Figure 5.

N-803 enhanced exPBNK cells numbers in vivo and the combination of exPBNK+N-803+ dinutuximab significantly inhibited OS cells growth and extended the survival of OS xenografted NSG mice. (A) After confirming tumor engraftment at day 7, 1×10⁷ exPBNK cells or 1×10⁷ exPBNK cells mixed with 0.2 mg/kg N-803 were intraperitoneally injected to each mouse once a week for 6 weeks. Two weeks after the last NK administration, blood was collected from the orbital sinus from each mouse and human NK cells were counted using flow cytometry. N-803 significantly enhanced human NK counts as compared with the mice injected with human NK cells without N-803 (each group n=4). (B) Whole mouse luciferase activity was measured once weekly at various time points. Photos at day 49 are shown in the left panel. photons emitted from luciferase-expression cells were measured in regions of interest that encompassed the entire body and quantified using the living image software. Signal intensities (total flux) are shown at the time points plotted as mean±SEM in the right panel (each group n=6). (C) 4×10⁶ of luciferase expression U2OS-Luc (OS) cells were subcutaneously injected in NSG mice on day 0. After confirming the tumor engraftment at day 7, 1×10⁷ exPBNK cells+15 ug IgG (n=5), 1×10⁷ exPBNK cells+15 µg dinutuzimab (n=5), 1×10⁷ exPBNK cells+0.2 mg/kg N-803 (n=5), 1×10⁷ exPBNK cells+15 µg dinutuzimab +0.2 mg/kg N-803 (n=9), 15 µg dinutuzimab +0.2 mg/kg N-803 (n=5), or PBS (n=5) 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. The tumor size was measured with a caliper once a week and plotted as the mean±SEM for each group. The OS xenografted mice treated with exPBNK cells+dinutuximab + N-803 have significantly smaller tumor sizes than other groups. (D) Photons emitted from luciferase-expression OS cells were measured in regions of interest that encompassed the entire body and quantified using the living image software. Signal intensities (total flux) are shown at the time points plotted as mean±SEM. The OS xenografted mice treated with exPBNK cells+dinutuximab + N-803 have significantly lower bioluminescence signal than other groups. (E) After different treatments, OS xenografted mice were followed until death. The Kaplan-Meier survival curves for all groups were generated following therapy initiation using animal sacrifice as the terminal event. Comparison of survival between groups is shown. The combination of exPBNK cells+dinutuximab + N-803 significantly extended the survival of U2OS-Luc mice as compared with other groups. *p<0.05, **P<0.01, ***p<0.001, Dinut=dinutuximab. OS=U2OS cell line. The data were generated from the pooled two independent experiments. exPBNK, expanded peripheral blood natural killer cell; NK, natural killer; OS, osteosarcoma; PBS, phosphate-buffered saline.