Abstract 378: Serum endoglin (CD105): Molecular heterogeneity and marked differences among different anti-endoglin monoclonal antibodies in the detection of metastasis and tumor progression

Abstract

Abstract Endoglin (ENG, CD105) is a proliferation-associated homodimeric cell membrane antigen of endothelial cells and leukemia cells. It is strongly expressed on angiogenic vascular endothelium in tumors and also expressed on lymphatic vessels in tumors. Furthermore, ENG is a TGF-β coreceptor and essential for angiogenesis. ENG represents a more specific marker for tumor angiogenesis and/or tumor progression than the commonly used pan-endothelial markers such as CD34 and CD31. Previously we developed a double antibody sandwich radioimmunoassay (DAS-RIA) using two anti-ENG monoclonal antibodies (mAbs) SN6h and SN6a to quantify nanograms/ml levels of serum ENG and found association of serum ENG levels with tumor metastasis (Clin Cancer Res, 2001, 7: 524). The objective of the present study is to characterize molecular nature of serum ENG and to test a hypothesis that sensitivity and specificity of DAS-RIA can be substantially improved by selecting appropriated pairs of anti-ENG mAbs. We generated 12 anti-ENG mAbs and epitopes defined by these mAbs were mapped. We selected 5 pairs of anti-ENG mAbs defining different epitopes to compare their sensitivity and specificity. Anti-ENG mAb SN6h was used as the capture antibody in each pair because SN6h possesses an extremely high antigen-binding avidity (association K = 1.38 × 1011 liters/mol) and it strongly binds both native and denatured forms of ENG. The order of sensitivity among the five pairs was determined to be SN6f/SN6h>> SN6g/SN6h> SN6a/SN6h ≈ SN6j/SN6h> SN6/SN6h. SN6f/SN6h and SN6g/SN6h pairs were particularly effective for distinguishing between Metastasis-positive (Meta+) and Meta− samples. In an additional study, we developed a sensitive DAS-ELISA using SN6f/SN6h and SN6g/SN6h to facilitate easier quantification of soluble ENG. Reasons for the different capacity of different pairs of anti-ENG mAbs to distinguish between Meta+ and Meta− samples will be attributable to molecular heterogeneity of serum ENG, different epitopes defined by individual mAbs, and differences in the antigen-binding avidity among these mAbs. Western blot of serum samples from cancer patients showed highly heterogeneous patterns of serum ENG. Furthermore, certain mAbs were able to detect only a fraction of the heterogeneous ENG molecules while others were capable of detecting a series of heterogeneous ENG molecules. Test results suggest that the major ENG component in the heterogeneous ENG corresponds to a complex of TGF-β1/TGF-β3, TGF-β receptor II and ENG fragment(s). Assay results of serum ENG will be strongly influenced by the molecular heterogeneity of serum ENG, different capacity of individual anti-ENG mAbs in detection of different components of serum ENG, and differences in the heterogeneous patterns of serum ENG among different cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 378.