A phase I/II study of preoperative letrozole, everolimus, and carotuximab in stage 2 and 3 hormone receptor-positive and Her2-negative breast cancer.

PURPOSE: In nonmetastatic hormone receptor-positive and Her2-negative breast cancer, preoperative endocrine therapies can yield outcomes similar with chemotherapy. We evaluated the tolerability and preliminary antitumor activity of preoperative letrozole, everolimus, and carotuximab, a monoclonal antibody targeting endoglin, in nonmetastatic breast cancer. METHODS: Eligible patients had newly diagnosed, stage 2 or 3, hormone receptor-positive and Her2/neu-negative breast cancer. Patients received escalating doses of everolimus; the dose of letrozole and carotuximab were fixed at 2.5 mg PO daily and 15 mg/kg intravenously every 2 weeks, respectively. The primary objective was to determine the safety and tolerability of the combination. Secondary objectives included pharmacokinetic and pharmacodynamic studies and assessments of antitumor activity. RESULTS: Fifteen patients enrolled. The recommended phase 2 dose of everolimus in combination with letrozole and carotuximab was 10 mg PO daily. The most frequent adverse events were headache (67%), fatigue (47%), facial flushing and swelling (47%), gingival hemorrhage (40%), epistaxis (33%), nausea and vomiting (27%). Headache constituted a dose-limiting toxicity. At least two signs of mucocutaneous telangiectasia developed in 92% of patients. Carotuximab accumulated in the extravascular space and accelerated the biodistribution and clearance of everolimus. All patients had residual disease. Gene expression analyses were consistent with downregulation of genes involved in proliferation and DNA repair. Among 6 patients with luminal B breast cancer, 5 converted to luminal A after one cycle of therapy. CONCLUSION: Letrozole, everolimus, and carotuximab were tolerated in combination at their single-agent doses. Pharmacokinetic studies revealed an interaction between everolimus and carotuximab. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov (Identifier: NCT02520063), first posted on August 11, 2015, and is active, not recruiting.

An Open Label Phase Ib Dose Escalation Study of TRC105 (Anti-Endoglin Antibody) with Axitinib in Patients with Metastatic Renal Cell Carcinoma.

BACKGROUND: TRC105 is an IgG1 endoglin monoclonal antibody that potentiates VEGF inhibitors in preclinical models. We assessed safety, pharmacokinetics, and antitumor activity of TRC105 in combination with axitinib in patients with metastatic renal cell carcinoma (mRCC). SUBJECTS, MATERIALS, AND METHODS: Heavily pretreated mRCC patients were treated with TRC105 weekly (8 mg/kg and then 10 mg/kg) in combination with axitinib (initially at 5 mg b.i.d. and then escalated per patient tolerance to a maximum of 10 mg b.i.d.) until disease progression or unacceptable toxicity using a standard 3 + 3 phase I design. RESULTS: Eighteen patients (median number of prior therapies = 3) were treated. TRC105 dose escalation proceeded to 10 mg/kg weekly without dose-limiting toxicity. Adverse event characteristics of each drug were not increased in frequency or severity when the two drugs were administered concurrently. TRC105 and axitinib demonstrated preliminary evidence of activity, including partial responses (PR) by RECIST in 29% of patients, and median progression-free survival (11.3 months). None of the patients with PR had PR to prior first-line treatment. Lower baseline levels of osteopontin and higher baseline levels of TGF-ß receptor 3 correlated with overall response rate. CONCLUSION: TRC105 at 8 and 10 mg/kg weekly was well tolerated in combination with axitinib, with encouraging evidence of activity in patients with mRCC. A multicenter, randomized phase II trial of TRC105 and axitinib has recently completed enrollment (NCT01806064). IMPLICATIONS FOR PRACTICE: TRC105 is a monoclonal antibody to endoglin (CD105), a receptor densely expressed on proliferating endothelial cells and also on renal cancer stem cells that is implicated as a mediator of resistance to inhibitors of the VEGF pathway. In this Phase I trial, TRC105 combined safely with axitinib at the recommended single agent doses of each drug in patients with renal cell carcinoma. The combination demonstrated durable activity in a VEGF inhibitor-refractory population and modulated several angiogenic biomarkers. A randomized Phase II trial testing TRC105 in combination with axitinib in clear cell renal cell carcinoma has completed accrual.

Endoglin: a novel target for therapeutic intervention in acute leukemias revealed in xenograft mouse models.

Endoglin (CD105), a receptor of the transforming growth factor-ß superfamily, has been reported to identify functional long-term repopulating hematopoietic stem cells, and has been detected in certain subtypes of acute leukemias. Whether this receptor plays a functional role in leukemogenesis remains unknown. We identified endoglin expression on the majority of blasts from patients with acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Using a xenograft model, we find that CD105+ blasts are endowed with superior leukemogenic activity compared with the CD105- population. We test the effect of targeting this receptor using the monoclonal antibody TRC105, and find that in AML, TRC105 prevented the engraftment of primary AML blasts and inhibited leukemia progression following disease establishment, but in B-ALL, TRC105 alone was ineffective due to the shedding of soluble CD105. However, in both B-ALL and AML, TRC105 synergized with reduced intensity myeloablation to inhibit leukemogenesis, indicating that TRC105 may represent a novel therapeutic option for B-ALL and AML.

Targeting angiogenesis for radioimmunotherapy with a 177Lu-labeled antibody.

PURPOSE: Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. METHODS: The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. RESULTS: Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p < 0.001, all groups). In most study groups (all except the nonspecific IgG group), the body weights of the mice did not decrease by more than 10%, indicating the safety of the injected agents. Serum alanine transaminase levels remained nearly constant indicating no damage to the liver (a primary clearance organ of the agent), and this was confirmed by ex vivo histological analyses. CONCLUSION: 177Lu-DTPA-TRC105, when administered at a sufficient dose, is able to curtail tumor growth and provide a significant survival benefit without off-target toxicity. Thus, this targeted agent could be used in combination with other treatment options to slow tumor growth allowing the other agents to be more effective.

86/90Y-Based Theranostics Targeting Angiogenesis in a Murine Breast Cancer Model.

Angiogenesis is widely recognized as one of the hallmarks of cancer. Therefore, imaging and therapeutic agents targeted to angiogenic vessels may be widely applicable in many types of cancer. To this end, the theranostic isotope pair, 86Y and 90Y, were used to create a pair of agents for targeted imaging and therapy of neovasculature in murine breast cancer models using a chimeric anti-CD105 antibody, TRC105. Serial positron emission tomography imaging with 86Y-DTPA-TRC105 demonstrated high uptake in 4T1 tumors, peaking at 9.6 ± 0.3%ID/g, verified through ex vivo studies. Additionally, promising results were obtained in therapeutic studies with 90Y-DTPA-TRC105, wherein significantly ( p < 0.05) decreased tumor volumes were observed for the targeted treatment group over all control groups near the end of the study. Dosimetric extrapolation and tissue histological analysis corroborated trends found in vivo. Overall, this study demonstrated the potential of the pair 86/90Y for theranostics, enabling personalized treatments for cancer.

Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.

ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF-/-) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

High Yield Production and Radiochemical Isolation of Isotopically Pure Arsenic-72 and Novel Radioarsenic Labeling Strategies for the Development of Theranostic Radiopharmaceuticals.

Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched (72)Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure (72)As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming (72)Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation.

Novel Preparation Methods of (52)Mn for ImmunoPET Imaging.

(52)Mn (t1/2 = 5.59 d, ß(+) = 29.6%, Eßave = 0.24 MeV) shows promise in positron emission tomography (PET) and in dual-modality manganese-enhanced magnetic resonance imaging (MEMRI) applications including neural tractography, stem cell tracking, and biological toxicity studies. The extension to bioconjugate application requires high-specific-activity (52)Mn in a state suitable for macromolecule labeling. To that end a (52)Mn production, purification, and labeling system is presented, and its applicability in preclinical, macromolecule PET is shown using the conjugate (52)Mn-DOTA-TRC105. (52)Mn is produced by 60 µA, 16 MeV proton irradiation of natural chromium metal pressed into a silver disc support. Radiochemical separation proceeds by strong anion exchange chromatography of the dissolved Cr target, employing a semiorganic mobile phase, 97:3 (v:v) ethanol:HCl (11 M, aqueous). The method is 62 ± 14% efficient (n = 7) in (52)Mn recovery, leading to a separation factor from Cr of (1.6 ± 1.0) × 10(6) (n = 4), and an average effective specific activity of 0.8 GBq/µmol (n = 4) in titration against DOTA. (52)Mn-DOTA-TRC105 conjugation and labeling demonstrate the potential for chelation applications. In vivo images acquired using PET/CT in mice bearing 4T1 xenograft tumors are presented. Peak tumor uptake is 18.7 ± 2.7%ID/g at 24 h post injection and ex vivo (52)Mn biodistribution validates the in vivo PET data. Free (52)Mn(2+) (as chloride or acetate) is used as a control in additional mice to evaluate the nontargeted biodistribution in the tumor model.

Effects of the combination of TRC105 and bevacizumab on endothelial cell biology.

Endoglin, or CD105, is a cell membrane glycoprotein that is overexpressed on proliferating endothelial cells (EC), including those found in malignancies and choroidal neovascularization. Endoglin mediates the transition from quiescent endothelium, characterized by the relatively dominant state of Smad 2/3 phosphorylation, to active angiogenesis by preferentially phosphorylating Smad 1/5/8. The monoclonal antibody TRC105 binds endoglin with high avidity and is currently being tested in phase 1b and phase 2 clinical trials. In this report, we evaluated the effects of TRC105 on primary human umbilical vascular endothelial cells (HUVEC) as a single agent and in combination with bevacizumab. As single agents, both TRC105 and bevacizumab efficiently blocked HUVEC tube formation, and the combination of both agents achieved even greater levels of inhibition. We further assessed the effects of each drug on various aspects of HUVEC function. While bevacizumab was observed to inhibit HUVEC viability in nutrient-limited medium, TRC105 had little effect on HUVEC viability, either alone or in combination with bevacizumab. Additionally, both drugs inhibited HUVEC migration and induced apoptosis. At the molecular level, TRC105 treatment of HUVEC lead to decreased Smad 1/5/8 phosphorylation in response to BMP-9, a primary ligand for endoglin. Together, these results indicate that TRC105 acts as an effective anti-angiogenic agent alone and in combination with bevacizumab.

In vivo tumor vasculature targeted PET/NIRF imaging with TRC105(Fab)-conjugated, dual-labeled mesoporous silica nanoparticles.

Multifunctional mesoporous silica nanoparticles (MSN) with well-integrated multimodality imaging properties have generated increasing research interest in the past decade. However, limited progress has been made in developing MSN-based multimodality imaging agents to image tumors. We describe the successful conjugation of, copper-64 ((64)Cu, t1/2 = 12.7 h), 800CW (a near-infrared fluorescence [NIRF] dye), and TRC105 (a human/murine chimeric IgG1 monoclonal antibody) to the surface of MSN via well-developed surface engineering procedures, resulting in a dual-labeled MSN for in vivo targeted positron emission tomography (PET) imaging/NIRF imaging of the tumor vasculature. Pharmacokinetics and tumor targeting efficacy/specificity in 4T1 murine breast tumor-bearing mice were thoroughly investigated through various in vitro, in vivo, and ex vivo experiments. Dual-labeled MSN is an attractive candidate for future cancer theranostics.

Imaging tumor angiogenesis in breast cancer experimental lung metastasis with positron emission tomography, near-infrared fluorescence, and bioluminescence.

The goal of this study was to develop a molecular imaging agent that can allow for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of CD105 expression in metastatic breast cancer. TRC105, a chimeric anti-CD105 monoclonal antibody, was labeled with both a NIRF dye (i.e., IRDye 800CW) and (64)Cu to yield (64)Cu-NOTA-TRC105-800CW. Flow cytometry analysis revealed no difference in CD105 binding affinity/specificity between TRC105 and NOTA-TRC105-800CW. Serial bioluminescence imaging (BLI) was carried out to non-invasively monitor the lung tumor burden in BALB/c mice, after intravenous injection of firefly luciferase-transfected 4T1 (i.e., fLuc-4T1) murine breast cancer cells to establish the experimental lung metastasis model. Serial PET imaging revealed that fLuc-4T1 lung tumor uptake of (64)Cu-NOTA-TRC105-800CW was 11.9 ± 1.2, 13.9 ± 3.9, and 13.4 ± 2.1 %ID/g at 4, 24, and 48 h post-injection respectively (n = 3). Biodistribution studies, blocking fLuc-4T1 lung tumor uptake with excess TRC105, control experiments with (64)Cu-NOTA-cetuximab-800CW (which served as an isotype-matched control), ex vivo BLI/PET/NIRF imaging, autoradiography, and histology all confirmed CD105 specificity of (64)Cu-NOTA-TRC105-800CW. Successful PET/NIRF imaging of tumor angiogenesis (i.e., CD105 expression) in the breast cancer experimental lung metastasis model warrants further investigation and clinical translation of dual-labeled TRC105-based agents, which can potentially enable early detection of small metastases and image-guided surgery for tumor removal.

A phase 1 study of TRC102, an inhibitor of base excision repair, and pemetrexed in patients with advanced solid tumors.

INTRODUCTION: TRC102 potentiates the activity of cancer therapies that induce base excision repair (BER) including antimetabolite and alkylating agents. TRC102 rapidly and covalently binds to apurinic/apyrimidinic (AP) sites generated during BER, and TRC102-bound DNA causes topoisomerase II-dependent irreversible strand breaks and apoptosis. This study assessed the safety, maximum-tolerated dose (MTD), pharmacokinetics and pharmacodynamics of TRC102 alone and in combination with pemetrexed. PURPOSE: Patients with advanced solid tumors received oral TRC102 daily for 4 days. Two weeks later, patients began standard-dose pemetrexed on day 1 in combination with oral TRC102 on days 1 to 4. The pemetrexed-TRC102 combination was repeated every 3 weeks until disease progression. METHODS: Twenty-eight patients were treated with TRC102 at 15, 30, 60 or 100 mg/m(2)/d. The MTD was exceeded at 100 mg/m(2)/d due to grade 3 anemia in 50 % of patients. TRC102 exposure increased in proportion to dose with a mean t1/2 of 28 h. A pharmacodynamic assay confirmed that TRC102 binds to pemetrexed-induced AP sites at all doses studied. Stable disease or better was achieved in 15 of 25 patients evaluable for response (60 %), including one patient with recurrent metastatic oropharyngeal carcinoma that expressed high levels of thymidylate synthase, who achieved a partial response and was progression free for 14 months. CONCLUSIONS: When administered with pemetrexed, the maximum tolerated dose of oral TRC102 is 60 mg/m(2)/d for 4 days. Randomized controlled studies are planned to evaluate the clinical benefit of adding TRC102 to pemetrexed and other agents that induce BER.

PET imaging of CD105/endoglin expression with a 6¹/64Cu-labeled Fab antibody fragment.

PURPOSE: The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e., endoglin), and investigate its potential for PET imaging of tumor angiogenesis in a small-animal model after (61/64)Cu labeling. METHODS: TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and (61/64)Cu labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficiency and normal organ distribution of (61/64)Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression. RESULTS: TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu labeling of NOTA-TRC105-Fab was achieved with about 50 % yield (specific activity about 44 GBq/µmol). PET imaging revealed rapid uptake of (64)Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6 ± 0.4, 4.2 ± 0.5, 4.9 ± 0.3, 4.4 ± 0.7, and 4.6 ± 0.8 %ID/g at 0.5, 2, 5, 16, and 24 h after injection, respectively; n = 4). Since tumor uptake peaked soon after tracer injection, (61)Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h after injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination. CONCLUSION: We report PET imaging of CD105 expression using (61/64)Cu-NOTA-TRC105-Fab, which exhibited prominent and target-specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to radiolabeled intact antibody, which may be translated into same-day immunoPET imaging for clinical investigation.

Positron emission tomography imaging of tumor angiogenesis with a (61/64)Cu-labeled F(ab')(2) antibody fragment.

The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')(2) fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e., endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after (61/64)Cu-labeling. TRC105-F(ab')(2) of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. (61/64)Cu-labeling of NOTA-TRC105-F(ab')(2) (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of >75% (specific activity: ∼115 GBq/µmol). PET imaging revealed rapid tumor uptake of (64)Cu-NOTA-TRC105-F(ab')(2) in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7% ID/g at 0.5, 3, 16, 24, and 48 h postinjection respectively; n = 4). Since tumor uptake peaked at 3 h postinjection, (61)Cu-NOTA-TRC105-F(ab')(2) also gave good tumor contrast at 3 and 8 h postinjection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')(2) fragment led to more rapid tumor uptake (which peaked at 3 h postinjection) than radiolabeled intact antibody (which often peaked after 24 h postinjection), which may allow for same day immunoPET imaging in future clinical studies.

Positron emission tomography imaging of angiogenesis in a murine hindlimb ischemia model with 64Cu-labeled TRC105.

The goal of this study was to assess ischemia-induced angiogenesis with (64)Cu-NOTA-TRC105 positron emission tomography (PET) in a murine hindlimb ischemia model of peripheral artery disease (PAD). CD105 binding affinity/specificity of NOTA-conjugated TRC105 (an anti-CD105 antibody) was evaluated by flow cytometry, which exhibited no difference from unconjugated TRC105. BALB/c mice were anesthetized, and the right femoral artery was ligated to induce hindlimb ischemia, with the left hindlimb serving as an internal control. Laser Doppler imaging showed that perfusion in the ischemic hindlimb plummeted to ∼ 20% of the normal level after surgery and gradually recovered to near normal level on day 24. Ischemia-induced angiogenesis was noninvasively monitored and quantified with (64)Cu-NOTA-TRC105 PET on postoperative days 1, 3, 10, 17, and 24. (64)Cu-NOTA-TRC105 uptake in the ischemic hindlimb increased significantly from the control level of 1.6 ± 0.2 %ID/g to 14.1 ± 1.9 %ID/g at day 3 (n = 3) and gradually decreased with time (3.4 ± 1.9 %ID/g at day 24), which correlated well with biodistribution studies performed on days 3 and 24. Blocking studies confirmed the CD105 specificity of tracer uptake in the ischemic hindlimb. Increased CD105 expression on days 3 and 10 following ischemia was confirmed by histology and reverse transcription polymerase chain reaction (RT-PCR). This is the first report of PET imaging of CD105 expression during ischemia-induced angiogenesis. (64)Cu-NOTA-TRC105 PET may play multiple roles in future PAD-related research and improve PAD patient management by identifying the optimal timing of treatment and monitoring the efficacy of therapy.

Endoglin inhibitor TRC105 with or without bevacizumab for bevacizumab-refractory glioblastoma (ENDOT): a multicenter phase II trial.

BACKGROUND: Glioblastoma (GBM), the most lethal primary brain tumor, has limited treatment options upon recurrence after chemoradiation and bevacizumab. TRC105 (carotuximab), a chimeric anti-endoglin (CD105) antibody, inhibits angiogenesis and potentiates activity of VEGF inhibitor bevacizumab in preclinical models. This study sought to assess safety, pharmacokinetics, and efficacy of TRC105 for bevacizumab-refractory GBM. METHODS: We conducted a pre-registered (NCT01564914), multicenter, open-label phase II clinical trial (ENDOT). We administered 10 mg/kg TRC105 monotherapy (first cohort) in adults with GBM and radiographic progression following radiation, temozolomide and bevacizumab therapy. Primary outcome was median time-to-progression (TTP), amended after first cohort's enrollment to median overall survival (mOS). Secondary outcomes were objective response rate, safety and tolerability, and progression-free survival (PFS). RESULTS: 6 patients were enrolled in TRC105 monotherapy cohort. Median TTP and PFS of 5 evaluable patients receiving monotherapy was 1.4 months, in whom plasma VEGF-A levels were elevated post-therapy. Lack of response led to protocol amendment, and second cohort's addition of bevacizumab+TRC105 with primary endpoint of mOS. 16 patients were enrolled in bevacizumab+TRC105 cohort. mOS of 15 evaluable patients was 5.7 (95%CI: 4.2-9.8) months. All 22 patients had measurable disease at baseline. Median PFS of 14 evaluable patients receiving bevacizumab+TRC105 was 1.8 months (95%CI 1.2-2.1). Serum TRC105 was measurable above target concentration of 25 ug/mL in all evaluable patients. Study medications were well-tolerated in both cohorts. Combined administration did not potentiate known toxicities of either medication, with cerebral hemorrhage not observed. CONCLUSIONS: Single-agent TRC105 lacks activity in bevacizumab-refractory GBM, possibly secondary to upregulated VEGF-A expression. Meaningful mOS in bevacizumab+TRC105 cohort warrants further trials to investigate efficacy of combination therapy.

Glioblastoma is an aggressive and lethal brain tumor, with patients typically expected to survive for 14 to 16 months after diagnosis. Nearly all patients experience tumor recurrence once conventional treatment strategies fail, after which a drug called bevacizumab is used. However, subsequent treatment options are extremely limited. We performed a clinical trial in which we investigated how safe and effective a new drug called TRC105 (carotuximab) is in patients who no longer respond to chemotherapy, radiotherapy or bevacizumab. We tested TRC105 both with and without bevacizumab, since TRC105 might enhance the activity of bevacizumab. We found that patients survived for an average of 5.7 months when given TRC105 and bevacizumab in combination. These findings suggest that further clinical trials are needed to confirm whether or not this combination therapy is a useful approach in patients with glioblastoma recurrence.

Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Positron emission tomography and optical imaging of tumor CD105 expression with a dual-labeled monoclonal antibody.

CD105 (endoglin) is an independent prognostic marker for poor prognosis in >10 solid tumor types, including breast cancer. The goal of this study was to develop a CD105-specific agent for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging, which can have potential clinical applications in diagnosis and imaged-guided surgery of breast cancer. TRC105, a chimeric anti-CD105 monoclonal antibody, was labeled with both a NIRF dye (i.e., 800CW) and (64)Cu to yield (64)Cu-NOTA-TRC105-800CW. Flow cytometry analysis revealed no difference in CD105 binding affinity/specificity between TRC105 and NOTA-TRC105-800CW. Serial PET imaging revealed that the 4T1 murine breast tumor uptake of (64)Cu-NOTA-TRC105-800CW was 5.2 ± 2.7, 11.0 ± 1.4, and 13.0 ± 0.4% ID/g at 4, 24, and 48 h postinjection respectively. Tumor uptake as measured by ex vivo NIRF imaging exhibited a good linear correlation with the % ID/g values obtained from PET (R = 0.74). Biodistribution data were consistent with the PET/NIRF findings. Blocking experiments, control studies with dual-labeled cetuximab (an isotype-matched control antibody), and histology confirmed the CD105 specificity of (64)Cu-NOTA-TRC105-800CW. Successful PET/NIRF imaging of CD105 expression warrants further investigation and clinical translation of dual-labeled TRC105-based imaging agents.

Positron emission tomography imaging of tumor angiogenesis with a 66Ga-labeled monoclonal antibody.

The goal of this study was to develop a (66)Ga-based positron emission tomography (PET) tracer for noninvasive imaging of CD105 expression during tumor angiogenesis, a hallmark of cancer. (66)Ga was produced using a cyclotron with (nat)Zn or isotopically enriched (66)Zn targets. TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (66)Ga. No difference in CD105 binding affinity or specificity was observed between TRC105 and NOTA-TRC105 based on flow cytometry analysis. Reactivity of (66)Ga for NOTA, corrected to the end of bombardment, was between 74 and 222 GBq/µmol for both target enrichments with <2 ppb of cold gallium. (66)Ga-labeling was achieved with >80% radiochemical yield. Serial PET imaging revealed that the murine breast cancer 4T1 tumor uptake of (66)Ga-NOTA-TRC105 was 5.9 ± 1.6, 8.5 ± 0.6, and 9.0 ± 0.6% ID/g at 4, 20, and 36 h postinjection, respectively (n = 4). At the last time point, tumor uptake was higher than that of all organs, which gave excellent tumor contrast with a tumor/muscle ratio of 10.1 ± 1.1. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiment, control studies with (66)Ga-NOTA-cetuximab, as well as ex vivo histology all confirmed the in vivo target specificity of (66)Ga-NOTA-TRC105. Successful PET imaging with high specific activity (66)Ga (>700 GBq/µmol has been achieved) as the radiolabel opens many new possibilities for future PET research with antibodies or other targeting ligands.