Antiangiogenic therapy of established tumors in human skin/severe combined immunodeficiency mouse chimeras by anti-endoglin (CD105) monoclonal antibodies, and synergy between anti-endoglin antibody and cyclophosphamide. (2001)

Endoglin (EDG; CD105) is a proliferation-associated cell membrane antigen of endothelial cells and is strongly expressed on the tumor-associated angiogenic vascular endothelium. Furthermore, EDG is essential for angiogenesis and a component of the transforming growth factor (TGF)-beta receptor complex. The present three anti-EDG monoclonal antibodies (mAbs), SN6f, SN6j, and SN6k, react strongly with proliferating human endothelial cells but cross-react very weakly with murine endothelial cells. Analysis of Scatchard plot of direct binding of these mAbs to proliferating human umbilical vein endothelial cells showed equilibrium constants of 8.3 x 10(9), 3.1 x 10(9), and 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 antiangiogenic tumor therapy by these mAbs in animal models, we used human skin/severe combined immunodeficiency (SCID) mouse chimeras bearing tumors of MCF-7. Blood vessels in the chimeras were analyzed by immunostaining with species (human or mouse)-specific anti-CD31 and anti-EDG mAbs including an antihuman EDG mAb termed SN6h. Blood vessels in the completely healed grafted human 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 and s.c. tissues. Therefore, murine vessels infiltrate into the human skin grafts 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. Analyses of the grafted skins before and after tumor transplantation showed that SN6h reacted with tumor-induced angiogenic blood vessels but not with nonangiogenic vessels, whereas antihuman CD31 mAb reacted with both angiogenic and nonangiogenic vessels. The results show that SN6h is capable of distinguishing the tumor-induced angiogenic vasculature from the nonangiogenic vasculature in the present model. Antiangiogenic therapy of the chimeras bearing established MCF-7 tumors was carried out by i.v. administration of a mAb(s) via the tail vein of mice. SN6j and SN6k were effective for suppressing the established tumors, whereas tumor suppression was weaker with SN6f. The results indicate an absence of a direct correlation between antigen-binding avidity and in vivo antitumor efficacy of anti-EDG mAbs and suggest the importance of other factors (e.g., epitopes) in antitumor efficacy. No significant toxicity of the mAbs was detected. Combination of SN6f and SN6k that define mutually nonoverlapping epitopes showed an additive antitumor effect. Combination of SN6j and cyclophosphamide using an antiangiogenic schedule of drug dosing showed synergistic antitumor efficacy. The combination therapy 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 chimeras at the end of therapeutic experiment. The test showed that SN6j therapy resulted in complete suppression of human vessels in the tumors but resulted in only weak suppression of murine vessels. Cyclophosphamide was not effective for suppressing human vessels and only weakly suppressive against murine vessels. Combination of SN6j and cyclophosphamide was effective for completely suppressing human vessels and also effective for partial (i.e., 35%) suppression of murine vessels. The results show that systemic administration of naked antihuman EDG mAbs can suppress established tumors, and the 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.