PurposeThe purpose was to determine the safety and antitumor activity of a folate-tubulysin conjugate (EC0531) in a relevant preclinical animal model, dogs with naturally-occurring invasive urothelial carcinoma (iUC). Canine iUC is an aggressive cancer with high folate receptor (FR) expression similar to that in certain forms of human cancer.Experimental DesignA 3+3 dose escalation study of EC0531 (starting dose 0.2 mg/kg given intravenously at two-week intervals) was performed in dogs with iUC expressing high levels of FRs (>50% positive tumor cells). Pharmacokinetic (PK) analysis was performed, and the maximum tolerated dose (MTD) was determined. The dose cohort at the MTD was expanded to determine antitumor activity.ResultsThe MTD of EC0531 was 0.26 mg/kg every two weeks, with grade 3-4 neutropenia and gastrointestinal toxicity observed at higher doses. Treatment at the MTD was well tolerated. Clinical benefit was found in 20 of 28 dogs (71%), including three dogs with partial remission and 17 dogs with stable disease. Plasma EC0531 concentrations in the dogs far exceeded those required to inhibit proliferation of FR-expressing cell in vitro. Unlike human neutrophils, canine neutrophils were found to express FRs, which contributes to the neutropenia at higher doses of EC0531 in dogs.ConclusionEC0531 was well tolerated and had good antitumor activity in dogs with iUC. It is likely that humans will tolerate higher, potentially more effective doses of folate-tubulysin without myelotoxicity because of the absence of FRs on human neutrophils. The results clearly justify the evaluation of folate-tubulysin in human clinical trials.

Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long-term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non-canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma-bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression-free survival (P = 0.4) or overall survival (P = 0.1).