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In the present study, we investigated the mechanisms by which anti-endoglin (EDG; CD105) monoclonal antibodies (mAbs) suppress angiogenesis and tumor growth. Antihuman EDG mAb SN6j specifically bound to murine endothelial cells and was internalized into the cells in vitro. SN6j effectively suppressed angiogenesis in mice in the Matrigel plug assay. We found that SN6j is more effective for tumor suppression in immunocompetent mice than in SCID mice. We hypothesized that T cell immunity is important for effective antitumor efficacy of SN6j in vivo. To test this hypothesis, we investigated effects of CpG oligodeoxynucleotides (ODN) and depletion of CD4 1 T cells and/or CD8 1 T cells on antitumor efficacy of SN6j in mice. Systemic (i.v.) administration of a relatively small dose (0.6 lg/g body weight/dose) of SN6j suppressed growth of established s.c. tumors of colon-26 in BALB/c mice and improved survival of the tumor-bearing mice. Addition of CpG ODN to SN6j synergistically enhanced antitumor efficacy of SN6j. In contrast, such enhancing effects of CpG ODN were not detected in SCID mice. Antitumor efficacy of SN6j in BALB/c mice was abrogated when CD41 T cells and/or CD8 1 T cells were depleted; effect of CD8 1 T cell depletion was stronger. Interestingly, CD4-depletion decreased tumor growth while CD8-depletion enhanced tumor growth in the absence of SN6j. SN6j induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner which indicates an additional mechanism of antiangiogenesis by SN6j. ' 2008 Wiley-Liss, Inc.Key words: endoglin; antiangiogenesis; monoclonal antibody therapy; T cell immunity; synergy Angiogenesis is essential for tumor growth, progression and metastasis and antiangiogenic therapy has become a new promising strategy in the treatment of cancer patients.

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We generated a human/mouse chimeric antibody c-SN6j of human IgG1 isotype from a murine anti-human endoglin (EDG) monoclonal antibody (mAb) SN6j that suppressed angiogenesis, tumor growth and metastasis in mice. We determined pharmacokinetics (PKs) and immunogenicity of c-SN6j in monkeys after multiple i.v. injections. A dose-escalation study was performed by administration of c-SN6j into six monkeys at the dose of 1 mg, 3 mg and 10 mg per kg body weight. In addition, both c-SN6j (3 mg/kg) and doxorubicin (0.275 mg/kg) were injected into two monkeys. c-SN6j and doxorubicin were injected twice a week for 3 weeks. We developed a unique and sensitive ELISA by sequentially targeting the common and idiotypic epitopes of c-SN6j-Fv to quantify plasma c-SN6j. Application of the ELISA showed that increasing the c-SN6j dose resulted in a proportional increase in the circulating c-SN6j after the first injection. In addition, the estimated area under the curve (AUC) for the first injection of c-SN6j is proportional to dose. We carried out detailed analyses of PKs of c-SN6j during and after the repeated injections. Our model of PKs fitted the empirical data well. Addition of doxorubicin modulated the PK parameters. We developed two ELISAs to separately determine the immune responses to the murine part and the human part of c-SN6j in monkeys. Interestingly, the murine part induced a weaker immune response than the human part. Doxorubicin potentiated the immune responses. Increasing the dose of c-SN6j increased plasma levels of c-SN6j but did not increase the immune responses to c-SN6j.