Yuro Haruta

Endoglin-Targeted Cancer Therapy

Vascular-targeting antiangiogenic therapy (VTAT) of cancer can be advantageous over conventional tumor cell targeted cancer therapy if an appropriate target is found. Our hypothesis is that endoglin (ENG; CD105) is an excellent target in VTAT. ENG is selectively expressed on vascular and lymphatic endothelium in tumors. This allows us to target both tumor-associated vasculature and lymphatic vessels to suppress tumor growth and metastasis. ENG is essential for angiogenesis/vascular development and a co-receptor of TGF-β. Our studies of selected anti-ENG monoclonal antibodies (mAbs) in several animal models and in vitro studies support our hypothesis. These mAbs and/or their immunoconjugates (immunotoxins and radioimmunoconjugates) induced regression of preformed tumors as well as inhibited formation of new tumors. In addition, they suppressed metastasis. Several mechanisms were involved in the suppressive activity of the naked (unconjugated) anti-ENG mAbs. These include direct growth suppression of proliferating endothelial cells, induction of apoptosis, ADCC (antibody-dependent cell-mediated cytotoxicity) and induction of T cell immunity. To facilitate clinical application, we generated a human/mouse chimeric anti-ENG mAb termed c-SN6j and performed studies of pharmacokinetics, toxicology and immunogenicity of c-SN6j in nonhuman primates. No significant toxicity was detected by several criteria and minimal immune response to the murine part of c-SN6j was detected after multiple i.v. injections. The results support our hypothesis that c-SN6j can be safely administered in cancer patients. This hypothesis is supported by the ongoing phase 1 clinical trial of c-SN6j (also known as TRC105) in patients with advanced or metastatic solid cancer in collaboration with Tracon Pharma and several oncologists (NCT00582985).

Antiangiogenic radioimmunotherapy of human solid tumors in SCID mice using 125I-labeled anti-endoglin monoclonal antibodies

Endoglin (CD105), which is a component of the TGF‐β receptor complex, is highly expressed at the surface of proliferating human endothelial cells such as those of tumor vessels. In the present study, we tested the antitumor efficacy of 125I‐labeled anti‐endoglin monoclonal antibodies (MAbs), SN6f and SN6j, against s.c. tumors of MCF‐7 human breast cancer cells in SCID mice by i.v. administration. SN6f and SN6j cross‐react weakly with mouse endothelial cells, but show no significant reactivity with MCF‐7 tumor cells. These MAbs are effectively internalized into the cells after binding to the cell surface antigen of endothelial cells. Four groups of SCID mice (n = 10 or 9 in each group) inoculated s.c. with 8 × 106 MCF‐7 cells were treated with 125I‐SN6f (10 μCi), 125I‐SN6j (10 μCi), a 125I‐labeled isotype‐matched control IgG (10 μCi) or PBS. The systemic therapy was performed in 2 series, i.e., on days 3, 5, 7 and days 58, 60, 62. Both 125I‐SN6f and 125I‐SN6j showed significant growth suppression of the tumors, whereas the 125I‐labeled control IgG did not show any significant antitumor efficacy. No significant toxicity or weight loss was observed in mice treated with either 125I‐SN6f or 125I‐SN6j. After 100 days of observation, autopsies revealed no significant organ damage. Our results show the possible usefulness of antiangiogenic radioimmunotherapy using 125I‐labeled anti‐endoglin MAbs. Int. J. Cancer 82:737–742, 1999.

Facilitation of endoglin-targeting cancer therapy by development/utilization of a novel genetically engineered mouse model expressing humanized endoglin (CD105)

Endoglin (ENG) is a TGF‐β coreceptor and essential for vascular development and angiogenesis. A chimeric antihuman ENG (hENG) monoclonal antibody (mAb) c‐SN6j (also known as TRC105) shows promising safety and clinical efficacy features in multiple clinical trials of patients with various advanced solid tumors. Here we developed a novel genetically engineered mouse model to optimize the ENG‐targeting clinical trials. We designed a new targeting vector that contains exons 4–8 of hENG gene to generate novel genetically engineered mice (GEMs) expressing functional human/mouse chimeric (humanized) ENG with desired epitopes. Genotyping of the generated mice confirmed that we generated the desired GEMs. Immunohistochemical analysis demonstrated that humanized ENG protein of the GEMs expresses epitopes defined by 7 of our 8 anti‐hENG mAbs tested. Surprisingly the homozygous GEMs develop normally and are healthy. Established breast and colon tumors as well as metastasis and tumor microvessels in the GEMs were effectively suppressed by systemic administration of anti‐hENG mAbs. Additionally, test result indicates that synergistic potentiation of antitumor efficacy can be induced by simultaneous targeting of two distinct epitopes by anti‐hENG mAbs. Sorafenib and capecitabine also showed antitumor efficacy in the GEMs. The presented novel GEMs are the first GEMs that express the targetable humanized ENG. Test results indicate utility of the GEMs for the clinically relevant studies. Additionally, we generated GEMs expressing a different humanized ENG containing exons 5–6 of hENG gene, and the homozygous GEMs develop normally and are healthy.

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.