We previously reported that eBAT, an EGF-targeted angiotoxin, was safe and improved overall survival for dogs with splenic hemangiosarcoma when added to the standard of care in a single cycle of three administrations in the minimal residual disease setting. Our objective for the SRCBST-2 trial was to assess whether increased dosing through multiple cycles of eBAT would be well tolerated and further enhance the benefits of eBAT. Eligibility was expanded to dogs with stage-3 hemangiosarcoma, provided that gross lesions could be surgically excised. The interval between eBAT and the start of chemotherapy was reduced, and the experimental therapy was expanded to three cycles, each administered at the biologically active dose (50 μg/kg) on a Monday/Wednesday/Friday schedule following splenectomy, and scheduled one week prior to the first, second, and fifth doxorubicin chemotherapy. Twenty-five dogs were enrolled; six experienced acute hypotension with two requiring hospitalization. Self-limiting elevation of ALT was observed in one dog. A statistically significant survival benefit was not seen in this study in eBAT-treated dogs compared to a Contemporary comparison group of dogs with stages 1-3 hemangiosarcoma treated with standard of care alone. Our results indicate that repeated dosing cycles of eBAT starting one week prior to doxorubicin chemotherapy led to greater toxicity and reduced efficacy compared to a single cycle given between surgery and a delayed start of chemotherapy. Further work is needed to understand the precise mechanisms of action of eBAT in order to optimize its clinical benefits in the treatment of canine hemangiosarcoma and other tumors. IACUC Protocols 1110A06186 and 1507-32804A. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.