A Novel Feeder cell based on 4-1BBL and Membrane-bound IL-21/ IL-15 Induce Highly Expansion and Anti-tumor Effect of Natural Killer Cells

A Novel Feeder cell based on 4-1BBL and Membrane-bound IL-21/ IL-15 Induce Highly Expansion and Anti-tumor Effect of 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 Method Article A Novel Feeder cell based on 4-1BBL and Membrane-bound IL-21/ IL-15 Induce Highly Expansion and Anti-tumor Effect of Natural Killer Cells Sha Gong, Nan Mei, Jun Wang, Junsheng Zhu, Lu Wang, Xiaohong Lu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4741998/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Natural killer (NK) cell immunotherapy is a promising approach for cancer treatment. However, its extensive clinical application was limited to the large-scale clinical-grade expansion of NK cells. In this study, we expanded NK cells from healthy donor’s peripheral blood mononuclear cells (PBMCs) using a newly designed K562 feeder cell line. Methods The feeder cells were generated by transducing K562 cells with lentiviral particles carrying 4-1BBL and mbIL-21/-15. NK cells were expanded from PBMCs with these genetically modified, frozen-thawed and irradiated K562 feeder cells in the presence of IL-2. The purity, quantity, and receptors expression of the expanding NK cells were dynamically monitored. Furthermore, their anti-tumor efficacy was evaluated both in vitro and in vivo following a two-week expansion period. Results The K562-4-1BBL-mbIL-21/-15 feeder cells induced highly-efficient NK cells expansion from PBMC (17902-fold) within two weeks. There was a notable upregulation in the expression of activating receptors including NKG2D, NKp30, NKp44, and NKp46 during the expansion process. Moreover, the expanded NK cells displayed enhanced cytotoxicity against a variety of hematological (K562, MOLM-13, OCI-AML-3, THP-1) and solid (Hep-G2, OVCAR3) cancer cell lines in vitro. In the humanized U937 xenograft mouse model, the NK cells extended the median survival time of the AML-bearing mice from 19.40 to 28.25 days. Conclusions We have successfully established a highly-efficient, cost-effective and rapid NK cell expansion platform from PBMC utilizing K562-4-1BBL-mbIL-21/-15 feeder cells, which also significantly improved the cytotoxicity both in vitro and in vivo, presenting a significant advancement in the field of NK cell-based immunotherapy. NK cells expansion Feeder cells mbIL-21 mbIL-15 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Natural killer (NK) cell immunotherapy has emerged as a new frontier in cancer treatment. Various therapeutic modalities, such as adoptive NK cell transfer, chimeric antigen receptor (CAR)-NK cells and NK cell engagers, are carried out and being investigated in clinical trials in a wide array of solid and hematological malignancies. Particularly noteworthy are anti-CD19 CAR-NK cells, which have demonstrated remarkable success in treating CD19 + B-cell malignancies [ 1 , 2 ] , earning FDA approval and triggering a wave of research on NK cell-based therapies. However, the widespread clinical application of NK cell therapy has been limited by the challenge in manufacturing large-scale and clinical-grade expansion of NK cells, which remains a critical bottleneck in the development of “off-the-shelf” NK cell immunotherapies. Generating adequate numbers of functional NK cells in vitro is a major challenge, as NK cells constitute only 10–20% of peripheral blood mononuclear cells (PBMC) [ 3 , 4 ] . In addition, the in vivo lifespan of NK cells is relatively short, generally limited to around two weeks [ 5 ] . Given the modest initial quantity of NK cells and the constraints on their proliferation time, it is essential to undertake a thorough in vitro expansion process to achieve the necessary therapeutic dosage. While several methods for NK cells expansion have been described, each with its specific advantages and limitations. Cytokine-based regimens demonstrate a low expansion fold (< 30-fold expansion within 3 weeks) due to NK cell senescence [ 6 – 8 ] , which was insufficient to meet the dosage requirements for patient treatment. In contrast, feeder cell-based approaches, particularly using irradiated K562 cells engineered to express activation signals (4-1BBL or OX40L) and membrane-bound (mb) cytokines (mbIL-21or mbIL-15 or mbIL-18), have demonstrated robust NK cell proliferation, with potential expansion rates approximately tens of thousands of folds [ 9 – 13 ] . However, these methods generally needed prolonged culture periods ranging from 3 to 5 weeks. Evidence suggested that mbIL-21 could block the process of NK cell senescence [ 10 ] , and mbIL-15 could enhance the NK cell survival and augment their cytotoxicity towards tumor cells in vitro and vivo [ 14 ] . Yet, the combined effects of mbIL-21 and mbIL-15 within feeder cell design was rarely reported. The aim of this study is to design a novel feeder cell simultaneously co-expressing mbIL-21, mbIL-15 and 4-1BBL to expand NK cells in vitro from PBMCs, thereby creating a rapid, highly efficient, cost-efficient, and straightforward platform for NK cell proliferation. We have recently utilized this platform to construct and manufacture NKG2D-CAR-NK cells for acute myeloid leukemia. It successfully completed the preclinical research and will start phase 1 clinical trial as soon. Materials and Methods Cell lines K562, THP-1 cells were purchased from Procell (Wu Han, China). OCI-AML3, U937, MOLM-13, Hep-G2, OVCAR3, 293T cells were provided by the Hematology Lab or Gene and Cell Therapy Research Institute at The First Affiliated Hospital of Xi'an Jiaotong University. K562, THP-1, OCI-AML3, U937 cells were maintained in PRMI-1640 medium (HyClone, USA) with 10% fetal bovine serum (FBS, ExCell Bio, China). Hep-G2 cells were maintained in MEM medium (HyClone, USA) with 10% FBS. OVCAR3 were maintained in PRMI-1640 medium and 10 µg/mL insulin with 20% FBS. 293T cells were used for lentiviral packaging and cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, USA) with 10% FBS. Mycoplasma testing was performed on all cell lines once every two weeks using the MycoAlert Mycoplasma Detection Kit (Lonza, Germany). Plasmid construction To construct mbIL-21, the coding sequence of mature human IL-21 was directly fused to the human CD8a hinge, transmembrane and cytoplasmic domains. The mbIL-15 was created by linking human IL-15 with its receptor IL-15RA. The codon-optimized sequences for mbIL-21 and mbIL-15 were synthesized and linked using the T2A self-cleaving peptide. This synthetic fusion sequence of mbIL-21/-15 was then cloned into the pLVX-EF1α-IRES-Neo vector (Miaoling Biology, China) using the EcoR1 and BamH1 restriction sites, resulting in the recombinant plasmid pLVX-EF1α-mbIL-21/-15-IRES-Neo (Fig. 1 a). Given that human 4-1BBL functions as a membrane protein, its codon-optimized sequence was also synthesized and subsequently inserted into the pSIN-EF1α-IRES-Puro vector (Miaoling Biology, China) to create the overexpression plasmid pSIN-EF1α-4-1BBL-IRES-Puro (Fig. 1 a). K562 feeder cells co-expressing 4-1BBL, mbIL-21 and mbIL-15 construction Lentiviral particles were produced by co-transfecting 293T cells with the respective lentiviral transfer plasmids (pLVX-EF1α-mbIL-21/-15-IRES-Neo or pSIN-EF1α-4-1BBL-IRES-Puro) along with the packaging plasmids (psPAX2 and pMD2.G). Three days following transfection, the viral supernatants were collected and concentrated by ultracentrifugation at 100,000 g for 2 hours. The concentrated viral particles were resuspended in RPMI-1640 medium and stored at -80°C until further use. To determine the viral titer, 293T cells were transduced with varying volumes of the viral solutions. After three days, the percentage of 4-1BBL + or IL-21/IL-15 + cells were estimated by flow cytometry. Titers was calculated using the following formula: Titer (IU/mL) = (Number of infected cells × Percentage of positive cells) / Volume of virus used (mL). K562 cells were seeded into 6-well plates at 2×10 5 cells/mL and subsequently transduced with the concentrated virus stocks containing mbIL-21/-15 and 4-1BBL at a multiplicity of infection (MOI) of 2 in the presence of protamine sulfate (10 µg/mL; Solarbio, China). Three days later, the transduced cells were selected with puromycin (2 µg/mL) and G418 (600 µg/mL) for two weeks. The transduction efficacy was analyzed by flow cytometry using APC-conjugated anti-human 4-1BBL monoclonal antibodies (mAb) (Biolegend, USA), PE-conjugated anti-human IL-21 and IL-15 mAbs (eBioscience, USA). The stable feeder cell line co-expressing 4-1BBL and mbIL-21/-15 (K562-4-1BBL-mbIL-21/-15) was maintained in RPMI 1640 with 10% FBS and low concentration of puromycin and G418. Upon reaching sufficient cell numbers, these feeder cells were irradiated with 100 Gy gamma rays by Cs-137 gamma to inhibit further cell division, then cryopreserved with 2×10 7 cells/vial in liquid nitrogen, creating a ready-to-use irradiated K562-4-1BBL-mbIL-21/-15 cell bank for future NK cell expansion endeavors. NK cells expansion using K562-4-1BBL-mbIL-21/-15 feeder cells PBMCs were isolated form the peripheral blood of healthy donors by Ficoll gradient centrifugation (GE Healthcare, USA). 2×10 7 PBMCs were directly co-cultured with frozen-thawed irradiated K562-4-1BBL-mbIL-21/-15 feeder cells at a ratio of 1:1 in a T75 flask containing 20 mL of fresh medium. The medium was composed of a serum-free medium (Hangzhou Zhongying Bio-Medical Technology, China) supplemented with 5% heat-inactivated autologous plasma and recombinant human IL-2 (200 U/mL; Jiangsu Kingsley Pharmaceutical Co., Ltd, China). Three days post-culture, the cell suspension was centrifuged at 350 g for 5 minutes, and then the supernatant was discarded and equal amount of fresh medium was added. Up until day 7, fresh medium was supplemented every two days to maintain the cell density between 1.0-1.5×10 6 cells/mL. On day 7, a second of frozen-thawed K562-4-1BBL-mbIL-21/-15 feeder cells were added for re-stimulation. Subsequently, the percentage of heat-inactivated autologous plasma in the medium was reduced from 5–1%, and the cell densities were adjusted to be within 1.5–2.5×10 6 cells/mL. Cells were harvest on day 13 or 14, and the expansion fold was calculated by the absolute number of NK cells on day 14 by the respective number on day 0. Flow cytometry analysis The purity of NK cells on days 0, 7 and 14 was examined by flow cytometry using FITC-conjugated anti-human CD3 mAb and APC- conjugated anti-human CD56 mAb (Biolegend, USA). The expression levels of various NK cell receptors were analyzed using PE- conjugated anti-human NKG2D, NKp30, NKp46 mAbs (BD Biosciences, USA), PE-Cy7 conjugated anti-human NKp44, CD69, CD158b mAbs (Biolegend, USA), and APC-Cy7 conjugated anti-human NKG2A and CD16 mAbs (Biolegend, USA). For all samples, PBMC or the expending cells (1.0×10 6 cells) were washed with FACS buffer (MACS rinsing solution with 0.2% BSA; Miltenyi Biotec, Germany) and resuspended with 100 µL FACS buffer in a 96 well U-bottom plate. To block non-specific binding, Human Fc-receptor Blocking Solution (Biolegend, USA) was applied to each sample for 5 minutes at room temperature. Subsequently, the samples were stained with the designated mAbs for 20 minutes at 4°C in the dark. Following staining, cells were washed twice with FACS buffer and then were resuspended with 100 µL FACS buffer. To assess cell viability, 10 µL Propidium Iodide (PI) were added to each sample, followed by incubation for 5 minutes at 4 ℃ in the dark. The data was required and analyzed using NoveExpress software. Cytotoxicity assay NK cell cytotoxicity was assessed using the One Glo Luciferase Assay System (Promega, USA). The stable luciferase-expressing cell lines (K562-Luc, Molm-13-Luc, THP-1-Luc, OCI-AML3-Luc, U937-Luc, Hep-G2-Luc, OVCAR-Luc) were prepared to serve as target cells. The unexpanded and expanded NK cells were co-cultured with each targeted cells at effector-to-target (E:T) ratio of 5:1, 2:1, 1:1 and 0.5:1 in a 384 well plate with total amount of 30 µL for 12–16 hours in triplicate. After co-culture, 30 µL of the luciferase ONE-Glo Reagent was added to each well containing the NK/target cell mixture. The plate was incubated at room temperature for 3 minutes to allow complete cell lysis. Finally, the luminescence was measured in a luminometer. The cytotoxicity was calculated using the following formula: Cytotoxicity (%) = [((Luminescence (target cells only) - Luminescence (effector cells only))- ((Luminescence (target cells with effector cells) - Luminescence (effector cells only))] / [Luminescence (target cells only) - Luminescence (effector cells only)] ×100%. CD107a degranulation assay The unexpanded and expanded NK cells were washed with phosphate-buffered saline (PBS) once and re-suspended into completed NK cells medium. Subsequently, they were seeded into a U-bottom 96-well plate at the concentration of 2×10^4 cells/well. APC-Cy7-conjugated CD107a antibody (Biolegend, USA) were added in and thoroughly mixed with the cells. NK cells were then cocultured at a 1:1 E:T ratio with target cells for 5 h. After 1 h of incubation, Golgi stop containing monensin (BD Biosciences) was added. Following 4 additional hours of incubation, the cells were used for anti-CD56 APC and anti-CD3 FITC antibody staining. The cells were resuspended with 100 µL FACS buffer for the detection of the CD107a surface expression on NK cells by NoveExpress software. In vivo functional studies of expanded NK cells in xenografted mice To evaluate the potential therapeutic effect of expanded NK cells for cancer therapy, an acute leukemia mouse model was employed. Female NSG mice, aged 6–8 weeks, were procured from the Saiye Model Biology Research Center Co. The mice were housed and maintained in individual ventilated cages under specific pathogen-free conditions, with adequate access to food and water. The mice were injected via the tail vein with U937 cells (1×10 6 U937 cells/mouse) mixed with either PBS or NK cells (1×10 7 ). Seven days post-injection, mice were injected with D-fluorescein potassium salt (150 mg/kg) and the bioluminescence signal was analyzed using an IVIS® Lumina II Multispectral Imaging System (PerkinElmer, USA) to verify the tumor cell engraftment. Tumor progression was monitored weekly through bioluminescence imaging (BLI). Statistics Group differences were analyzed using one-way analysis of variance (ANOVA) or Student’ s t -test. Each experiment was conducted with a minimum of three independent replicates. The survival distributions were estimated by the Kaplan-Meier method and compared with the use of log-rank test between groups. The data were performed using SPSS software (version 23.0). The following denotations for significance levels were used: *p < 0.05, **p < 0.01. Results Generation of K562-4-1BBL-mbIL-21/-15 feeder cells 4-1BBL, whose receptor is a strong co-stimulatory molecule expressed on NK cells, could trigger proliferation and activation of NK cells by receptor-ligand interactions. Surface expression of IL-21 and IL-15 is not only preventing NK cells form senescence, but also enhancing their survival and expansion in vitro. The K562 cell line, characterized by its lack of MHC-1 expression, serves as an ideal feeder cell line for NK cell expansion, presenting a prime target for NK cell-mediated cytotoxicity due to its inherent characteristics. We engineered a novel NK feeder cell line by transducing K562 cells with mbIL-21/-15 and 4-1BBL. The construction of the final plasmid, incorporating 4-1BBL and mbIL-21/-15, was shown in Fig. 1 a. After transduction and antibiotic selection using two antibiotics (puromycin and G418), FACS analysis revealed that K562-4-1BBL-mbIL-21/-15 feeder cells had a high surface expression of 4-1BBL, IL-21, and IL-15, whereas the conventional K562 cells showed negligible expression of surface IL-21 and IL-15, only low expression of 4-1BBL (Fig. 1 b). K562-4-1BBL-mbIL-21/-15 feeder cells induced vigorous NK cell expansion The expansion protocol was shown in Fig. 2 a. To initiate NK cell expansion from peripheral blood, isolated PBMCs were co-cultured with frozen-thawed irradiated K562-4-1BBL-mbIL-21/-15 feeder cells, introduced at the onset of the culture period and again on day 7. The cell types during the expansion were analyzed by FACS according to the following gating strategy (Fig. 2 b): size (FSC/SSC), single cells (FSC-H/FSC-A), living lymphocytes (PI/CD45), NK cells (CD3-CD56+), T cells (CD3 + CD56-), NKT cells (CD3 + CD56+) and B cells (CD3-CD19+). After two-week culture, the median expansion fold of NK cells from 5 healthy donors’ PBMCs was 17902 (4534–31504) (Fig. 2 c). By day 14, the median purity of NK cells reached an average of 93.51 (95% CI: 90.97–95.64), with over 90% characterized as CD56 + CD16 + cells. CD3 + T cells constituted less than 5% of the cell population (95% CI: 0.09–3.18), and B cells were virtually undetectable by day 7. The residual NKT cells made up approximated 3% (95% CI: 0.27–5.15) and they showed a trend of first rising and then falling during expansion (Fig. 2 d). Expression levels of receptors on expanding NK cells As NK cell function was regulated by an array of activating and inhibitory receptors, we dynamically monitored the expression of activating receptors (NKG2D, NKp30, NKp44, NKp46, and CD226) and inhibitory receptors (NKG2A and CD158b), along with the activation marker CD69 (Fig. 3 ). These parameters were analyzed at the beginning and the end of expansion process. On day 14, NK cells showed increased expression of almost estimated activating receptors (NKG2D, NKp30, NKp44, NKp46) and the activation marker CD69 compared to baseline, indicating enhanced activation potential. Notably, the expression of the inhibitory receptor NKG2A also increased by day 14. Changes in the expression of CD226 (an activating receptor) and CD158b (an inhibitory receptor) were variable and had no statistical significance during the expansion period. Cytotoxicity of expanded NK cells in vitro The cytotoxicity of the expanded NK cells was evaluated using luciferase release assay with the results presented in Fig. 4 a. Compared with unexpanded NK cells, the expanded NK cells on day 14 demonstrated effective lysis against several hematological cancer cell lines, including K562, MOLM-13, OCI-AML3, THP-1 and U937. At the E:T-ratio of 5:1, the expanded NK cells showed extremely strong cytotoxicity (> 80%) against all of the estimated targeted cells after the overnight incubation. Even at the low E:T-ratio of 0.5:1, the expanded NK cells effectively killed more than 20% K562, MOLM-13 and OCI-AML-3 cells. In contrast, the cytotoxicity of the expanded NK cells on THP-1 were relatively weaker, but also more than 40% at the low E:T-ratio of 1:1. Only U937 cells, could not be efficiently killed by expanded NK cells at the lower E:T-ratio of 1:1 and 0.5:1. The expanded NK cells exhibited potent lytic activity specifically targeting solid cancer cell lines, including Hep-G2 and OVCAR3. The cytotoxicity was more than 50% even at the low E:T-ratio of 0.5:1. Meanwhile, NK cells displayed greater degranulation (CD107a) against their targets ( solid and hematological cancer cell lines) after expansion (Fig. 4 b). Expanded NK cells reduced tumor burden and prolonged the survival of U937-engrafted NSG mice The anti-leukemia effect of the expanded NK cells was analyzed using a humanized U937 xenograft mouse model. This model was chosen, as the cytotoxicity of NK cells against U937 NK cells was comparatively weaker at low effector-to-target (E:T) ratios in vitro. This selection criterion aimed to provide a robust challenge to the expanded NK cells, with the rationale that demonstrating enhanced in vivo outcomes against U937 xenografts could more accurately reflect the functional capabilities of NK cells. NSG mice were intravenously infused with a simultaneous injection of 1×10 6 U937-Luc cells and 1×10 7 of the ex vivo expanded NK cells. Remarkably, we not only observed a reduction of tumor burden with NK cells administration, but the median survival time of the AML-bearing mice in NK cells group (28.25 days) was significantly extended compared to the control group (19.40 days) (p < 0.01) (Fig. 5 a-d). By day 7, human NK cells were successfully detected within the U937 xenograft mice and was 6.80% ± 4.28. However, the persistence of NK cells was lower and it could not be detected by day 14. Discussion In this study, we introduced an innovative, efficient, and cost-effective NK cell expansion platform—K562-4-1BBL-mbIL-21/-15 feeder cells, which could expand NK cells starting directly from PBMCs and manufacture sufficient clinical-grade NK cells within just two weeks. It is important to note that the expanded NK cells exhibited potent anti-tumor activity both in vitro and in vivo. Since most of the previous regimens expanded NK cells from isolated purified NK cells, the rapid and robust NK cells expansion from PBMC in this study significantly reduced the healthy donors’ amount of blood collection, as well as saving time and high cost of magnetic beads sorting. In addition, the ability to produce therapeutic NK cells within a two-week timeframe could expedite treatment delivery, substantially shortening the waiting period for patients in need of NK cell-based immunotherapy. Evelyn Ullrich’s group reported a cytokine-based expansion protocol combining IL-15 and IL-21 improved both the proliferation and cytotoxicity of NK cells [ 7 ] . Their protocol initiated with IL-15 to drive early NK cell expansion, followed by a brief exposure to IL-21, which notably increased NK cell cytotoxicity. This cytokine-based method demonstrated the potent synergistic effect of IL-15 and IL-21 in augmenting NK cell expansion. However, an intriguing discrepancy arises concerning the role of IL-21. Ullrich’s group observed that the long-term presence of IL-21 negatively influenced NK cell proliferation capacity. This view may be true in cytokine-based method. Nevertheless, the application of mbIL-21 in feeder cell-based NK cell expansion has been reliably demonstrated to be effective [ 10 , 17 ] . This suggested that the delivery form of IL-21—soluble or membrane-bound—plays different role in influencing NK cell behavior. While the quantity and purity of NK cells in the final product are critical metrics of success, the pivotal determinant of therapeutic efficacy lies in their activation and subsequent anti-tumor immunity. Our findings reveal that the expanded NK cells exhibited upregulated activating receptors (Fig. 3 ), especially for NKG2D, which was the most important activating receptor of NK cells and implicated stronger cytotoxic response to malignant cells [ 18 ] . However, an increase in the expression of the inhibitory receptor NKG2A was also noted. Although several studies suggested that the silencing or knock-out of NKG2A was an alternative strategy for caner immunotherapy [ 19 , 20 ] , a novel role of NKG2A was recently reported. Obinna Chijioke’s findings showed that NKG2A played a crucial role in preserving the expansion capacity of NK cells by moderating their proliferative activity and mitigating excessive activation-induced cell death [ 21 ] . Similarly, in a study utilizing PC 3 PSCA-IL-2-4-1BBL-mbbIL-15d feeder cells for NK cell expansion, Achim Temme observed an increase in NKG2A expression, highlighting its importance in maintaining NK cell tolerance to self [ 15 ] . These observations could illustrate the reason why NKG2A increased in the process of NK cells expansion. More importantly, the balance between amplifying NK cell numbers and their anti-tumor activity associated with NKG2A need to consider in future cancer immunotherapy studies. Consistent with the observed upregulation of activating receptors, our expanded NK cells demonstrated potent anti-tumor effects against hematological and solid cancers in subsequent in vitro experiments (Fig. 4 ). Moreover, these cells exhibited significant anti-tumor activity in vivo. Notably, we utilized a xenograft mouse model established with U937 cells—a cell line not highly sensitive to NK cell-mediated cytotoxicity in vitro. This further underscores the robustness of the anti-tumor response elicited by the expanded NK cells, highlighting their potential therapeutic efficacy even against targets that are less inherently susceptible to NK cell attack. In conclusion, in this study we explored a novel platform for expanding NK cells from healthy donors’ PBMCs using K562-4-1BBL-mbIL-21/-15 feeder cell, providing a highly efficient, rapid, straightforward, and cost-effective method for NK cell expansion. We demonstrated that NK cells expanded through this regimen exhibit potent anti-tumor effects both in vitro and in vivo. Our findings highlight the potential of this ex vivo NK cell expansion platform to produce large quantities of highly active, clinical-grade NK cells, thereby significantly promoting the development of NK cell-based immunotherapy. Declarations Ethics approval and consent to participate The experiment was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University in Xi'an, China (Approval no. XJTUAE2023-1883). Consent for publication All authors have read and approved the final manuscript. Competing interests The authors declare no competing interests. Funding This study was supported by Natural Science Foundation of Shaanxi Province (grant no. 2024SF-YBXM-148) and Bethune Charity Foundation (grant no. BCF-IBW-XY-20231023-08). Author Contribution Huaiyu Wang and Xiaohu Fan contributed to the study conception, design and methodology. Sha Gong and Nan Mei performed the material preparation, data collection and analysis. Sha Gong, Nan Mei, Jun Wang, Lu Wang and Junsheng Zhu written the first draft of the manuscript. Wenjuan Wang, Jincheng Wang, Xiaohong Lu, Pengcheng He, Weiwei Chen, Lei Xi, Yingying Bao and David N. Wald performed interpretation of data. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of data and materials The data are available from the corresponding author on reasonable request. References Liu E, Marin D, Banerjee P et al (2020) Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors[J]. 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J Immunother 36(7):373–381 Li X, He C, Liu C et al (2015) Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21.[J]. Int J Oncol 47(1):335–342 Caignard A (2019) NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment[J]. Front Immunol, 10 Albinger N, Bexte T, Buchinger L et al (2022) CRISPR/Cas9 Gene Editing of Immune Checkpoint Receptor NKG2A Improves the Efficacy of Primary CD33-CAR-NK Cells Against AML[J]. Blood 140(Supplement 1):4558–4559 André P, Denis C, Soulas C et al (2018) Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.[J]. Cell 175(7):1731–1743e13 Kaulfuss M, Mietz J, Fabri A et al (2023) The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells.[J]. Sci Rep 13(1):10555 Additional Declarations No competing interests reported. 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Wald","email":"","orcid":"","institution":"Case Western Reserve University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"N.","lastName":"Wald","suffix":""},{"id":335086000,"identity":"ca022caf-ae08-4bec-a1a6-dd165f81e22e","order_by":11,"name":"Xiaohu Fan","email":"","orcid":"","institution":"Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohu","middleName":"","lastName":"Fan","suffix":""},{"id":335086001,"identity":"7eb89658-0516-43c2-b9e4-0dcdcf650452","order_by":12,"name":"Huaiyu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACCQbGBww8DAzsM4AcHiK1MBsw8ADRDRK0sEkwMJCixeB2j1k1j8yfxB7pBsYHb9sY5M0JarlzxuzmDB6DxB6ZA8yGc9sYDHc2ENBidiPH7MYHoJb9Egls0rxtDAkGB4jQUpAAskUigf030VoYPkC0sDETpcX+Rlqx5AweY+MeicRmyTnnJAw3ENIiOSN542feHjnZHonkgx/elNnIE7SFgYHDgIGxB8RgbGAARRMRgP0BA8MPYhSOglEwCkbBiAUA4Fg86WbJ/VUAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Huaiyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-07-15 09:51:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4741998/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4741998/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62142712,"identity":"15e503c9-6799-4293-b12b-d3466c4f7516","added_by":"auto","created_at":"2024-08-09 17:51:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":631174,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of K562-4-1BBL-mbIL-21/-15 feeder cell line. (A) Schematic representation of 4-1BBL, mbIL-21 and mbIL-15 coding sequences in the lentiviral vector. (B) The surface expression of 4-1BBL, mbIL-21 and mbIL-15 was determined by flow cytometry analysis.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/d754d9c1b0a38f5b551b82d9.png"},{"id":62142410,"identity":"c0057c75-6f9e-4352-87a5-60cc5610cf7a","added_by":"auto","created_at":"2024-08-09 17:43:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":748771,"visible":true,"origin":"","legend":"\u003cp\u003eExpansion of primary NK cells from PBMC with K562-4-1BBL-mbIL-21/-15 feeder cell line. (A) Schema of the NK cell expansion platform. (B) The purity of the expanded NK cells. Representative flow plots depicting purity of NK cells on day 0 and day 14. (C) Fold expansion of NK cells within 14 days, n=5. (D) Relative fractions of cell types during two-week expansion, n=5.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/8a4258c0d28b227556cbf011.png"},{"id":62142411,"identity":"a8b22c81-eade-495a-a75d-dde067bf335c","added_by":"auto","created_at":"2024-08-09 17:43:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":468203,"visible":true,"origin":"","legend":"\u003cp\u003eThe NK cell activating and inhibitory receptors changes in cell surface phenotype (*p \u0026lt; 0.05, **p \u0026lt; 0.01, n=5).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/09e3d21cdce9d9100a72588d.png"},{"id":62142413,"identity":"04373902-a315-4a6d-88ef-51deebd6c7e9","added_by":"auto","created_at":"2024-08-09 17:43:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":470622,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of the unexpanded and expanded NK cells against tumor cells at different E:T ratios, and 12-16 h coculture in vitro (n = 3). (A) Killing rates of the NK cells against solid and hematological tumor cell lines (including K562, MOLM-13, OCI-AML-3, THP-1, U937, Hep-G2 and OVCAR3). (B) Bar plots summarize the CD107a degranulation by unexpanded NK cells (blue bars), expanded NK cells (red bars) after coculture at a 1:1 E:T ratio with target cells for 5 hours (n=3). Statistical significance is indicated as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.01; bars represent mean values with standard deviation.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/baeacee064da9e7a036d1333.png"},{"id":62142412,"identity":"b44805f0-f5ba-4de7-a5d4-3445e86c3729","added_by":"auto","created_at":"2024-08-09 17:43:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1011916,"visible":true,"origin":"","legend":"\u003cp\u003eExpanded NK cells exhibited in vivo anti-leukemic activity. (A) Schematic diagram of a mouse experiment assessing the antitumor effects of expanded NK cells in immune-deficient NSG mice, n = 5. (B) Tumor growth was assessed by bioluminescence imaging (BLI) on a weekly basis. (C) Tumor burden over time by BLI after the treatment. (D) Kaplan-Meier analysis of survival, statistical analysis of survival between groups was performed using the log rank test.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/7b8f8290d0d90beb796bdb8f.png"},{"id":66598673,"identity":"efb9ee27-0ef7-47aa-b7d3-60b564f87b9e","added_by":"auto","created_at":"2024-10-14 16:20:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4403295,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4741998/v1/d6928dc0-a72a-4e88-8904-30c2a888982c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Feeder cell based on 4-1BBL and Membrane-bound IL-21/ IL-15 Induce Highly Expansion and Anti-tumor Effect of Natural Killer Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNatural killer (NK) cell immunotherapy has emerged as a new frontier in cancer treatment. Various therapeutic modalities, such as adoptive NK cell transfer, chimeric antigen receptor (CAR)-NK cells and NK cell engagers, are carried out and being investigated in clinical trials in a wide array of solid and hematological malignancies. Particularly noteworthy are anti-CD19 CAR-NK cells, which have demonstrated remarkable success in treating CD19\u0026thinsp;+\u0026thinsp;B-cell malignancies\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, earning FDA approval and triggering a wave of research on NK cell-based therapies. However, the widespread clinical application of NK cell therapy has been limited by the challenge in manufacturing large-scale and clinical-grade expansion of NK cells, which remains a critical bottleneck in the development of \u0026ldquo;off-the-shelf\u0026rdquo; NK cell immunotherapies.\u003c/p\u003e \u003cp\u003eGenerating adequate numbers of functional NK cells in vitro is a major challenge, as NK cells constitute only 10\u0026ndash;20% of peripheral blood mononuclear cells (PBMC)\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In addition, the in vivo lifespan of NK cells is relatively short, generally limited to around two weeks\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Given the modest initial quantity of NK cells and the constraints on their proliferation time, it is essential to undertake a thorough in vitro expansion process to achieve the necessary therapeutic dosage.\u003c/p\u003e \u003cp\u003eWhile several methods for NK cells expansion have been described, each with its specific advantages and limitations. Cytokine-based regimens demonstrate a low expansion fold (\u0026lt;\u0026thinsp;30-fold expansion within 3 weeks) due to NK cell senescence\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, which was insufficient to meet the dosage requirements for patient treatment. In contrast, feeder cell-based approaches, particularly using irradiated K562 cells engineered to express activation signals (4-1BBL or OX40L) and membrane-bound (mb) cytokines (mbIL-21or mbIL-15 or mbIL-18), have demonstrated robust NK cell proliferation, with potential expansion rates approximately tens of thousands of folds\u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, these methods generally needed prolonged culture periods ranging from 3 to 5 weeks. Evidence suggested that mbIL-21 could block the process of NK cell senescence\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, and mbIL-15 could enhance the NK cell survival and augment their cytotoxicity towards tumor cells in vitro and vivo\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Yet, the combined effects of mbIL-21 and mbIL-15 within feeder cell design was rarely reported.\u003c/p\u003e \u003cp\u003eThe aim of this study is to design a novel feeder cell simultaneously co-expressing mbIL-21, mbIL-15 and 4-1BBL to expand NK cells in vitro from PBMCs, thereby creating a rapid, highly efficient, cost-efficient, and straightforward platform for NK cell proliferation. We have recently utilized this platform to construct and manufacture NKG2D-CAR-NK cells for acute myeloid leukemia. It successfully completed the preclinical research and will start phase 1 clinical trial as soon.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eK562, THP-1 cells were purchased from Procell (Wu Han, China). OCI-AML3, U937, MOLM-13, Hep-G2, OVCAR3, 293T cells were provided by the Hematology Lab or Gene and Cell Therapy Research Institute at The First Affiliated Hospital of Xi'an Jiaotong University. K562, THP-1, OCI-AML3, U937 cells were maintained in PRMI-1640 medium (HyClone, USA) with 10% fetal bovine serum (FBS, ExCell Bio, China). Hep-G2 cells were maintained in MEM medium (HyClone, USA) with 10% FBS. OVCAR3 were maintained in PRMI-1640 medium and 10 \u0026micro;g/mL insulin with 20% FBS. 293T cells were used for lentiviral packaging and cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, USA) with 10% FBS. Mycoplasma testing was performed on all cell lines once every two weeks using the MycoAlert Mycoplasma Detection Kit (Lonza, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eTo construct mbIL-21, the coding sequence of mature human IL-21 was directly fused to the human CD8a hinge, transmembrane and cytoplasmic domains. The mbIL-15 was created by linking human IL-15 with its receptor IL-15RA. The codon-optimized sequences for mbIL-21 and mbIL-15 were synthesized and linked using the T2A self-cleaving peptide. This synthetic fusion sequence of mbIL-21/-15 was then cloned into the pLVX-EF1α-IRES-Neo vector (Miaoling Biology, China) using the EcoR1 and BamH1 restriction sites, resulting in the recombinant plasmid pLVX-EF1α-mbIL-21/-15-IRES-Neo (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Given that human 4-1BBL functions as a membrane protein, its codon-optimized sequence was also synthesized and subsequently inserted into the pSIN-EF1α-IRES-Puro vector (Miaoling Biology, China) to create the overexpression plasmid pSIN-EF1α-4-1BBL-IRES-Puro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eK562 feeder cells co-expressing 4-1BBL, mbIL-21 and mbIL-15 construction\u003c/h2\u003e \u003cp\u003eLentiviral particles were produced by co-transfecting 293T cells with the respective lentiviral transfer plasmids (pLVX-EF1α-mbIL-21/-15-IRES-Neo or pSIN-EF1α-4-1BBL-IRES-Puro) along with the packaging plasmids (psPAX2 and pMD2.G). Three days following transfection, the viral supernatants were collected and concentrated by ultracentrifugation at 100,000 g for 2 hours. The concentrated viral particles were resuspended in RPMI-1640 medium and stored at -80\u0026deg;C until further use. To determine the viral titer, 293T cells were transduced with varying volumes of the viral solutions. After three days, the percentage of 4-1BBL\u0026thinsp;+\u0026thinsp;or IL-21/IL-15\u0026thinsp;+\u0026thinsp;cells were estimated by flow cytometry. Titers was calculated using the following formula: Titer (IU/mL) = (Number of infected cells \u0026times; Percentage of positive cells) / Volume of virus used (mL).\u003c/p\u003e \u003cp\u003eK562 cells were seeded into 6-well plates at 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL and subsequently transduced with the concentrated virus stocks containing mbIL-21/-15 and 4-1BBL at a multiplicity of infection (MOI) of 2 in the presence of protamine sulfate (10 \u0026micro;g/mL; Solarbio, China). Three days later, the transduced cells were selected with puromycin (2 \u0026micro;g/mL) and G418 (600 \u0026micro;g/mL) for two weeks. The transduction efficacy was analyzed by flow cytometry using APC-conjugated anti-human 4-1BBL monoclonal antibodies (mAb) (Biolegend, USA), PE-conjugated anti-human IL-21 and IL-15 mAbs (eBioscience, USA). The stable feeder cell line co-expressing 4-1BBL and mbIL-21/-15 (K562-4-1BBL-mbIL-21/-15) was maintained in RPMI 1640 with 10% FBS and low concentration of puromycin and G418. Upon reaching sufficient cell numbers, these feeder cells were irradiated with 100 Gy gamma rays by Cs-137 gamma to inhibit further cell division, then cryopreserved with 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/vial in liquid nitrogen, creating a ready-to-use irradiated K562-4-1BBL-mbIL-21/-15 cell bank for future NK cell expansion endeavors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNK cells expansion using K562-4-1BBL-mbIL-21/-15 feeder cells\u003c/h2\u003e \u003cp\u003ePBMCs were isolated form the peripheral blood of healthy donors by Ficoll gradient centrifugation (GE Healthcare, USA). 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e PBMCs were directly co-cultured with frozen-thawed irradiated K562-4-1BBL-mbIL-21/-15 feeder cells at a ratio of 1:1 in a T75 flask containing 20 mL of fresh medium. The medium was composed of a serum-free medium (Hangzhou Zhongying Bio-Medical Technology, China) supplemented with 5% heat-inactivated autologous plasma and recombinant human IL-2 (200 U/mL; Jiangsu Kingsley Pharmaceutical Co., Ltd, China). Three days post-culture, the cell suspension was centrifuged at 350 g for 5 minutes, and then the supernatant was discarded and equal amount of fresh medium was added. Up until day 7, fresh medium was supplemented every two days to maintain the cell density between 1.0-1.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. On day 7, a second of frozen-thawed K562-4-1BBL-mbIL-21/-15 feeder cells were added for re-stimulation. Subsequently, the percentage of heat-inactivated autologous plasma in the medium was reduced from 5\u0026ndash;1%, and the cell densities were adjusted to be within 1.5\u0026ndash;2.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. Cells were harvest on day 13 or 14, and the expansion fold was calculated by the absolute number of NK cells on day 14 by the respective number on day 0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eThe purity of NK cells on days 0, 7 and 14 was examined by flow cytometry using FITC-conjugated anti-human CD3 mAb and APC- conjugated anti-human CD56 mAb (Biolegend, USA). The expression levels of various NK cell receptors were analyzed using PE- conjugated anti-human NKG2D, NKp30, NKp46 mAbs (BD Biosciences, USA), PE-Cy7 conjugated anti-human NKp44, CD69, CD158b mAbs (Biolegend, USA), and APC-Cy7 conjugated anti-human NKG2A and CD16 mAbs (Biolegend, USA). For all samples, PBMC or the expending cells (1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells) were washed with FACS buffer (MACS rinsing solution with 0.2% BSA; Miltenyi Biotec, Germany) and resuspended with 100 \u0026micro;L FACS buffer in a 96 well U-bottom plate. To block non-specific binding, Human Fc-receptor Blocking Solution (Biolegend, USA) was applied to each sample for 5 minutes at room temperature. Subsequently, the samples were stained with the designated mAbs for 20 minutes at 4\u0026deg;C in the dark. Following staining, cells were washed twice with FACS buffer and then were resuspended with 100 \u0026micro;L FACS buffer. To assess cell viability, 10 \u0026micro;L Propidium Iodide (PI) were added to each sample, followed by incubation for 5 minutes at 4 ℃ in the dark. The data was required and analyzed using NoveExpress software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assay\u003c/h2\u003e \u003cp\u003eNK cell cytotoxicity was assessed using the One Glo Luciferase Assay System (Promega, USA). The stable luciferase-expressing cell lines (K562-Luc, Molm-13-Luc, THP-1-Luc, OCI-AML3-Luc, U937-Luc, Hep-G2-Luc, OVCAR-Luc) were prepared to serve as target cells. The unexpanded and expanded NK cells were co-cultured with each targeted cells at effector-to-target (E:T) ratio of 5:1, 2:1, 1:1 and 0.5:1 in a 384 well plate with total amount of 30 \u0026micro;L for 12\u0026ndash;16 hours in triplicate. After co-culture, 30 \u0026micro;L of the luciferase ONE-Glo Reagent was added to each well containing the NK/target cell mixture. The plate was incubated at room temperature for 3 minutes to allow complete cell lysis. Finally, the luminescence was measured in a luminometer. The cytotoxicity was calculated using the following formula:\u003c/p\u003e \u003cp\u003eCytotoxicity (%) = [((Luminescence (target cells only) - Luminescence (effector cells only))- ((Luminescence (target cells with effector cells) - Luminescence (effector cells only))] / [Luminescence (target cells only) - Luminescence (effector cells only)] \u0026times;100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCD107a degranulation assay\u003c/h2\u003e \u003cp\u003eThe unexpanded and expanded NK cells were washed with phosphate-buffered saline (PBS) once and re-suspended into completed NK cells medium. Subsequently, they were seeded into a U-bottom 96-well plate at the concentration of 2\u0026times;10^4 cells/well. APC-Cy7-conjugated CD107a antibody (Biolegend, USA) were added in and thoroughly mixed with the cells. NK cells were then cocultured at a 1:1 E:T ratio with target cells for 5 h. After 1 h of incubation, Golgi stop containing monensin (BD Biosciences) was added. Following 4 additional hours of incubation, the cells were used for anti-CD56 APC and anti-CD3 FITC antibody staining. The cells were resuspended with 100 \u0026micro;L FACS buffer for the detection of the CD107a surface expression on NK cells by NoveExpress software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo functional studies of expanded NK cells in xenografted mice\u003c/h2\u003e \u003cp\u003eTo evaluate the potential therapeutic effect of expanded NK cells for cancer therapy, an acute leukemia mouse model was employed. Female NSG mice, aged 6\u0026ndash;8 weeks, were procured from the Saiye Model Biology Research Center Co. The mice were housed and maintained in individual ventilated cages under specific pathogen-free conditions, with adequate access to food and water.\u003c/p\u003e \u003cp\u003eThe mice were injected via the tail vein with U937 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e U937 cells/mouse) mixed with either PBS or NK cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e). Seven days post-injection, mice were injected with D-fluorescein potassium salt (150 mg/kg) and the bioluminescence signal was analyzed using an IVIS\u0026reg; Lumina II Multispectral Imaging System (PerkinElmer, USA) to verify the tumor cell engraftment. Tumor progression was monitored weekly through bioluminescence imaging (BLI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eGroup differences were analyzed using one-way analysis of variance (ANOVA) or Student\u0026rsquo; s \u003cem\u003et\u003c/em\u003e-test. Each experiment was conducted with a minimum of three independent replicates. The survival distributions were estimated by the Kaplan-Meier method and compared with the use of log-rank test between groups. The data were performed using SPSS software (version 23.0). The following denotations for significance levels were used: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of K562-4-1BBL-mbIL-21/-15 feeder cells\u003c/h2\u003e \u003cp\u003e4-1BBL, whose receptor is a strong co-stimulatory molecule expressed on NK cells, could trigger proliferation and activation of NK cells by receptor-ligand interactions. Surface expression of IL-21 and IL-15 is not only preventing NK cells form senescence, but also enhancing their survival and expansion in vitro. The K562 cell line, characterized by its lack of MHC-1 expression, serves as an ideal feeder cell line for NK cell expansion, presenting a prime target for NK cell-mediated cytotoxicity due to its inherent characteristics. We engineered a novel NK feeder cell line by transducing K562 cells with mbIL-21/-15 and 4-1BBL. The construction of the final plasmid, incorporating 4-1BBL and mbIL-21/-15, was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. After transduction and antibiotic selection using two antibiotics (puromycin and G418), FACS analysis revealed that K562-4-1BBL-mbIL-21/-15 feeder cells had a high surface expression of 4-1BBL, IL-21, and IL-15, whereas the conventional K562 cells showed negligible expression of surface IL-21 and IL-15, only low expression of 4-1BBL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eK562-4-1BBL-mbIL-21/-15 feeder cells induced vigorous NK cell expansion\u003c/h2\u003e \u003cp\u003eThe expansion protocol was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. To initiate NK cell expansion from peripheral blood, isolated PBMCs were co-cultured with frozen-thawed irradiated K562-4-1BBL-mbIL-21/-15 feeder cells, introduced at the onset of the culture period and again on day 7. The cell types during the expansion were analyzed by FACS according to the following gating strategy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb): size (FSC/SSC), single cells (FSC-H/FSC-A), living lymphocytes (PI/CD45), NK cells (CD3-CD56+), T cells (CD3\u0026thinsp;+\u0026thinsp;CD56-), NKT cells (CD3\u0026thinsp;+\u0026thinsp;CD56+) and B cells (CD3-CD19+). After two-week culture, the median expansion fold of NK cells from 5 healthy donors\u0026rsquo; PBMCs was 17902 (4534\u0026ndash;31504) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). By day 14, the median purity of NK cells reached an average of 93.51 (95% CI: 90.97\u0026ndash;95.64), with over 90% characterized as CD56\u0026thinsp;+\u0026thinsp;CD16\u0026thinsp;+\u0026thinsp;cells. CD3\u0026thinsp;+\u0026thinsp;T cells constituted less than 5% of the cell population (95% CI: 0.09\u0026ndash;3.18), and B cells were virtually undetectable by day 7. The residual NKT cells made up approximated 3% (95% CI: 0.27\u0026ndash;5.15) and they showed a trend of first rising and then falling during expansion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExpression levels of receptors on expanding NK cells\u003c/h2\u003e \u003cp\u003eAs NK cell function was regulated by an array of activating and inhibitory receptors, we dynamically monitored the expression of activating receptors (NKG2D, NKp30, NKp44, NKp46, and CD226) and inhibitory receptors (NKG2A and CD158b), along with the activation marker CD69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These parameters were analyzed at the beginning and the end of expansion process. On day 14, NK cells showed increased expression of almost estimated activating receptors (NKG2D, NKp30, NKp44, NKp46) and the activation marker CD69 compared to baseline, indicating enhanced activation potential. Notably, the expression of the inhibitory receptor NKG2A also increased by day 14. Changes in the expression of CD226 (an activating receptor) and CD158b (an inhibitory receptor) were variable and had no statistical significance during the expansion period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity of expanded NK cells in vitro\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of the expanded NK cells was evaluated using luciferase release assay with the results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Compared with unexpanded NK cells, the expanded NK cells on day 14 demonstrated effective lysis against several hematological cancer cell lines, including K562, MOLM-13, OCI-AML3, THP-1 and U937. At the E:T-ratio of 5:1, the expanded NK cells showed extremely strong cytotoxicity (\u0026gt;\u0026thinsp;80%) against all of the estimated targeted cells after the overnight incubation. Even at the low E:T-ratio of 0.5:1, the expanded NK cells effectively killed more than 20% K562, MOLM-13 and OCI-AML-3 cells. In contrast, the cytotoxicity of the expanded NK cells on THP-1 were relatively weaker, but also more than 40% at the low E:T-ratio of 1:1. Only U937 cells, could not be efficiently killed by expanded NK cells at the lower E:T-ratio of 1:1 and 0.5:1. The expanded NK cells exhibited potent lytic activity specifically targeting solid cancer cell lines, including Hep-G2 and OVCAR3. The cytotoxicity was more than 50% even at the low E:T-ratio of 0.5:1. Meanwhile, NK cells displayed greater degranulation (CD107a) against their targets ( solid and hematological cancer cell lines) after expansion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExpanded NK cells reduced tumor burden and prolonged the survival of U937-engrafted NSG mice\u003c/h2\u003e \u003cp\u003eThe anti-leukemia effect of the expanded NK cells was analyzed using a humanized U937 xenograft mouse model. This model was chosen, as the cytotoxicity of NK cells against U937 NK cells was comparatively weaker at low effector-to-target (E:T) ratios in vitro. This selection criterion aimed to provide a robust challenge to the expanded NK cells, with the rationale that demonstrating enhanced in vivo outcomes against U937 xenografts could more accurately reflect the functional capabilities of NK cells. NSG mice were intravenously infused with a simultaneous injection of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e U937-Luc cells and 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e of the ex vivo expanded NK cells. Remarkably, we not only observed a reduction of tumor burden with NK cells administration, but the median survival time of the AML-bearing mice in NK cells group (28.25 days) was significantly extended compared to the control group (19.40 days) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). By day 7, human NK cells were successfully detected within the U937 xenograft mice and was 6.80% \u0026plusmn; 4.28. However, the persistence of NK cells was lower and it could not be detected by day 14.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we introduced an innovative, efficient, and cost-effective NK cell expansion platform\u0026mdash;K562-4-1BBL-mbIL-21/-15 feeder cells, which could expand NK cells starting directly from PBMCs and manufacture sufficient clinical-grade NK cells within just two weeks. It is important to note that the expanded NK cells exhibited potent anti-tumor activity both in vitro and in vivo. Since most of the previous regimens expanded NK cells from isolated purified NK cells, the rapid and robust NK cells expansion from PBMC in this study significantly reduced the healthy donors\u0026rsquo; amount of blood collection, as well as saving time and high cost of magnetic beads sorting. In addition, the ability to produce therapeutic NK cells within a two-week timeframe could expedite treatment delivery, substantially shortening the waiting period for patients in need of NK cell-based immunotherapy.\u003c/p\u003e \u003cp\u003eEvelyn Ullrich\u0026rsquo;s group reported a cytokine-based expansion protocol combining IL-15 and IL-21 improved both the proliferation and cytotoxicity of NK cells\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Their protocol initiated with IL-15 to drive early NK cell expansion, followed by a brief exposure to IL-21, which notably increased NK cell cytotoxicity. This cytokine-based method demonstrated the potent synergistic effect of IL-15 and IL-21 in augmenting NK cell expansion. However, an intriguing discrepancy arises concerning the role of IL-21. Ullrich\u0026rsquo;s group observed that the long-term presence of IL-21 negatively influenced NK cell proliferation capacity. This view may be true in cytokine-based method. Nevertheless, the application of mbIL-21 in feeder cell-based NK cell expansion has been reliably demonstrated to be effective\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This suggested that the delivery form of IL-21\u0026mdash;soluble or membrane-bound\u0026mdash;plays different role in influencing NK cell behavior.\u003c/p\u003e \u003cp\u003eWhile the quantity and purity of NK cells in the final product are critical metrics of success, the pivotal determinant of therapeutic efficacy lies in their activation and subsequent anti-tumor immunity. Our findings reveal that the expanded NK cells exhibited upregulated activating receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), especially for NKG2D, which was the most important activating receptor of NK cells and implicated stronger cytotoxic response to malignant cells\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, an increase in the expression of the inhibitory receptor NKG2A was also noted. Although several studies suggested that the silencing or knock-out of NKG2A was an alternative strategy for caner immunotherapy\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, a novel role of NKG2A was recently reported. Obinna Chijioke\u0026rsquo;s findings showed that NKG2A played a crucial role in preserving the expansion capacity of NK cells by moderating their proliferative activity and mitigating excessive activation-induced cell death\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Similarly, in a study utilizing PC\u003csup\u003e3\u003c/sup\u003ePSCA-IL-2-4-1BBL-mbbIL-15d feeder cells for NK cell expansion, Achim Temme observed an increase in NKG2A expression, highlighting its importance in maintaining NK cell tolerance to self \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. These observations could illustrate the reason why NKG2A increased in the process of NK cells expansion. More importantly, the balance between amplifying NK cell numbers and their anti-tumor activity associated with NKG2A need to consider in future cancer immunotherapy studies.\u003c/p\u003e \u003cp\u003eConsistent with the observed upregulation of activating receptors, our expanded NK cells demonstrated potent anti-tumor effects against hematological and solid cancers in subsequent in vitro experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, these cells exhibited significant anti-tumor activity in vivo. Notably, we utilized a xenograft mouse model established with U937 cells\u0026mdash;a cell line not highly sensitive to NK cell-mediated cytotoxicity in vitro. This further underscores the robustness of the anti-tumor response elicited by the expanded NK cells, highlighting their potential therapeutic efficacy even against targets that are less inherently susceptible to NK cell attack.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study we explored a novel platform for expanding NK cells from healthy donors\u0026rsquo; PBMCs using K562-4-1BBL-mbIL-21/-15 feeder cell, providing a highly efficient, rapid, straightforward, and cost-effective method for NK cell expansion. We demonstrated that NK cells expanded through this regimen exhibit potent anti-tumor effects both in vitro and in vivo. Our findings highlight the potential of this ex vivo NK cell expansion platform to produce large quantities of highly active, clinical-grade NK cells, thereby significantly promoting the development of NK cell-based immunotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e The experiment was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University in Xi'an, China (Approval no. XJTUAE2023-1883).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e All authors have read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by Natural Science Foundation of Shaanxi Province (grant no. 2024SF-YBXM-148) and Bethune Charity Foundation (grant no. BCF-IBW-XY-20231023-08).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHuaiyu Wang and Xiaohu Fan contributed to the study conception, design and methodology. Sha Gong and Nan Mei performed the material preparation, data collection and analysis. Sha Gong, Nan Mei, Jun Wang, Lu Wang and Junsheng Zhu written the first draft of the manuscript. Wenjuan Wang, Jincheng Wang, Xiaohong Lu, Pengcheng He, Weiwei Chen, Lei Xi, Yingying Bao and David N. Wald performed interpretation of data. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe data are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu E, Marin D, Banerjee P et al (2020) Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors[J]. N Engl J Med 382(6):545\u0026ndash;553\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarin D, Li Y, Basar R et al (2024) Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19(+) B cell tumors: a phase 1/2 trial.[J]. Nat Med 30(3):772\u0026ndash;784\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCooley S, Parham P, Miller JS (2018) Strategies to activate NK cells to prevent relapse and induce remission following hematopoietic stem cell transplantation[J]. Blood 131(10):1053\u0026ndash;1062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDogra P, Rancan C, Ma W et al (2020) Tissue Determinants of Human NK Cell Development, Function, and Residence[J]. Cell 180(4):749\u0026ndash;763e13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimasaki N, Jain A, Campana D (2020) NK cells for cancer immunotherapy[J]. Nat Rev Drug Discov 19(3):200\u0026ndash;218\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi YH, Lim EJ, Kim SW et al (2019) IL-27 enhances IL-15/IL-18-mediated activation of human natural killer cells[J]. J Immunother Cancer 7(1):168\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner J, Pfannenstiel V, Waldmann A et al (2017) A Two-Phase Expansion Protocol Combining Interleukin (IL)-15 and IL-21 Improves Natural Killer Cell Proliferation and Cytotoxicity against Rhabdomyosarcoma[J]. Front Immunol 8:676\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGranzin M, Wagner J, K\u0026ouml;hl U et al (2017) Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation.[J]. Front Immunol 8:458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOjo EO, Sharma AA, Liu R et al (2019) Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells[J]. Sci Rep 9(1):14916\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenman CJ, Senyukov VV, Somanchi SS et al (2012) Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells[J]. PLoS ONE 7(1):e30264\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThangaraj JL, Phan MTT, Kweon S et al (2022) Expansion of cytotoxic natural killer cells in multiple myeloma patients using K562 cells expressing OX40 ligand and membrane-bound IL-18 and IL-21[J]. Cancer Immunol Immunother 71(3):613\u0026ndash;625\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKweon S, Phan MTT, Chun S et al (2019) Expansion of Human NK Cells Using K562 Cells Expressing OX40 Ligand and Short Exposure to IL-21.[J]. Front Immunol 10:879\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomanchi SS, Lee DA (2016) Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21[J]. Methods Mol Biol 1441:175\u0026ndash;193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImamura M, Shook D, Kamiya T et al (2014) Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15[J]. Blood 124(7):1081\u0026ndash;1088\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichen S, Frosch J, F\u0026uuml;ssel M et al (2020) Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self[J]. Cytotherapy 22(7):354\u0026ndash;368\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn YO, Kim S, Kim TM et al (2013) Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro[J]. J Immunother 36(7):373\u0026ndash;381\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, He C, Liu C et al (2015) Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21.[J]. Int J Oncol 47(1):335\u0026ndash;342\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaignard A (2019) NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment[J]. Front Immunol, 10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbinger N, Bexte T, Buchinger L et al (2022) CRISPR/Cas9 Gene Editing of Immune Checkpoint Receptor NKG2A Improves the Efficacy of Primary CD33-CAR-NK Cells Against AML[J]. Blood 140(Supplement 1):4558\u0026ndash;4559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndr\u0026eacute; P, Denis C, Soulas C et al (2018) Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.[J]. Cell 175(7):1731\u0026ndash;1743e13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaulfuss M, Mietz J, Fabri A et al (2023) The NK cell checkpoint NKG2A maintains expansion capacity of human NK cells.[J]. Sci Rep 13(1):10555\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NK cells expansion, Feeder cells, mbIL-21, mbIL-15","lastPublishedDoi":"10.21203/rs.3.rs-4741998/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4741998/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNatural killer (NK) cell immunotherapy is a promising approach for cancer treatment. However, its extensive clinical application was limited to the large-scale clinical-grade expansion of NK cells. In this study, we expanded NK cells from healthy donor\u0026rsquo;s peripheral blood mononuclear cells (PBMCs) using a newly designed K562 feeder cell line.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe feeder cells were generated by transducing K562 cells with lentiviral particles carrying 4-1BBL and mbIL-21/-15. NK cells were expanded from PBMCs with these genetically modified, frozen-thawed and irradiated K562 feeder cells in the presence of IL-2. The purity, quantity, and receptors expression of the expanding NK cells were dynamically monitored. Furthermore, their anti-tumor efficacy was evaluated both in vitro and in vivo following a two-week expansion period.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe K562-4-1BBL-mbIL-21/-15 feeder cells induced highly-efficient NK cells expansion from PBMC (17902-fold) within two weeks. There was a notable upregulation in the expression of activating receptors including NKG2D, NKp30, NKp44, and NKp46 during the expansion process. Moreover, the expanded NK cells displayed enhanced cytotoxicity against a variety of hematological (K562, MOLM-13, OCI-AML-3, THP-1) and solid (Hep-G2, OVCAR3) cancer cell lines in vitro. In the humanized U937 xenograft mouse model, the NK cells extended the median survival time of the AML-bearing mice from 19.40 to 28.25 days.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe have successfully established a highly-efficient, cost-effective and rapid NK cell expansion platform from PBMC utilizing K562-4-1BBL-mbIL-21/-15 feeder cells, which also significantly improved the cytotoxicity both in vitro and in vivo, presenting a significant advancement in the field of NK cell-based immunotherapy.\u003c/p\u003e","manuscriptTitle":"A Novel Feeder cell based on 4-1BBL and Membrane-bound IL-21/ IL-15 Induce Highly Expansion and Anti-tumor Effect of Natural Killer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 17:43:32","doi":"10.21203/rs.3.rs-4741998/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0081e6a-683c-40d9-8304-d136290fbbd6","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-31T17:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 17:43:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4741998","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4741998","identity":"rs-4741998","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}