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Background Recurrent osteosarcoma (OSA) has a dismal prognosis, largely due to therapy resistance within the tumor microenvironment (TME). NK cell immunity in solid tumors can be improved by increasing NK cell number, function and persistence. 1 We have developed the antigen-presenting cell line, K562-mbIL-21-41BBL, and demonstrated significant expansion of NK cells by 30-35,000-fold.2 Dinutuximab is an FDA approved mAb against GD2, which is overexpressed in OSA. NKTR-255 is an investigational polymer-conjugated IL-15 agonist that activates, proliferates and expands NK and CD8+T-cells, promotes survival, and expands memory CD8+ T cells.3 Here we investigate expanded NK cell in combination with NKTR-255 and dinutuximab to overcome the OSA TME resistance.Methods PBMCs were expanded into NK cells using K562-mbIL-21-41BBL feeder cells with IL-2 (50 IU/mL). NK cell cytotoxicity was evaluated by luciferase-based cytotoxicity assay. IFN-γ and perforin secretion were analyzed by ELISA. An orthotopic mouse model generated by implanting OSA 143B cells into the flanks of NSG mice was utilized for evaluating in vivo tumor growth and animal survival. NK cells were injected intraperitoneally (IP) at a dose of 0.5-1x10e7 cells/animal once every week for 5 weeks. Dinutuximab was administered via IP injection at 15 ug/animal twice a week. NKTR-255 was injected via IP injection at 0.3 mg/kg once every two weeks.Results We found that expanded NK cells were highly cytotoxic to OSA cells (U2OS and 143B) at various target to effector ratios (T:E=20:1, 10:1, 5:1, 2:1, 1:1) (p<0.01 and p<0.05). Compared to NK cells alone, combining with dinutuximab or NKTR-255 significantly enhanced NK cell cytotoxicity against OSA cells in vitro (p<0.01 and p<0.05). Furthermore, NK cells combined with both NKTR-255 and dinutuximab had further significantly enhanced in vitro cytotoxicity compared to NK+dinutuximab or NK+NKTR-255 (p<0.05) ( figure 1A and 1B). NK cells combined with dinutuximab and NKTR-255 secreted significantly higher levels of IFN-γ and perforin than NK cells alone (p<0.01 and p<0.05). In the OSA xenograft mouse model, we observed significantly decreased tumor growth (p<0.001) and significantly improved animal survival (p<0.0001) in mice treated with the combination of NK+dinutuximab+NKTR-255 compared to the control (figure 1C and 1D).Conclusions Our data demonstrated enhanced anti-tumor efficacy of expanded NK cells combined with NKTR-255 and dinutuximab against OSA in vitro and in vivo. NK cells in combination with immune modulators that enhance NK function and persistence such as anti-GD2 and IL-15 agonist constitute potential novel effective immunotherapies for patients with recurrent OSA.Acknowledgements This study was supported by the NCI Cancer Moonshot U54 Grant (CA232561-01A1), St. Baldrick’s Foundation, Pediatric Cancer Research Foundation, Children’s Cancer Fund. We thank Nektar Therapeutics for generously providing NKTR-255.References Nayyar G, Chu Y, Cairo MS. Overcoming resistance to natural killer cell based immunotherapies for solid tumors. Front Oncol. 2019;9:51. PMC6378304, https://www.ncbi.nlm.nih.gov/pubmed/30805309Denman CJ, Senyukov VV, Somanchi SS, Phatarpekar PV, Kopp LM, Johnson JL, Singh H, Hurton L, Maiti SN, Huls MH, Champlin RE, Cooper LJ, Lee DA. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS One. 2012;7(1):e30264.Miyazaki T, Maiti M, Hennessy M, et al. NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy, Journal for Immunotherapy of Cancer. 2021;9.Ethics Approval All animal experiments were approved by the Institutional Animal Care and Use Committee at New York Medical College (Protocol #15112) and performed in accordance with the ethical standards of the institutional research committee.Abstract 310 Figure 1Expanded NK cells combined with dinutuximab and NKTR-255 significantly enhanced NK cytotoxicity in vitro (A and B), decreased tumor growth (C) and improved animal survival (D) in OS xenograft mouse model