, \u003cem\u003eDAP10\u003c/em\u003e, \u003cem\u003eCCL19\u003c/em\u003e, \u003cem\u003eCCR2B\u003c/em\u003e, \u003cem\u003eCD16\u003c/em\u003e, and \u003cem\u003eIL-15\u003c/em\u003e, have the following functions: infiltration and migration into cancer tissue and cancer cells,\u003csup\u003e7, 29)\u003c/sup\u003e recruitment of the patient's own immune cells for activation of the immune system in the tumor microenvironment and enhancement of cytotoxic activity, \u003csup\u003e4, 5)\u003c/sup\u003e, and maintenance of cell activation and persistence.\u003csup\u003e9, 30)\u003c/sup\u003e Based on the \u003cem\u003ein vivo\u003c/em\u003e distribution characteristics of cell therapy products, it is expected that systemically administered eNK cells will accumulate in the lungs. \u003csup\u003e31, 32, 33)\u003c/sup\u003e The present results suggest that eNK cells have similar properties. In addition, it was found that the number of eNK cells decreased after reaching a peak at 24 h following intravenous administration. This reduced level was maintained for approximately one week. Subsequently the number of eNK cells increased, likely due to proliferation, and eventually returned to the level observed at 24 h post administration, where it remained for up to 28 days. The persistence of eNK concentrations is considered to contribute to adequate efficacy.\u003c/p\u003e\u003cp\u003eThe CDX model, in which intravenously administered cancer cells become engrafted in an animal\u0026rsquo;s lungs,\u003csup\u003e34)\u003c/sup\u003e is an orthotopic lung cancer engraftment model. Intravenously administered eNK cells clearly inhibited tumor growth in this model. The remarkable efficacy of eNK cells administered via the clinical route in an orthotopic lung cancer model indicates their strong potential for clinical application. Since eNK cells were primarily localized in the lungs after intravenous administration, we believe that local intratumoral administration of eNK cells in subcutaneously transplanted PDX tumors can be used to predict the results of systemic administration. Therefore, the fact that eNK cells demonstrated antitumor effects in PDX models, which have high predictive value clinically, \u003csup\u003e35, 36)\u003c/sup\u003e suggests their usefulness in clinical applications.\u003c/p\u003e\u003cp\u003eTargeting EGFR is an important treatment modality for many solid tumors including NSCLC. Cmab and Nmab are monoclonal antibodies containing the constant region of human IgG1 with high affinity to EGFR and exhibiting ADCC activity,\u003csup\u003e14, 37, 38)\u003c/sup\u003e It was reported that the addition of Cmab to chemotherapy in the first-line treatment improves overall survival of patients with EGFR-expressing advanced NSCLC.\u003csup\u003e39)\u003c/sup\u003e But no clear combined effects\u003csup\u003e40)\u003c/sup\u003e and toxicity concerns were reported despite the improvement in antitumor effects when Cmab and chemotherapy were combined. \u003csup\u003e41, 42)\u003c/sup\u003e Moreover, retrospective analyses revealed that mutational status and copy number of EGFR were not predictive for cetuximab benefit.\u003csup\u003e43)\u003c/sup\u003e Nmab also was unable to achieve sufficient efficacy when combined with chemotherapy because Nmab showed adverse effects in combination therapy.\u003csup\u003e38)\u003c/sup\u003e Therefore, Cmab or Nmab and eNK cells are useful in clinical applications, since eNK cells show anti-tumor effects and the combination of eNK cells with Cmab and Nmab enhances these effects through ADCC activity.\u003c/p\u003e\u003cp\u003eCurrently, treatment options are limited, and in refractory cases of lung cancer, new treatment modalities, such as gene therapy and cell therapy, are actively developed. Because cell therapy is thought to be very useful for treating lung cancer based on cell dynamics studies showing that most cells are distributed in lung tissue, \u003csup\u003e44, 45)\u003c/sup\u003e lung cancer was selected as the first solid tumor target for systemic treatment, and the results of evaluation have demonstrated the significant efficacy of systemic treatment. eNK cell therapy is a new concept. eNK cells are gene-transduced iPS cell-derived NK cells that improve immune function in the tumor microenvironment, immune cell homing and migration to tumor sites, and the distribution of immune cells in the targets of treatment (lung tissue and lung cancer lesions). Leveraging these properties, eNK cells have tumor damaging activity that is different from the antitumor activity engendered by CAR-T cells, and we believe that its antitumor effects on solid tumors and lung cancer are groundbreaking.\u003c/p\u003e\u003cp\u003eIn conclusion, eNK cell therapy is a novel and promising treatment option for refractory lung cancer. However, the antitumor effect of eNK cells alone is limited in preclinical models, so further evaluation is necessary to optimize therapeutic outcomes. Combination strategies with other antibody-based drugs or refined dosing schedules may be essential to maximize efficacy and ensure the translational potential of this approach.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eH.K. is Executive Officer of HEALIOS K.K.; K.T. is a former CSO of HEALIOS K.K.; F.N. is a former employee of HEALIOS K.K.; Y.S., K.G., K.M., N.U., Y.-s.T., Y.N., R.T., R.S., Y.T., K.K., M.Y., and Y.H. are current employees of HEALIOS K.K.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.S. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing\u0026ndash;original draft; K.G. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing \u0026ndash; review \u0026amp; editing; S.Y. : Resources, Performed the experiments, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Supervision; K.M. , N.U. , Y.-s.T. and Y.N. : Performed the experiments, Methodology, Data curation, Data analysis, Visualization, Writing \u0026ndash; review \u0026amp; editing; R.T. and R.S. : Performed the experiments, Data curation, Data analysis; Y.T. and M.Y. : Methodology, Writing \u0026ndash; review \u0026amp; editing, Project administration, Supervision; K. K. : Resources, Writing \u0026ndash; review \u0026amp; editing, Project administration, Supervision; Y. H. and F. N. : Writing \u0026ndash; review \u0026amp; editing, Project administration; H. K. : Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Project administration, Supervision; A. H. : Resources, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Supervision; K. T. : Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Supervision, Funding acquisition.All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Oriental Bio Service, Kobe BM Laboratory (Kobe, Japan) for animal housing and care. We also thank Dr. Takashi Murakami (Saitama Medical University) for kindly providing the NCI-H1975-Luc and NCI-H460-Luc cell lines. PDX model experiments were conducted at Mediford Corporation (Tokyo, Japan) (formerly LSI Medience Corporation, Tokyo, Japan), and we are grateful for their technical support. Hematoxylin and eosin (H\u0026amp;E) staining and Ki-67 immunostaining were performed at Sapporo General Pathology Laboratory Co., Ltd. (Sapporo, Japan). We thank Sumisho Pharma International Co., Ltd. (Tokyo, Japan) for their support with IVIS imaging analysis. The National Cancer Center J-PDX library used in this study is partly supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number 17pc0101011h0001, and by the National Cancer Center Research and Development Fund, Japan. English proofreading services were provided by ASCA Corporation (Osaka, Japan). The authors would like to thank all HEALIOS lab members for their technical assistance and helpful discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbelda SM (2024) CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn. Nat Reviews Clin Oncol 21:47\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1038/s41571-023-00832-4\u003c/span\u003e\u003cspan address=\"10.1038/s41571-023-00832-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUslu U, June CH (2024) Beyond the blood: expanding CAR T cell therapy to solid tumors. 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Stem Cells Int Article ID 7397819. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1155/2023/7397819\u003c/span\u003e\u003cspan address=\"10.1155/2023/7397819\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"iPSC (induced pluripotent stem cell), NK (natural killer) cells, Genetic engineering, Cell therapy, Lung cancer, PDX (Patient-derived xenograft)","lastPublishedDoi":"10.21203/rs.3.rs-8246785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8246785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVarious therapeutic approaches have been developed for lung cancer, including chemotherapy, radiation therapy, and immune checkpoint inhibitors. However, none of these approaches, including chimeric antigen receptor (CAR)-T cell therapy, have been effective against solid tumors.\u003c/p\u003e\u003cp\u003eTo enhance the therapeutic effect, we focused on the multiple effects of a new modality of cell therapy and created engineered natural killer (eNK) cells, which are gene-engineered induced pluripotential stem cell (iPSC)-derived NK cells armed with CC motif ligand 19 (CCL19), CC chemokine receptor type 2B (CCR2B), high-affinity cluster of differentiation 16 (CD16), interleukin (IL)-15, and natural killer group 2, member D (NKG2D)-DNAX-activating protein 10 (DAP10) complex. \u003cem\u003eIn vitro\u003c/em\u003e studies showed that eNK cells showed significant long-lasting cytotoxicity and antibody dependent cell-mediated cytotoxicity (ADCC) against human lung cancer cell lines. Intravenous and intra-tumoral treatment with eNK cells almost completely inhibited tumor growth in both orthotopically and subcutaneously transplanted cell line-derived xenograft (CDX) models, respectively. Moreover, intra-tumoral eNK cells administered as a single dose or in combination with cetuximab in the patient-derived xenograft (PDX) model of lung cancer showed clear tumor growth inhibition. This study demonstrates that eNK cells exhibit significant antitumor effects in both CDX and PDX models, and that these effects are further enhanced via ADCC.\u003c/p\u003e\u003cp\u003eWe successfully demonstrated that eNK cells having the ability to migrate to tumor cells and become activated in the tumor microenvironment and having the ability to induce tumor-specific killing activity without targeting CAR, are an innovative mode of treating lung cancer in pre-clinical studies.\u003c/p\u003e","manuscriptTitle":"An innovative treatment for lung cancer using gene-engineered human-induced pluripotent stem cell- derived natural killer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 07:15:37","doi":"10.21203/rs.3.rs-8246785/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T10:38:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T01:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235478667072471143612686736764487824899","date":"2025-12-09T09:41:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T03:54:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105371484541635927439516189040716313212","date":"2025-12-04T14:45:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330948316910010940273485303350843254663","date":"2025-12-04T13:39:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-04T09:00:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T11:41:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-03T11:40:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-12-01T06:27:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82d0528e-bf93-4fa1-a3ee-e5a1b9444efe","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:06:48+00:00","versionOfRecord":{"articleIdentity":"rs-8246785","link":"https://doi.org/10.1007/s00262-026-04370-7","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2026-03-31 15:59:25","publishedOnDateReadable":"March 31st, 2026"},"versionCreatedAt":"2025-12-09 07:15:37","video":"","vorDoi":"10.1007/s00262-026-04370-7","vorDoiUrl":"https://doi.org/10.1007/s00262-026-04370-7","workflowStages":[]},"version":"v1","identity":"rs-8246785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8246785","identity":"rs-8246785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}