Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin

Abstract

In the present study, we developed an antitumor immunoconjugate that appears to be promising as a novel curative antitumor agent against a variety of human solid tumors. We generated a new antihuman endoglin (EDG) monoclonal antibody (mAb) K4-2C10 (or termed SN6f) that cross-reacts with mouse endothelial cells. Such cross-reactive anti-EDG mAbs have not been reported previously. This mAb was used to target tumor-associated vasculature in SCID mice inoculated with human tumors. No anti-EDG mAb or its immunoconjugates have previously been successfully used for targeting vasculature in vivo. In this study, MCF-7 human breast cancer cells were inoculated s.c. into SCID mice. K4-2C10 did not react with the MCF-7 cells but showed a weak reactivity with mouse endothelial cells. The mAb reacted with the proliferating endothelial cells more strongly than with the quiescent endothelial cells. The mAb exhibited much stronger reactivity (>10-fold) with human endothelial cells than with mouse endothelial cells and reacted strongly with vascular endothelium of tumor-associated blood vessels in a variety of human malignant tissues. Conjugates of K4-2C10 with ricin A chain (RA) and deglycosylated ricin A chain (dgRA) showed a weak but specific cytotoxic activity against murine endothelial cells in vitro; the 50% inhibitory dose of the RA and dgRA conjugates was 54 nm and 29 nm, respectively. Remarkable antitumor efficacy was observed when a small amount (a total of 60 microgram corresponding to 24% of the LD50 dose) of the dgRA conjugate was administered i.v. into SCID mice that had been inoculated s.c. with MCF-7. Unconjugated mAb K4-2C10 was not significantly effective in the inhibition of the tumor growth. The immunotoxin (IT) completely inhibited growth of the tumor in all of the treated mice (n = 8). Furthermore, similar antitumor efficacy was observed when the IT was administered i.v. into the tumor-inoculated SCID mice that had been pretreated with unconjugated K4-2C10 to block the potentially available weak binding sites of normal tissues. The strong therapeutic effects of the IT were reproduced in another set of therapeutic experiments. No significant side effects were observed in the mice. The differences in the tumor growth between the control group and the IT-treated groups were statistically significant. The IT showed antiangiogenic activity in the dorsal air sac method. The results indicate that K4-2C10 IT effectively treated the tumor-bearing mice by selectively inhibiting the tumor-associated blood vessels and by disrupting tumor-associated angiogenesis. The strong antitumor efficacy of the K4-2C10 IT is remarkable in view of the fact that K4-2C10 and its IT showed only a weak reactivity with mouse endothelial cells, and a relatively small amount of the IT was administered i.v. to treat s.c. tumors. We anticipate that the K4-2C10 IT will show much stronger antitumor efficacy and antiangiogenic activity in patients with solid tumors and other angiogenesis-associated diseases. The present results demonstrate for the first time that an anti-EDG mAb or its immunoconjugate can effectively target tumor-associated vasculature in vivo.