Antiangiogenic therapy of established tumors in human skin/severe combinedimmunodeficiency mouse chimeras by anti-endoglin (CD105) monoclonal antibodies, and synergy between anti-endoglin antibody and cyclophosphamide

Endoglin (EDG; CD105) is a proliferation-associated cell membrane antigen o f endothelial cells and is strongly expressed on the tumor-associated angio genic vascular endothelium. Furthermore, EDG is essential for angiogenesis and a component of the transforming growth factor (TGF)-beta receptor compl ex. The present three anti-EDG monoclonal antibodies (mAbs), SN6f, SN6j, an d SN6k, react strongly with proliferating human endothelial cells but cross -react very weakly with murine endothelial cells. Analysis of Scatchard plo t of direct binding of these mAbs to proliferating human umbilical vein end othelial cells showed equilibrium constants of 8.3 x 10(9), 3.1 x 10(9), an d 1.0 X 10(9) liter/mol, respectively, for SN6f, SN6j, and SN6k. These mAbs did not react with MCF-7 human breast cancer cells. To facilitate antiangi ogenic tumor therapy by these mAbs in animal models, we used human skin/sev ere combined immunodeficiency (SCID) mouse chimeras bearing tumors of MCF-7 . Blood vessels in the chimeras were analyzed by immunostaining with specie s (human or mouse)-specific anti-CD31 and anti-EDG mAbs including an antihu man EDG mAb termed SN6h. Blood vessels in the completely healed grafted hum an skins consisted of a mixture of human (43.5%) and murine (56.5%) vessels , whereas only murine vessels were detected in the adjacent murine skins an d s.c. tissues. Therefore, murine vessels infiltrate into the human skin gr afts from the adjacent murine tissues, whereas the growth of human vessels is limited within the boundary of human skins. Growth of human MCF-7 tumors in the human skin grafts increased the ratio of human:murine vessels. Anal yses of the grafted skins before and after tumor transplantation showed tha t SN6h reacted with tumor-induced angiogenic blood vessels but not with non angiogenic vessels, whereas antihuman CD31 mAb reacted with both angiogenic and nonangiogenic vessels. The results show that SN6h is capable of distin guishing the tumor-induced angiogenic vasculature from the nonangiogenic va sculature in the present model. Antiangiogenic therapy of the chimeras bear ing established MCF-7 tumors was carried out by i.v. administration of a mA b(s) via the tail vein of mice. SN6j and SN6k were effective for suppressin g the established tumors, whereas tumor suppression was weaker with SN6f. T he results indicate an absence of a direct correlation between antigen-bind ing avidity and in vivo antitumor efficacy of anti-EDG mAbs and suggest the importance of other factors (e.g., epitopes) in antitumor efficacy. No sig nificant toxicity of the mAbs was detected. Combination of SN6f and SN6k th at define mutually nonoverlapping epitopes showed an additive antitumor eff ect. Combination of SN6j and cyclophosphamide using an antiangiogenic sched ule of drug dosing showed synergistic antitumor efficacy. The combination t herapy induced lasting complete regression of the established tumors in two of the eight treated chimeras. We examined human and murine blood vessels in large human tumors from the c himeras at the end of therapeutic experiment. The test showed that SN6j the rapy resulted in complete suppression of human vessels in the tumors but re sulted in only weak suppression of murine vessels. Cyclophosphamide was not effective for suppressing human vessels and only weakly suppressive agains t murine vessels. Combination of SN6j and cyclophosphamide was effective fo r completely suppressing human vessels and also effective for partial (i.e. , 35%) suppression of murine vessels. The results show that systemic admini stration of naked antihuman EDG mAbs can suppress established tumors, and t he efficacy is markedly enhanced by combining a chemotherapeutic drug using an antiangiogenic schedule of drug dosing. These mAbs should show stronger antitumor efficacy in patients whose tumors depend entirely on human blood vessels.