U2OS (OS), M059K (GBM) and SKNFI (NB) cell lines were purchased from the American Type Culture Collection, Gaithersburg, Maryland. K526-mbIL21-41BBL cells were generously provided by Dean A. Lee, MD/PhD from Nationwide Children’s Hospital, Columbus, Ohio.²² Dinutuximab was generously provided by United Therapeutics, Silver Springs, Maryland. N-803 was generously provided by Hing Wong, PhD, Peter R. Rhode, PhD, John H. Lee, MD, and Jeffrey T. Safrit, PhD from ImmunityBio/Altor Bioscience, Culver City, California. Leukocytes were obtained after informed consent from healthy donors at the New York Blood Center, New York, New York. Peripheral blood mononuclear cells (PBMNCs) were obtained by Ficoll gradient (Amersham Biosciences, Piscataway, New Jersey, USA) separation as we previously described.17 U2OS, M059K and SKNFI cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics penicillin and streptomycin (100 µg/mL). K526-mbIL21-41BBL cells were cultured in complete medium (RPMI1640 medium supplemented with 10% FBS and penicillin and streptomycin (100 µg/mL)). NK cells were cultured in complete medium with 50 IU IL-2.

PBMNCs were stimulated with irradiated genetically modified K562-mbIL21 - 41BBL cells for 2 weeks as we previously described.22 Expanded PBNK cells were isolated by negative selection using Miltenyi NK cell isolation kit (Miltenyi Biotec, Cambridge, Massachusetts) as we have previously described.17

Bioluminescence (BLI) based in vitro cytotoxicity assays were performed as previously described with minor modification.23 Luciferase-expressing tumor cells were placed in 96–well flat bottom plates at a concentration of 3×10⁵ cells/mL. Subsequently, effector cells were added at different effector-to-target (E:T) ratios with or without N-803 and dinutuximab and incubated at 37°C for 2–3 days. After incubation, the samples in the plates were spun and 75 µg/mL D-firefly luciferin potassium salt (PerkinElmer, Massachusetts, USA) with fresh media added to the cell pellets in each well. BLI was measured with a luminometer (Molecular Devices Multifilter F5 plate reader) as relative light units (RLU). Cells were treated with 1% Triton X-100 as a measure of maximal killing. Target cells incubated without effector cells were used to measure spontaneous death RLU. Percent lysis was calculated from the data with the following equation: % specific lysis = 100×(spontaneous death RLU – test RLU)/(spontaneous death RLU – maximal killing RLU). All tests were run in quadruplicate.

PBMNCs were stimulated with irradiated genetically modified K562-mbIL21 - 41BBL cells for 2–3 weeks. The same number of purified exPBNK cells were cultured in medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803, the same molar dose of immunoglobulin G (IgG), or medium only for 3 days. CellTiter 96 AQueous one solution cell proliferation assay (Promega, Madison, Wisconsin, USA)18 was used to determine the number of proliferating viable cells following the manufacturer’s instructions. Briefly, 10⁴ NK cells/well were seeded in culture medium containing N-803, IgG or medium at 37°C and 5% CO₂. At the end of 3 days incubation, CellTiter 96 AQueous one solution was added to each well and cells were incubated for 4 hours at 37°C and 5% CO₂. Finally, spectrophotometrical absorbance was measured using a multifilter plate reader (Molecular Device, San Jose, California, USA) at OD490.

Intracellular proteins and phosphoproteins were measured as we have previous described.18 Fixed and permeabilized cells were stained with fluorescent-dye conjugated anti-human antibodies: phospho-p38 MAPK-PE (ebioscience, #12-9078-41), phospho-Akt1-APC (ebioscience, #17-9715-41), phospho-Stat3-FITC (ebioscience, #11-9033-41), or phospho-Stat5-PE (ebioscience, #12-9033-41). Cells were analyzed using MACSQuant Analyzer (Miltenyi Biotec Cambridge, Massachusetts, USA). No stain, or isotype controls were used for gating.

Cell culture supernatants were collected after 3 days culture and were stored at −80 °C. The concentrations of cytokines/chemokines/growth factors were measured by the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay (Bio-Rad Laboratories, Hercules, California, USA) according to the manufacturer’s instructions. In brief, 50 µL aliquot of sample was diluted 1:4 with sample diluent, incubated with antibody-coupled beads, biotinylated secondary antibodies, and followed by streptavidin-phycoerythrin. The beads were read on a Luminex System (Bioplex 200, Bio-Rad) and the data were analyzed using Bioplex Manager Software.

Platelet-derived growth factor (PDGF)-AA (Raybiotech, # ELH-PDGFAA-1), PDGF-BB (Raybiotech, # ELH-PDGFBB-1), interferon (IFN)-γ (eBioscience, # KHC4021), and Perforin (Abcam, # ab46068) concentrations were analyzed by ELISA according to the manufacturer’s instructions. Briefly, recombinant standards were run with serial dilutions. Cell culture supernatants were diluted at 1:1 or 1:4 with assay diluent. 100 uL of diluted samples and standard were added to microwells simultaneously and incubated for 2–2.5 hours at room temperature. Biotin conjugated anti-human PDGF-AA, PDGF-BB, IFN-γ, or perforin antibody was used and incubated for 1 hour at room temperature. After washing, streptavidin-HRP solution was added for 30 min at room temperature. ELISA plates were developed with 100 uL TMB substrate reagents. TMB Stop Solution was added to halt the reaction. The absorbance at 450 nm was measured on a Molecular Devices Multifilter F5 plate reader.

The exPBNK cells under different conditions were analyzed for phenotypic expression of inhibitory NK receptors (CD94, NKG2A), inhibitory NK killer cell immunoglobulin-like receptors (KIR) (CD158a, CD158b, CD158e), activating NK KIR (KIR2DSA), activating C-lectin NK receptors (NKG2C, NKG2D), and activating natural cytotoxicity receptors (Nkp46, NKp30, NKp44) by flow cytometry as we have previous described.17 Cells were analyzed using MACSQuant Analyzer (Miltenyi Biotec, Cambridge, Massachusetts, USA). No stain, or isotype controls were used for gating.

Six to eight weeks old NOD/SCID/γ-chain-/- (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). The experimental protocol was conducted in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at New York Medical College is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Luciferase expressing U2OS-Luc (OS), M059K-Luc (GBM) and SKNFI-Luc (NB) cells were generated as we have previously described.17 4×10⁶ of U2OS-Luc, M059K-Luc cells, or SKNFI-Luc cells were subcutaneously injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 1×10⁷ exPBNK cells+15 µg IgG, 1×10⁷ exPBNK cells+15 µg dinutuximab, 1×10⁷ exPBNK cells+0.2 mg/kg N-803, 1×10⁷ exPBNK cells+15 µg dinutuximab +0.2 mg/kg N-803, 15 µg dinutuximab +0.2 mg/kg N-803, or phosphate-buffered saline (PBS) was intraperitoneally injected to each mouse. NK cells were administered once a week for 3 weeks and IgG, dinutuximab and N-803 were given twice a week for 6 weeks. Tumor engraftment and progression were evaluated using the Xenogen IVIS-200 system (PerkinElmer, Shelton, Connecticut) as we have previously described.17 Tumor size was estimated according to the following formula: tumor size (cm³)=length (cm) × width2 (cm) × 0.5. Mice were followed until death or sacrificed if any tumor size reached 2 cm³ or larger.

Statistical analyses were performed using the InStat statistical program (GraphPad, San Diego, California, USA). Average values were reported as the mean±SEM. Results were compared using the one-tailed unpaired Student’s t-test with p<0.05 considered as significant. Probability of survival in animal studies was determined by the Kaplan-Meier method using the Prism program V.8.0 (GraphPad Software).

Expanded PBNK cells (exPBNK) were generated by coculturing PBMNCs with irradiated genetically modified K562-mbIL21 - 41BBL feeder cells for 2 weeks and isolated by negative selection using Miltenyi NK cell isolation kit as we previously described.22 To investigate if N-803 stimulates exPBNK cells viability and proliferation as compared with the same molar dose of IgG, the purified exPBNK cells without any feeder cells were cultured in medium with 0.35 ng/mL (low) or 3.5 ng/mL (high) N-803 or molar equivalent dose of IgG for 3 or 7 days. The exPBNK cells with N-803 at 0.35 ng/mL or 3.5 ng/mL had significantly higher viability as compared with IgG or medium controls (p<0.001). Furthermore, N-803 at 3.5 ng/mL significantly stimulated the proliferation of exPBNK cells as compared with N-803 at 0.35 ng/mL at day 3 (figure 1A) (p<0.001) and day 7 (figure 2 (online supplemental file 1)) by MTS assays. We also observed that sustained viability and proliferation of exPBNK cells stimulated by N-803 at 3.5 ng/mL with morphological changes in NK cell shape, size, and number which correlate to NK proliferation and activation as compared with NK cells cultured with IgG (figure 1B). Consistent with enhanced exPBNK proliferation and similar to IL-15, N-803 at 3.5 ng/mL significantly enhanced the phosphorylation of Stat 3, AKT1 and p38MAPK as compared with IgG (p<0.01) at day 3 (figure 1C).

The expression of receptors on viable exPBNK cells was compared by flow cytometry analysis. Purified non-expanded NK (PBNK) cells were used as controls. N-803 at 3.5 ng/mL significantly enhanced the expression of NK activating receptors: NKG2D, NKp30, NKp44, NKp46 at day 10 as compared with IgG (p<0.001) (figure 1D). N-803 at 3.5 ng/mL also significantly enhanced the expression of CD16, known as FcγRIII, at day 10 as compared with IgG (p<0.001) (figure 1D), supporting in part the rationale for the combinatorial immunotherapy of monoclonal antibody with expanded NK cells and N-803.

Since N-803 stimulated exPBNK cells to express high level of CD16 (figure 1D) and dinutuximab is an IgG1 type monoclonal antibody, we investigated whether the combination of N-803 and dinutuximab significantly stimulates the antibody dependent cellular cytotoxicity (ADCC) of exPBNK cells against OS, GBM, and NB. Tumor cell lines: U2OS, M059K, SKNFI cells express GD2 (figure 3 (online supplemental file 1)) and were treated with 1 µg/mL IgG +exPBNK, 3.5 ng/mL N-803 +exPBNK, 1 µg/mL dinutuximab +exPBNK or 3.5 ng/mL N-803 +1 µg/mL dinutuximab +exPBNK cells at E:T ratio=1:1 and 3:1. We found that the combination of N-803, dinutuximab and exPBNK significantly killed U2OS, M059K and SKNFI cells (p<0.001) as compared with other groups (figure 2A) in an E:T ratio dependent manner. The enhanced in vitro cytotoxicity of exPBNK cells was associated with significantly enhanced secretion of perforin (figure 2B) and IFN-γ (figure 2C) from exPBNK cells as compared with all other groups against U2OS, M059K and SKNFI cells (p<0.001) at E:T=3:1.

To investigate the cytokines and growth factors that are significantly secreted by exPBNK cells stimulated by N-803 and dinutuximab against tumor cells, exPBNK cells were cultured with N-803, dinutuximab or the combination of N-803 and dinutuximab with or without U2OS cells at E:T=3:1 for 3 days. The concentrations of cytokines/chemokines/growth factors in the supernatants were measured by the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay. For the factors that are secreted by U2OS tumor cells such as tumor factor-related apoptosis-inducing ligand (TRAIL), platelet-derived growth factor-BB (PDGF-BB), and stem cell growth factor beta (SCGF-β), the combination of exPBNK +N-803+dinutuximab significantly reduced the secretion of these factors from U2OS tumor cells as compared with U2OS alone (p<0.001) and exPBNK +U2OS (p<0.01, p<0.05 and p<0.001, respectively) (figure 3A). For the factors that are secreted by exPBNK cells and can be further enhanced by N-803 and dinutuximab such as regulated on activation, normal T cell expressed and presumably secreted (RANTES) and stromal cell-derived factor-1 alpha (SDF-1α), U2OS significantly inhibited the secretion of these factors from exPBNK cells (p<0.001) (figure 3B). Monokine induced by gamma interferon (MIG) and interferon gamma-induced protein 10 (IP-10) are important ligands of CXCR3 that are pivotal for NK-cell migration towards tumor cells.24 We found that without U2OS tumor cells, exPBNK cells or exPBNK cells incubated with N-803 +dinutuximab do not secrete MIG. However, U2OS significantly increased the secretion of MIG and IP-10 from exPBNK cells (p<0.001) (figure 3C). Macrophage inflammatory proteins (MIP) 1 alpha and beta are members of the C-C motif subfamily of chemokines and both are ligands of C-C chemokine receptor type 5 receptor, which is essential for NK trafficking in host defense.25 We found that the combination of U2OS, N-803 and dinutuximab significantly enhanced the secretion of MIP-1beta from exPBNK cells (p<0.001) (figure 3D), while MIP-1alpha secretion by exPBNK cells was significantly enhanced by U2OS alone, N-803+ dinutuximab, or U2OS+N-803+dinutuximab (p<0.001) as compared with exPBNK cells (figure 3E).

We further confirmed our findings using M059K cells as tumor targets. We found that the combination of exPBNK +N-803+dinutuximab reduced the secretion of TRAIL (p<0.05), and significantly reduced the secretion of PDGF-BB (p<0.01), and SCGF-β (p<0.05) from M059K tumor cells as compared with exPBNK +M059K (figure 4A). M059K significantly inhibited the secretion of SDF-1α from exPBNK cells (p<0.001) (figure 4B) but significantly enhanced MIG secretion from exPBNK cells (p<0.001) (figure 4C). The combination of M059K, N-803 and dinutuximab significantly enhanced the secretion of MIP-1beta from exPBNK cells (p<0.001) as compared with all other groups (figure 4D).

PDGF-BB is one of the five ligands of PDGF Receptor (PDGFR) α and PDGFRβ. PDGFRs and their ligands have been found to be overexpressed or mis-regulated in many cancers, such as gliomas and sarcomas26 and PDGFRs are also expressed on the non-cancerous cells of the tumor microenvironment to support the growth of cancer cells.27 These findings were similar when we assessed PDGF-AA that the combination of exPBNK +N-803+dinutuximab significantly reduced the secretion of PDGF-AA (p<0.001) from U2OS or M059K tumor cells as compared with the NK, NK +N-803, or NK +dinutuximab (figure 4E).

To investigate if N-803 stimulates the proliferation of exPBNK in vivo and has anti-tumor effect with exPBNK cells, we generated luciferase expressing U2OS-Luc cells and xenografted U2OS-Luc cells to immunodeficient NSG mice.18 We found that the mice treated with exPBNK cells+N-803 have a significantly higher number of human NK cells as compared with the mice treated with exPBNK alone (figure 5A). Tumor burden was significantly reduced in mice treated with exPBNK cells+N-803 as compared with mice treated with exPBNK cells alone (figure 5B).

We further confirmed the anti-tumor effects of N-803 combined with dinutuximab and exPBNK cells in human U2OS cells xenografted NSG mice. The U2OS xenografted mice treated with exPBNK cells+dinutuximab + N-803 had significantly smaller tumor sizes (figure 5C) and BLI signals than other groups (figure 5D), and significantly longer survival (figure 5E).

To confirm that the effect of combination therapy was not specific to OS disease, we investigated if the combination of N-803 with dinutuximab and exPBNK significantly enhances the overall survival of NSG mice with GBM or NB diseases. NSG mice were xenografted with tumor cell line M059K or SKNFI. We found that the combination of exPBNK cells+dinutuximab + N-803 (n=7) significantly extended the survival of M059K mice as compared with the control groups which were treated with PBS (n=4, p<0.001), exPBNK +IgG (n=5, p<0.001), exPBNK +N-803 (n=5, p<0.001), exPBNK +dinutuximab (n=4, p<0.001), and dinutuximab +N-803 (n=4, p<0.01) (figure 6A). Moreover, the combination of exPBNK cells+dinutuximab + N-803 (n=8) significantly extended the survival of SKNFI mice as compared with the control groups which were treated with PBS (n=6, p<0.001), exPBNK +IgG (n=5, p<0.01), exPBNK +N-803 (n=7, p<0.01), exPBNK +dinutuximab (n=7, p<0.05), and dinutuximab +N-803 (n=6, p<0.01) (figure 6B).