Abstract Osteosarcoma (OS) is a highly aggressive bone malignancy that predominantly affects children and young adults. There have been no major changes in patient survival in the past four decades. Therefore, new therapeutic interventions are needed. We have developed an enhanced conditionally replicative canine oncolytic adenovirus, CAV2-AU-M3, armed with an anti-PD1 heavy-chain antibody (HcAb), and evaluated its efficacy against osteosarcoma across four canine cell lines. CAV2-AU-M3 was chara