J. S. Park1, Z. Wang2, S. K. Grossenbacher2, J. I. Luna2, I. Sturgill2, S. S. Withers3, M. S. Kent3, M. Chen4, W. T. Culp3, R. Rebhun3, A. M. Monjazeb5, W. J. Murphy2, R. J. Canter1 1UC Davis,Surgical Oncology/Surgery,Sacramento, CA, USA 2UC Davis,Dermatology,Sacramento, CA, USA 3UC Davis School Of Veterinary Medicine,Surgical And Radiological Sciences,Davis, CA, USA 4UC Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA 5UC Davis,Radiation Oncology,Sacramento, CA, USA
Introduction: We have previously shown that radiotherapy (RT) increases natural killer (NK) cytotoxicity and homing in pre-clinical models of human solid cancers, including sarcomas. Since canine clinical trials are a valuable resource for novel immunotherapy protocols and sarcomas commonly afflict dogs, we hypothesized that dog PBMC-derived NK cells would be effective in canine models of sarcoma, including adoptive transfer in a canine RT/NK clinical trial.
Methods: Canine NK cells were isolated from 15 mls of fresh whole blood using Ficoll separation and CD5 depletion. Isolated NK cells were then expanded with irradiated K562c9IL21 for 2-3 weeks. Using 6-month metastasis-free survival as the primary endpoint, we are evaluating RT and adoptive NK immunotherapy in a canine clinical trial. For this trial, treatment consists of palliative RT weekly x4 followed by two intra-lesional injections of autologous NK cells. In correlative studies, including dog patient-derived xenografts (PDX), we assessed NK homing using eFluor 670 cell proliferation dye and NK function by expression of activation markers IFNγ, granzyme B, and perforin.
Results: We have treated 8 of planned 14 dogs with osteosarcoma on protocol with a median of 76×10^6 cells (92% viable). Of 3 evaluable dogs who have reached the 6-month primary endpoint, we have observed 1 partial response and 2 are metastasis-free, including 1 dog with complete resolution of a suspicious 3 mm pulmonary nodule. In dog patients on trial, phenotyping of expanded NK cells from all patients showed > 90% granzyme B and IFNγ expression prior to adoptive transfer. Tagging experiments 1 week after intratumoral injection revealed that 11 – 60% of CD45+ cells are eFluor 670 positive, confirming persistence of injected NK cells post injection. Analysis of unactivated circulating PBMCs post-injection demonstrated a significant increase in granzyme B expression (2.25X ± 0.42, P<0.01). Dog PDX studies demonstrate that focal RT increases NK homing to sarcomas on average 3.8X±0.3 (P<0.001) compared to unirradiated controls. Immunohistochemical analysis of tissue samples post RT shows a significant increase in CD3+ tumor-infiltrating lymphocytes post RT (P<0.05, see figure). Co-culture experiments of dog PDX sarcomas ex vivo with allogeneic NK cells shows RT-induced sensitization to NK killing at doses of 10 – 20 Gy (P<0.01)
Conclusion: RT and NK immunotherapy appear synergistic in dog models of sarcoma. Preliminary results from a canine clinical trial of palliative RT and autologous NK transfer for osteosarcoma are promising, including possible abscopal effects. Further evaluation of this novel radio-immunotherapy approach is warranted.
