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Background Barriers that limit natural killer (NK) cells as a cellular therapy for cancer include limited numbers in peripheral blood and optimization of activation and inhibitory signals. Companion canines have spontaneous malignancies resembling human malignancies and need advances in therapeutic options. Prior work by us and others has established feasibility of autologous NK cell infusions in canines, however, autologous products are limited by dysfunctional immunity and up to a 3-week manufacturing process that delays care. Allogeneic NK cells offer the possibility of ‘off-the-shelf’ therapy to be administered from healthy donors but need further optimization for therapeutic use.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy canine blood donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 along with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15 on days 0 and 7 ( figure 1). NK cells were defined as CD3-, NKp46+ and T cells were defined as CD3+, NKp46- by flow cytometry. In vitro potency was assessed via co-culture with the D17-mKate2+ canine osteosarcoma cell line at various effector-to-target (E:T) ratios using live cell imaging via Incucyte. Three canines were enrolled in a phase 1 trial testing infusion of ex vivo expanded allogeneic NK cells after lymphodepletion with fludarabine and cyclophosphamide. Canines were given subcutaneous recombinant human IL-2 in vivo to support NK activation.Results Flow cytometric analysis confirmed successful expansion of canine allogeneic NK cells with up to 50% of cells demonstrating NKp46+ after 14 days. Residual T cell numbers varied based on donor (range of 2.5-30%) leading to incorporation of a T cell depletion step. Live cell imaging demonstrated potency with increasing osteosarcoma cell death correlated with higher E:T ratios. Three canines with metastatic/refractory malignancies were successfully lymphodepleted and infused with 150-500,000 allogeneic NK cells per kilogram in an outpatient setting. All canines tolerated lymphodepletion and allogeneic NK infusions well, with no grade 3 or 4 toxicities noted and no graft-versus-host-disease. By study design, efficacy was not formally evaluated although all subjects were euthanized off study due to progressive disease.Conclusions Canine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and importantly safety in vivo. We will continue to optimize the NK cell product and escalate dosing until reaching the maximally tolerated dose. These experiments serve as feasibility for infusing allogeneic NK cells and will inform translation to human clinical trials.Ethics Approval This study was approved by the University of Wisconsin Institutional Animal Care and Use Committee; approval number V006877.Abstract 348 Figure 1NK cell expansion protocol