Hilda Tsai

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).

Synergy between anti-endoglin (CD105) monoclonal antibodies and TGF-β in suppression of growth of human endothelial cells

Endoglin (CD105) is a proliferation‐associated cell membrane antigen of endothelial cells and strongly expressed in the angiogenic vasculature of solid tumors. Endoglin is essential for angiogenesis/vascular development and an ancillary transforming growth factor β (TGF‐β) receptor. Certain anti‐endoglin monoclonal antibodies (mAbs), termed SN6 series mAbs, inhibited angiogenesis, tumor growth and metastasis in mice. We investigated the mechanisms by which anti‐endoglin mAbs suppress growth of proliferating endothelial cells. We found that 4 SN6 series mAbs suppressed growth of human umbilical vein endothelial cells (HUVECs) in a dose‐dependent manner in the absence of any effector cells or complement. Significant differences in the growth suppression between the 4 anti‐endoglin mAbs defining different epitopes were observed. These differences were not determined by antigen‐binding avidities of the mAbs. Combination of TGF‐β1 and each of the 4 anti‐endoglin mAbs exerted synergistic growth suppression of HUVECs. Binding of anti‐endoglin mAbs to endoglin‐expressing cells did not block the subsequent binding of TGF‐β1. Conversely, preincubation of HUVECs with TGF‐β1 did not change cell surface expression of endoglin. The present results suggest that direct suppression of the endothelial cell growth by SN6 series mAbs is one of the underlying mechanisms by which anti‐endoglin mAbs exert antiangiogenic and tumor‐suppressive activity in vivo. The results further suggest that TGF‐β1 plays an important role in the in vivo antiangiogenic efficacy of anti‐endoglin mAbs by synergistically enhancing the activity of these mAbs. Further studies of the present novel findings may provide valuable information about the functional roles of endoglin and anti‐endoglin mAbs in the TGF‐β‐mediated cell regulation.

Anti-endoglin monoclonal antibodies are effective for suppressing metastasis and the primary tumors by targeting tumor vasculature

Anti‐metastatic activity of an antitumor agent is exceedingly important because metastasis is the primary cause of death for most solid cancer patients. In this report, we show that 3 anti‐endoglin (ENG) monoclonal antibodies (mAbs) SN6a, SN6j and SN6k which define individually distinct epitopes of ENG of tumor vasculature are capable of suppressing tumor metastases in the multiple metastasis models. The metastasis models were generated by i.v., s.c. (into flank) or mammary gland fat pad injection of 4T1 murine mammary carcinoma cells and splenic injection of two types of colon26 murine colorectal carcinoma cells. Individual mAbs were injected i.v. via the tail vein of mice. SN6a and SN6j effectively suppressed the formation of metastatic colonies of 4T1 in the lung in all of the three 4T1 metastatic models. In addition, these mAbs were effective for suppressing the primary tumors of 4T1 in the skin and mammary fat pad. These mAbs effectively suppressed microvessel density and angiogenesis in tumors as measured by the Matrigel plug assay in mice. No significant side effects of the administered mAbs were detected. Furthermore, SN6a and SN6j extended survival of the tumor‐bearing mice. SN6j, SN6k and their immunoconjugates with deglycosylated ricin A‐chain were all effective for suppressing hepatic metastasis of colon26. The findings in the present study are clinically relevant in view of the ongoing clinical trial of a humanized (chimerized) form of SN6j.

Anti-tumor activity of an anti-endoglin monoclonal antibody is enhanced in immunocompetent mice.

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+ T cells and/or CD8+ T cells on antitumor efficacy of SN6j in mice. Systemic (i.v.) administration of a relatively small dose (0.6 μg/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 CD4+ T cells and/or CD8+ T cells were depleted; effect of CD8+ 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.

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.

Abstract 3278: Novel knock-in mice expressing human/mouse chimeric endoglin (ENG; CD105) develop normally and will be valuable for evaluating clinical application of anti-human ENG antibodies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The major objective of this study is to develop a novel animal model to facilitate optimal clinical application of anti-human ENG (huENG) monoclonal antibodies (mAbs) to treat cancer patients. ENG 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 in lymphatic vessels in tumors. Furthermore, ENG is a TGF-β coreceptor and essential for angiogenesis/vascular development. Mice lacking ENG die from defective vascular development. Mutations in the huENG gene are associated with hereditary hemorrhagic telangiectasia type 1 (HHT1) which is characterized by vascular malformations and recurrent bleeding. huENG shows a relatively restricted expression pattern in tissues and cells. We hypothesized that huENG is a safe and effective target in vascular-targeting antiangiogenic therapy (VTAT). Clinical trials of a chimeric anti-huENG mAb c-SN6j (also known as TRC105) are in progress (ClinicalTrials.gov Identifier: [NCT00582985][1] and [NCT01090765][2]). The major obstacle in animal model studies of anti-huENG mAbs is that most anti-huENG mAbs do not react with mouse ENG or mouse endothelial cells. To overcome this obstacle, we generated knock-in mice expressing human/mouse chimeric ENG. We designed the chimeric vectors to contain huENG gene segments of desired location and length so that the expressed huENG protein contains desired epitopes. Nevertheless, the major concern was that the generated knock-in mice might suffer from defective vascular development or HHT-like symptoms if the chimeric ENG does not function properly in mice. We generated two types of knock-in mice containing different sizes of huENG gene. In both cases, mice develop normally and show no sign of HHT-like symptoms in the homozygous mice. Tumors of 4T1 breast cancer cells and Colon26 colon cancer cells were generated in the knock-in mice to test expression of chimeric ENG protein in the tumor microvessels. Immunohistochemical staining of the tumors using several anti-huENG mAbs showed that the tumor microvessels express desired epitopes of huENG protein which are candidate targets for VTAT. The epitopes defined by these mAbs have been mapped. This novel knock-in mouse model will be useful for evaluating antitumor efficacy of many anti-huENG mAbs. In addition, this model will be useful for evaluating appropriate combination of an anti-huENG mAb with other anticancer agents to achieve optimal antitumor efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2011-3278 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00582985&atom=%2Fcanres%2F71%2F8_Supplement%2F3278.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01090765&atom=%2Fcanres%2F71%2F8_Supplement%2F3278.atom