Ben K. Seon

A Phase I First-in-Human Study of TRC105 (Anti-Endoglin Antibody) in Patients with Advanced Cancer

Purpose: TRC105 is a chimeric IgG1 monoclonal antibody that binds CD105 (endoglin). This first-in-human, phase I, open-label study assessed safety, pharmacokinetics, and antitumor activity of TRC105 in patients with advanced refractory solid tumors. Patients and Methods: Patients received escalating doses of intravenous TRC105 until disease progression or unacceptable toxicity using a standard 3 + 3 phase I design. Results: Fifty patients were treated with escalating doses of TRC105. The maximum tolerated dose (MTD) was exceeded at 15 mg/kg every week because of dose-limiting hypoproliferative anemia. TRC105 exposure increased with increasing dose, and continuous serum concentrations that saturate CD105 receptors were maintained at 10 mg/kg weekly (the MTD) and 15 mg/kg every 2 weeks. Common adverse events including anemia, telangiectasias, and infusion reactions reflected the mechanism of action of the drug. Antibodies to TRC105 were not detected in patients treated with TRC105 from Chinese hamster ovary cells being used in ongoing phase Ib and phase II studies. Stable disease or better was achieved in 21 of 45 evaluable patients (47%), including two ongoing responses at 48 and 18 months. Conclusion: TRC105 was tolerated at 10 mg/kg every week and 15 mg/kg every 2 weeks, with a safety profile that was distinct from that of VEGF inhibitors. Evidence of clinical activity was seen in a refractory patient population. Ongoing clinical trials are testing TRC105 in combination with chemotherapy and VEGF inhibitors and as a single agent in prostate, ovarian, bladder, breast, and hepatocellular cancer. Clin Cancer Res; 18(17); 4820–9. ©2012 AACR.

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.

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.

An Open-Label Phase Ib Dose-Escalation Study of TRC105 (Anti-Endoglin Antibody) with Bevacizumab in Patients with Advanced Cancer

Purpose: Endoglin, an endothelial cell membrane receptor expressed on angiogenic tumor vessels, is essential for angiogenesis and upregulated in the setting of VEGF inhibition. TRC105 is an anti-endoglin IgG1 monoclonal antibody that potentiates VEGF inhibitors in preclinical models. This study assessed safety, pharmacokinetics, and antitumor activity of TRC105 in combination with bevacizumab. Experimental Design: Patients (n = 38) with advanced solid tumors, Eastern Cooperative Group performance status 0–1, and normal organ function were treated with escalating doses of TRC105 plus bevacizumab until disease progression or unacceptable toxicity using a standard 3 + 3 phase I design. Results: TRC105 and bevacizumab were well tolerated at their recommended single-agent doses (10 mg/kg) when the initial dose of TRC105 was delayed by one week and divided over 2 days to limit the frequency of headache. The concurrent administration of bevacizumab and TRC105 did not otherwise potentiate known toxicities of TRC105 or bevacizumab. Hypertension and proteinuria were observed, though not at rates expected for single-agent bevacizumab. Several patients who had previously progressed on bevacizumab or VEGF receptor tyrosine kinase inhibitor (VEGFR TKI) treatment experienced reductions in tumor volume, including two partial responses by RECIST, and 6 remained without progression for longer periods than during their prior VEGF inhibitor therapy. Conclusions: TRC105 was well tolerated with bevacizumab and clinical activity was observed in a VEGF inhibitor–refractory population. Ongoing clinical trials are testing TRC105 in combination with bevacizumab in glioblastoma and with VEGFR TKIs in renal cell carcinoma, hepatocellular carcinoma, and soft tissue sarcoma. Clin Cancer Res; 20(23); 5918–26. ©2014 AACR.

Expression of endoglin (CD105) in tumor blood vessels

Dear Sir, I read with interest the report of Balza et al.1 describing the expression of endoglin (EDG) in human tissues and cultured cell lines using a new monoclonal antibody termed A11. Although the report contains interesting data, I wish to address and clarify obvious deficiencies and questionable conclusions. These problems are important because they are concerned with the potential clinical application of anti-EDG monoclonal antibodies (MAbs) for antiangiogenic therapy of patients with various angiogenesis-associated diseases including cancer. First, one needs to include an isotype-matched control IgG (or MAb) as a negative control when a MAb is used in various immunoassays. However, the authors used cells treated with fresh medium as a negative control in their immunoassay. Such a control is inadequate. The cells should have been treated with medium containing an isotype-matched control murine IgG (i.e., IgG1). The authors did not report use of an isotypematched control IgG in any of their immunoassays. Use of such a proper control is particularly important when a weak reactivity of a MAb with tissues and cells is tested. It is commonly observed that IgGs bind to tissues and cells via Fc receptors and other interactions. Therefore, some of the reactivities of A11 with normal tissues and cell lines could be background reactivities or weaker reactions than they observed. Second, reactivity of an anti-EDG MAb with cultured cell lines does not represent reactivity of the MAb with corresponding fresh tissues, although the authors used such reactivity of A11 as evidence for the lack of specificity of anti-EDG MAbs in general. Anti-EDG MAbs reacted with vascular endothelium but not with tumor cells per se of solid tumor tissues in our tests of several anti-EDG MAbs with more than 100 malignant tissue samples.2,3 However, these and other anti-EDG MAbs reacted with many malignant epithelial cell lines.4,5 Therefore, reactivity of anti-EDG MAbs with epithelial cell lines is not relevant to the reactivity of the MAbs in vivo. In contrast, the reactivity of anti-EDG MAbs with hematologic cell lines is clinically relevant because the expression profile of EDG is consistent between the cultured cell lines and fresh leukemia/ lymphoma cells of the corresponding phenotypes, e.g., between immature B-lineage leukemia cells and cell lines.6 Third, there are substantial differences among different antiEDG MAbs in their specificity for tumor vasculature compared with normal tissue vasculature. In our experience, certain antiEDG MAbs defining different epitopes show differences in fine specificity.7 Balza et al.1 studied reactivity of a single antiEDG MAb A11 and emphasized the lack of specificity of EDG expression for tumor blood vessels. Despite this assertion, the reactivity of A11 with tumor vasculature was stronger than that with the blood vessels of normal tissues.1 They stated that clinical use of EDG as a target marker should be reevaluated. However, their report does not present new data that may lead us to reevaluate our approach to potential clinical application of our anti-EDG MAbs. In addition, the previously reported reactivity of anti-EDG MAbs with several minor cells4 may not preclude selected anti-EDG MAbs from therapeutic application. We are aware of the lack of strict specificity of anti-EDG MAbs for tumor vasculature. We recently7 stated that EDG is not a tumor-specific marker and that it is expressed in varying degrees in the vasculature of normal tissues. Despite this limitation, we could effectively target tumor-associated vasculature using selected anti-EDG MAbs and immunoconjugates.2,3,7,8 We believe that this effective targeting of tumorassociated vascular endothelium is attributable to the combined effect of the following: 1. Certain anti-EDG MAbs show a highly restricted reactivity among different tissues and cells. 2. EDG is a proliferation-associated antigen on endothelial cells and its expression is upregulated in tumor-associated vascular endothelium.2,7,9–12 3. Turnover of endothelial cells of normal adult tissue vasculature is very slow (e.g., more than 1,000 days), whereas these endothelial cells undergo rapid proliferation during spurts of angiogenesis in tumors.13,14 Therefore, the rapidly dividing endothelial cells of tumor vasculature are much more susceptible to killing by anti-EDG MAbs and immunoconjugates than the quiescent vascular endothelium of normal tissues. Other, undefined, factors may be involved as well. I would like to point out that most, if not all, of the MAbs that are being successfully applied for cancer therapy in patients are not tumor-specific MAbs. These MAbs include rituximab (antiCD20 MAb), trastuzumab (anti-HER2 MAb) and C225 (antiepidermal growth factor receptor MAb). In the therapeutic application of a MAb, we need to find an appropriate therapeutic window in which the MAb can achieve therapeutic efficacy without exerting severe side effects in the host. We were able to find such therapeutic windows for our selected anti-EDG MAbs in animal studies.2,3,7,8 We need to find such windows in patients for the clinical application of these MAbs.

A Phase II Clinical Trial of TRC105 (Anti-Endoglin Antibody) in Adults With Advanced/Metastatic Urothelial Carcinoma

&NA; TRC105 is a chimeric monoclonal antibody that targets CD105 (endoglin). Heavily pretreated patients with metastatic urothelial carcinoma received TRC105 at 15 mg/m2 every 2 weeks on a 28‐day cycle. Treatment was not associated with significant toxicities, but did not improve 6‐month progression‐free survival. Exploratory analyses suggest interplay between immunosuppressive subsets and TRC105, which warrants further study. Background: In this trial we assessed the efficacy and tolerability of TRC105, a chimeric monoclonal antibody that targets CD105 (endoglin) in patients with advanced, previously treated urothelial carcinoma (UC). Patients and Methods: Patients received TRC105 15 mg/kg every 2 weeks on days 1 and 15 of each 28‐day cycle. The primary end point was progression‐free survival (PFS) at 6 months. Secondary end points included safety, toxicity, and overall survival (OS). CD105 expression was evaluated using immunohistochemistry (IHC) in a separate cohort of 50 UC patients. Biomarker studies included immune subsets, circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPs), and osteopontin. Results: Of 13 patients enrolled, 12 were evaluable for OS and PFS. The 3‐month PFS probability was 18.2% (median PFS, 1.9 months [95% confidence interval (CI), 1.8‐2.1 months). This met the criterion for ending accrual on the basis of the 2‐stage design. Median OS was 8.3 months (95% CI, 3.3‐17.0 months). IHC for CD105 scores was not associated with T stage (P = .26) or presence of lymph nodes (P = .64). Baseline levels of regulatory T and B cells, CEPs, and changes in CEC level after TRC105 exhibited trends toward an association with PFS or OS. CTCs pre‐ and post‐TRC105 were detected in 4 of 4 patients. Conclusion: Although TRC105 was well tolerated, it did not improve 6‐month PFS in heavily pretreated patients with advanced UC. CD105 staining was present in 50% of UC tumors at different intensities. Our observations on the pharmacodynamic significance of immune subsets, CECs, and CTCs warrant further study.

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.