Simple SummaryImmune checkpoint inhibitors (ICIs) are promising treatment options for spontaneous cancers in dogs. To optimize the clinical benefit of ICIs, combination therapy with radiation has been proposed for human cancers; however, the safety and efficacy of the combination approach have not yet been reported in dogs. The retrospective analysis of dogs bearing pulmonary metastatic oral malignant melanoma suggested that the use of previous (≤8 weeks) hypofractionated radiation therapy is associated with a better clinical response to anti-PD-L1 immunotherapy. Radiotherapy before the initiation of anti-PD-L1 therapy can be an effective approach to enhance antitumor immunity in dogs.AbstractAlthough immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n= 20, free from the effect of RT), previous RT (n= 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n= 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p< 0.05 vs. no RT group), and 20%/129 days (p> 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.