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66Ga-Labeled PEGylated nano-graphene oxide (GO) covalently linked to NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody TRC105

, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: June 28, 2012.

Chemical name:66Ga-Labeled PEGylated nano-graphene oxide (GO) covalently linked to NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody TRC105
Abbreviated name:[66Ga]-NOTA-GO-TRC105
Agent Category:Antibody
Target:CD105 (endoglin) antigen
Target Category:Antigen
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:66Ga
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



The CD105 antigen (endoglin) is a hypoxia-inducible, 180-kDa, disulfide-linked, homodimeric transmembrane glycoprotein that is a co-receptor for the transforming growth factor β (TGF-β) (1). Both CD105 and TGF-β are expressed at low levels in resting endothelial cells, but they are overexpressed in cancerous lesions and play a significantly proangiogenic role in remodeling the vasculature of malignant tumors (2). It has been shown that the levels of CD105 in endothelial tissues correlate well with the degree of cell proliferation and that the antigen is a suitable biomarker to quantify tumor angiogenesis and can be used to determine the prognostic outcome for cancer patients (3). The biological activity of CD105 has been discussed in detail by Seon et al. (4). Investigators have demonstrated that immunotoxins and radioimmunoconjugates generated with anti-CD105 monoclonal antibodies (mAbs) can inhibit angiogenesis and prevent the growth and metastasis of cancerous tumors (4). For translation to the clinic, a human/mouse chimeric anti-CD105 mAb (designated c-SNj6 or TRC105) was generated and shown to have suitable pharmacokinetic, toxicological, and immunogenicity characteristics for use in non-human primates (5). Currently, a clinical trial is in progress to evaluate the use of TRC105 for the treatment of metastatic breast cancer.

TRC105 has been labeled with 64Cu (6) and 89Zr (7), respectively, and has been reported to detect the expression of CD105 with positron emission tomography (PET) imaging in xenograft tumors in mice. In another study, TRC105 was conjugated to IRDye 800CW, a near-infrared fluorescent (NIRF) dye, and the expression of CD105 in tumors was visualized with NIRF imaging (8). TRC105 has also been conjugated with 64Cu and IRDye 800CW to develop a dual-modality (PET/NIRF) imaging agent that can be used to detect murine breast cancer 4T1 cell tumors in mice (9). Recently, TRC105 was conjugated to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with 66Ga, and the radioimmunoconjugate was shown to be suitable to visualize the expression of CD105 with PET in 4T1 cell tumors in mice (10).

Investigators have recently become interested in the use of PEGylated graphene oxide (GO; to read about the physical and chemical properties of graphene, see Feng and Liu (11)), a carbon-based material, in biomedical applications such as drug delivery (12), cancer therapy (13), and preclinical in vivo imaging and ablation of tumors in mice (14). Hong et al. used PET to evaluate 66Ga-labeled PEGylated nano-GO covalently linked to NOTA-conjugated TRC105 ([66Ga]-NOTA-GO-TRC105) to visualize and target tumors that express the CD105 antigen in mice (15).



The 66Ga used to perform the work described in this chapter was produced by the bombardment of either natZn or 66Zn as described by Hong et al. (15). When natZn was used to produce 66Ga, the final preparation of the nuclide contained <5% 67Ga (t1/2 = 78.3 h) as a contaminant. The purity of 66Ga produced from 66Zn was reported to be >99.9%.

NOTA-GO and NOTA-GO-TRC105 were produced for radiolabeling with 66Ga and utilized in the PET imaging and biodistribution studies as described elsewhere (15). Fluorescein isothiocyanate (FITC)-labeled GO (FITC-GO) and FITC-GO-TRC105 were synthesized to investigate the in vitro CD105 binding affinity and specificity of the complexes with fluorescence techniques (15). The average number of NOTA or TRC105 molecules covalently linked to a sheet of GO was not reported. The average small sheet size range of NOTA-GO and NOTA-GO-TRC105 was 10–50 nm, and this size range was confirmed with dynamic light scattering (DLS) analysis. The average diameters of the two GO conjugates were 21.9 ± 0.6 nm and 27.0 ± 0.9 nm, respectively, as determined with DLS (15). The zeta-potentials of NOTA-GO and NOTA-GO-TRC105 were determined to be −9.46 ± 4.74 mV and 0.08 ± 5.35 mV, respectively.

NOTA-GO and NOTA-GO-TRC105 were respectively labeled with 66Ga, and the radiolabeled products were purified with size-exclusion chromatography using phosphate-buffered saline containing 0.25 M ammonium acetate (pH 7.2) as the mobile phase (15). The final radiolabeled preparations were passed through a 0.22-μm filter for use in in vivo studies. The radiochemical yields, radiochemical purities, and specific activities of [66Ga]-NOTA-GO and [66Ga]-NOTA-GO-TRC105 were not reported.

In Vitro Studies: Testing in Cells and Tissues


The stability of [66Ga]-NOTA-GO and [66Ga]-NOTA-GO-TRC105 was investigated by incubating the tracers in complete mouse serum at 37°C for up to 24 h, and the incubation mixtures were sampled at various time points to determine the percentage of radioactivity retained on the GO conjugates (15). Both 66Ga-labeled GO conjugates were reported to retain >97% radioactivity at all the time points, suggesting that the radiolabeled complexes would have excellent in vivo stability.

Flow cytometric analysis of HUVEC cells (human umbilical vein endothelial cells that have a high expression of CD105) exposed to 50 μg/mL FITC-GO-TRC105 (based on GO content) showed that the cells had >600-fold higher fluorescence intensity compared with the untreated cells (15). Cells blocked with 3.33 nmol TRC105 and then treated with FITC-GO-TRC105 as above had ~100-fold lower fluorescence intensity compared with cells exposed to FITC-GO-TRC105 alone. This indicated that FITC-GO-TRC105 bound specifically to the CD105 antigen on the HUVEC cells. Minimal fluorescence was observed in MCF-7 cells (a human breast cancer epithelial cell line that does not express CD105; used a negative control in this study), indicating that FITC-GO-TRC105 exhibited low non-specific binding to the cells.

Animal Studies



The biodistribution of [66Ga]-NOTA-GO and [66Ga]-NOTA-GO-TRC105 was investigated in mice bearing murine breast cancer 4T1 cell tumors as described by Hong at al (15). The animals (n = 4 mice/group) were given an injection of the tracer (dose not mentioned) through the tail vein and euthanized at 3 h postinjection (p.i.). All the major organs, including the tumors, were retrieved from the animals to determine the amount of radioactivity accumulated in the various tissues. Data obtained from the study were presented as percent of injected dose per gram tissue (% ID/g). With both radiolabeled GO conjugates, radioactivity was detected mainly in the liver (~10.5% ID/g), followed by the blood (~7.5% ID/g), spleen (~12% ID/g and ~7.5% ID/g with [66Ga]-NOTA-GO-TRC105 and [66Ga]-NOTA-GO, respectively), and the tumors (~6.5% ID/g and ~3.5% ID/g with [66Ga]-NOTA-GO-TRC105 and [66Ga]-NOTA-GO, respectively; P < 0.05) at 3 h p.i. All other organs showed a radioactivity uptake of <2.5% ID/g with either tracer. Tumors of mice injected with [66Ga]-NOTA-GO-TRC105 showed a significantly higher uptake of label (~4.0% ID/g; P < 0.05) compared with the amount of radioactivity detected in the tumors of mice injected with [66Ga]-NOTA-GO (~2.5% ID/g). Another group of mice (n = 4 animals) that were given a blocking dose of NOTA-GO-TRC105 (33.33 nmol/kg body weight) before injecting the radiolabeled GO conjugate showed a significantly lower accumulation of radioactivity in the tumors at 24 h p.i. (~1.5% ID/g) compared with the lesions of animals injected with [66Ga]-NOTA-GO-TRC105 alone (~4.5% ID/g; P < 0.05). This indicated that the 66Ga-labeled NOTA-GO-TRC105 complex had a high binding specificity for the CD105 antigen (15).

For PET imaging of mice bearing 4T1 cell tumors (n = 4 animals), the rodents were injected with 5–10 MBq (135–270 μCi) [66Ga]-NOTA-GO-TRC105 or [66Ga]-NOTA-GO through the tail vein, and static whole-body images of the animals were acquired at 0.5 h, 3 h, 7 h, and 24 h p.i (15). Quantitative data were obtained from the images with region-of-interest analysis as described by Hong et al. (15). The amount of tracer in the blood was 10.9 ± 1.4% ID/g, 7.5 ± 1.2% ID/g, 5.3 ± 1.1% ID/g, and 3.4 ± 0.4% ID/g at 0.5, 3, 7, and 24 h p.i., respectively, and the amount of label in the liver was 10.7 ± 1.7% ID/g, 11.4 ± 1.5% ID/g, 10.4 ± 1.3% ID/g, and 8.0 ± 1.1% ID/g at 0.5, 3, 7, and 24 h p.i. respectively. The tumors were clearly visible at 0.5 h p.i. and showed an uptake of 3.8 ± 0.4% ID/g, 4.5 ± 0.4% ID/g, 5.8 ± 0.3% ID/g, and 4.5 ± 0.4% ID/g at 0.5, 3, 7, and 24 h p.i. respectively. The accumulation of radioactivity in the muscles with [66Ga]-NOTA-GO-TRC105 was <0.5% ID/g at all the time points. In general, with [66Ga]-NOTA-GO the level and pattern of radioactivity uptake in the liver and blood were similar to those with [66Ga]-NOTA-GO-TRC105. With [66Ga]-NOTA-GO, the level of tracer in the tumors was high at all time points (~3.0% ID/g) but significantly lower (P < 0.05) than that observed with [66Ga]-NOTA-GO-TRC105. The investigators attributed the increased accumulation of radioactivity with [66Ga]-NOTA-GO in the 4T1 tumors to the enhanced permeability and retention effect in the lesions (15).

To determine the in vivo target-binding specificity of [66Ga]-NOTA-GO-TRC105, the animals (n = 4 mice) were injected with 13.3 nmol nonradioactive TRC105 2 h before administration of the 66Ga-labeled GO conjugate (15). PET images of the animals were acquired as before. The uptake of label in the tumors was 1.3 ± 0.3% ID/g, 1.5 ± 0.2% ID/g, 1.1 ± 0.3% ID/g, and 1.2 ± 0.1% ID/g at 0.5, 3, 7, and 24 h p.i., respectively, which was significantly lower (P < 0.05 at all the time points) than the accumulation observed in the lesions of animals injected with [66Ga]-NOTA-GO-TRC105 alone (for uptake values, see above). Although the amount of radioactivity in the liver of animals given the blocking dose was similar to that of animals injected with [66Ga]-NOTA-GO-TRC105 alone at all the time points (9.0 ± 1.8% ID/g, 10.8 ± 2.0% ID/g, 10.0 ± 1.9% ID/g, and 10.2 ± 1.9% ID/g at 0.5, 3, 7, and 24 h p.i. respectively), the level of tracer in the blood of animals pretreated with TRC105 was lower (6.0 ± 1.2% ID/g, 3.4 ± 0.8% ID/g, 2.0 ± 0.3% ID/g, and 2.0 ± 0.3% ID/g at 0.5, 3, 7, and 24 h p.i., respectively). Therefore, this study confirmed the in vivo CD105 binding specificity of the GO-radioimmunoconjugate.

To confirm the uptake of NOTA-GO-TRC105 in CD105-specific tissues of mice bearing 4T1 cell tumors, the animals (n = 3 mice) were injected with 5 mg/kg body weight of the non-radiolabeled GO-immunoconjugate and euthanized at 3 h p.i. to remove the tumors, liver, spleen, and muscles of the rodents (15). The harvested tissues were then frozen and cryo-sectioned for histological examination. High levels of NOTA-GO-TRC105 were observed as dark spots only in tissue sections of the tumors, liver, and spleen, but not in the muscle of these animals. However, NOTA-GO was observed only in the sections of liver and spleen tissues obtained from animals injected with the GO-chelating agent complex. This study demonstrated that NOTA-GO-TRC105 specifically targets the CD105 antigen in the tumor. Immunofluorescence staining of the tissue slices for CD31 (a biomarker for the vasculature), with anti-mouse CD31 as the primary antibody and for CD105 using the TRC105 within NOTA-GO-TRC105 as the primary antibody, showed that the uptake of NOTA-GO-TRC105 in the liver and spleen was due to the non-specific capture of the complex by the reticuloendothelial system and not due to the specific targeting of CD105 on the vasculature. Almost no uptake of NOTA-GO-TRC105 was observed in the muscles and other normal tissues of the animals.

From these studies, the investigators concluded that [66Ga]-NOTA-TRC105 is a suitable agent to detect tumors that express the CD105 antigen in rodents (15).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


No information is currently available.

NIH Support

This work was supported in part by a grant from the National Institutes of Health through the UW Radiological Sciences Training Program 5 T32 CA009206-32.


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