J. S. Park1, S. Grossenbacher2, J. Modiano3, J. Miller4, E. Ames2, S. Mac2, A. Monjazeb5, M. Kent6, W. Culp6, M. Chen7, W. J. Murphy2, R. Canter1 1University Of California – Davis,Surgical Oncology/Surgery,Sacramento, CA, USA 2University Of California – Davis,Dermatology,Sacramento, CA, USA 3University Of Minnesota,Veterinary Medicine,Minneapolis, MN, USA 4University Of Minnesota,Internal Medicine/Oncology,Minneapolis, MN, USA 5University Of California – Davis,Radiation Oncology,Sacramento, CA, USA 6University Of California – Davis,Veterinary Medicine,Sacramento, CA, USA 7University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA
Introduction: We have previously shown that combination radiotherapy (RT) and Natural Killer (NK) immunotherapy is effective in diverse pre-clinical models of human solid malignancies. Since bone and soft tissue sarcomas commonly afflict dogs, and canine clinical trials are a tremendous resource, particularly for novel immunotherapy protocols, we hypothesized that dog PBMC-derived NK cells could be similarly expanded and activated ex vivo as a precursor for a canine combination radioimmunotherapy clinical trial.
Methods: Dog NK cells were isolated from 10-15 mL fresh PBMCs using Ficoll separation and CD5 depletion. Isolated NK cells (CD3+, CD5dim, TCR-) were expanded via co-culture with irradiated (100Gy) K562-C9-mIL21 for 2-3 weeks in 100IU/mL recombinant human IL-2. Canine osteosarcoma (OSCA) tumor lines and fresh canine primary sarcomas were evaluated for susceptibility to NK killing before/after RT in vitro and in xenograft experiments with NSG mice. NK cytotoxicity was assessed in 4-16 hour killing assays by Flow cytometry using a BD Fortessa cell sorter (BD Biosciences) with 7-Aminoactinomycin as cell viability marker. Parametric and non-parametric statistical tests were performed as appropriate.
Results: NK expansion was successful in 14/20 donors (including 9 tumor-bearing dogs) from baseline 4.5(±1.9) x106 cells to 103.5(±29.1) x106, mean increase 23.2X (±2.3). Canine NK cells were also responsive to human cytokines (IL-2, IL-12, and IL-18), but expansions were lower (1.6-3.5 fold expansion over 14 days). NK cytotoxicity to OSCA78, OSCA32, and NK-sensitive CTAC cells in vitro increased in a dose-dependent fashion reaching 74 – 88% cytolysis at effector:target ratios of 10:1 – 20:1 (P<0.001). RT augmented NK cytotoxicity with greatest synergy at 2.5-5 Gy RT in 4-hour killing assays (1.3-3.4X increased killing, P<0.01). At doses of 10 Gy and/or 16-hour killing assays, only minor differences in overall killing were observed. Similar results were observed with RT sensitization to NK killing in primary canine sarcomas. In a dog sarcoma PDX model using focal RT, intravenous NK transfer, and hydrodynamic human IL-15 for in vivo NK support, focal RT increased NK homing to tumors by 3.8X±0.3 (P<0.001).
Conclusion: NK cell homing and effector functions are increased following RT in canine models of sarcoma. Dog sarcoma appears to be a valuable model to facilitate clinical translation of NK immunotherapy.
