Radiotherapy enhances natural killer cell cytotoxicity and localization in pre-clinical canine sarcomas and first-in-dog clinical trial

Background: We have previously shown that radiotherapy (RT) augments natural killer (NK) functions in preclinical models of human and mouse cancers, including sarcomas. Since dogs are an excellent outbred model for immunotherapy studies, we sought to assess RT plus local autologous NK transfer in canine sarcomas. Methods: Dog NK cells (CD5(dim), NKp46+) were isolated from PBMCs and expanded with irradiated K562-C9-mIL21 feeder cells and 100 IU/mL recombinant human IL-2. NK homing and cytotoxicity +/- RT were evaluated using canine osteosarcoma tumor lines and dog patient-derived xenografts (PDX). In a first-in-dog clinical trial for spontaneous osteosarcoma, we evaluated RT and intra-tumoral autologous NK transfer. Results: After 14 days, mean NK expansion and yield were 19.0-fold (+/- 8.6) and 258.9(+/- 76.1) x 10(6) cells, respectively. Post-RT, NK cytotoxicity increased in a dose-dependent fashion in vitro reaching similar to 80% at effector: target ratios of >= 10:1 (P < 0.001). In dog PDX models, allogeneic NK cells were cytotoxic in ex vivo killing assays and produced significant PDX tumor growth delay (P < 0.01) in vivo. After focal RT and intravenous NK transfer, we also observed significantly increased NK homing to tumors in vivo. Of 10 dogs with spontaneous osteosarcoma treated with focal RT and autologous NK transfer, 5 remain metastasis-free at the 6-month primary endpoint with resolution of suspicious pulmonary nodules in one patient. We also observed increased activation of circulating NK cells after treatment and persistence of labelled NK cells in vivo. Conclusions: NK cell homing and cytotoxicity are increased following RT in canine models of sarcoma. Results from a first-in-dog clinical trial are promising, including possible abscopal effects.