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64Cu-Labeled NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody linked to near-infrared dye IRDye 800CW

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

Created: ; Last Update: May 10, 2012.

Chemical name:64Cu-Labeled NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody linked to near-infrared dye IRDye 800CW
Abbreviated name:[64Cu]-NOTA-TRC105-800CW
Agent Category:Antibody
Target:CD105 (endoglin) antigen
Target Category:Antigen
Method of detection:Multimodal (positron emission tomography (PET); optical imaging (near-infrared (NIRF) imaging)
Source of signal / contrast:64Cu (PET); IRDye 800CW (NIRF)
  • 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 pro-angiogenic 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, that the antigen is a suitable biomarker to quantify tumor angiogenesis and can be used to determine the prognostic outcome for cancer patients (3). Investigators have reported that immunotoxins and radioimmunoconjugates generated with anti-CD105 monoclonal antibodies (mAbs) can inhibit angiogenesis and prevent the growth and metastasis of cancerous tumors (4). The biological activity of CD105 has been discussed by Seon et al. (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 was labeled with 64Cu (6) and 89Zr (7), respectively, and shown 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 could be visualized with NIRF imaging (8). Either PET or NIRF imaging can be used by itself for the in vivo detection of tumors, but the former has high sensitivity and can be used for whole-body scans, whereas the main drawback of this modality is that it has low spatial resolution. The latter imaging method generates fluorescence signals that can be used to visualize the target tissue in real time, but this imaging modality cannot be used for whole-body scans or for the precise quantification of the target molecule(s) (9). Therefore, the combination of PET and NIRF imaging capabilities within the same agent would greatly improve the noninvasive investigative potential of a probe at the preclinical or clinical stages because each modality will complement the deficiency of the other (10). In addition, a dual-modality PET/NIRF imaging probe can be utilized to identified to locate neoplastic tumors with a whole-body PET scan, and later NIRF imaging can be used by surgeons to visualize the lesions for surgical resection (11). On the basis of this idea, TRC105 was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), a metal chelating agent, and IRDye 800CW (an NIRF dye with excitation at 778 nm and emission at 806 nm). The double-conjugated mAb was then labeled with 64Cu (half-life = 12.7 h), and the multimodal agent ([64Cu]-NOTA-TRC105-800CW) was evaluated for the PET and NIRF detection of 4T1 cell tumors in mice (11).



TRC105 was obtained from a commercial source, and the mAb was conjugated with NOTA and IRDye 800CW, respectively, as described by Zhang et al. (11). NOTA-TRC105-800CW was purified on a PD-10 column and labeled with 64Cu as detailed elsewhere (11). The radiolabeled and NIRF-conjugated mAb was purified on a PD-10 column with phosphate-buffered saline (pH not reported) as the mobile phase and passed through a 0.22-μm filter for use in in vivo studies.

Cetuximab (a human/mouse chimeric mAb that targets the human epidermal growth factor receptor) was obtained from a pharmaceutical company and used to prepare [64Cu]-NOTA-cetuximab-800CW for use as control (11).

The total time taken to prepare the two purified 64Cu-labeled mAbs was reported to be 60 ± 10 min (n = 10 preparations), and both labeled mAbs had a radiochemical yield and radiochemical purity of >85% and >98%, respectively (11). The specific activity of the two 64Cu-labeled and 800CW-conjugated mAbs was ~1.3 GBq (35.1 mCi)/6.5 nmol (11). Ultraviolet spectrophotometric analysis revealed that on average ~5 NOTA and 0.9 800CW molecules were conjugated to each of the mAbs.

In Vitro Studies: Testing in Cells and Tissues


A fluorescence-assisted cell-sorting analysis of HUVEC cells (human umbilical vein endothelial cells that express CD105) and 4T1 cells (murine breast cancer cells that do not express CD105) with AlexaFluor488- and Cy3-labeled secondary antibodies showed that both TRC105 and NOTA-TRC105-800CW had similar affinities for the CD105 antigen (11). This indicated that conjugation of NOTA and IRDye 800CW to TRC105 did not alter its affinity or specificity for the antigen targeted on the cell surface. No blocking studies were reported.

Animal Studies



4T1 cells were used to generate tumors in mice because these cells do not express the CD105 antigen and angiogenesis in the tumor would be only with the endothelial cells that express this antigen. Therefore, any binding of [64Cu]-NOTA-TRC105-800CW in the tumor would be to the endothelial cells rather than to the tumor cells. Mice bearing 4T1 cell tumors (n = 3 animals/time point) were intravenously injected with 5–10 MBq (135–270 μCi; 300 pmol); the final concentration of [64Cu]-NOTA-TRC105-800CW or [64Cu]-NOTA-cetuximab-800CW used for the injections was adjusted with unlabeled NOTA-TRC105-800CW or NOTA-cetuximab-800CW (11). Five-minute PET scans were acquired from the animals at predetermined time points (4, 24, and 48 h postinjection (p.i.)), and the region-of-interest (ROI) analysis of each scan was performed with appropriate software to calculate the percentage of injected dose per gram of tissue (% ID/g). The tumors were faintly visible in the images at 4 h p.i. and were clearly distinguishable from the surrounding tissues at 24 h p.i. and 48 h p.i. The amount of radioactivity from [64Cu]-NOTA-TRC105-800CW in the blood, liver, and tumor at the different time points is shown in Table 1.

Table 1: Amount of radioactivity from [64Cu]-NOTA-TRC105-800CW in various tissues of mice bearing 4T1 cell tumors at the different time points (11).

Tissue% ID/g at various time points p.i.
4 h24 h48 h
Blood19.6 ± 3.0
(21.3 ± 2.5)
11.4 ± 1.8
(11.2 ± 1.3)
9.4 ± 0.4
(7.6 ± 0.4)
Liver16.4 ± 3.1
(17.5 ± 2.5)
10.8 ± 1.1
(11.3 ± 1.6)
11.7 ± 4.4
(11.4 ± 1.1)
Tumor5.2 ± 2.7
11.0 ± 1.4
13.0 ± 0.4

# Values derived from time-activity curves; *P < 0.01.

Numbers in parenthesis represent radioactivity accumulated in the tissues of animals (n = 3 mice/time point) pretreated with a blocking dose (~15 nmol) of unlabeled TRC105 2 h before the [64Cu]-NOTA-TRC105-800CW injection.

For the blocking study, the animals were injected with ~15 nmol unlabeled TRC105 2 h before administration of [64Cu]-NOTA-TRC105-800CW (11). At all the time points, the uptake of radioactivity in the blood, liver, and tumors from [64Cu]-NOTA-cetuximab-800CW (used as a negative control) was significantly less than that from [64Cu]-NOTA-TRC105-800CW (P < 0.05 at 4 h p.i.; P < 0.01 at 24 h and 48 h p.i.). Results obtained from this study indicated that the 64Cu- and NIRF-labeled TRC105 mAb had a high binding specificity for the CD105 antigen in vivo.

NIRF images of the animals (n = 3 mice/time point) were acquired immediately after the PET scans at the same time points as mentioned above (11). The tumor fluorescence signal intensity was 7.11 × 104 ± 0.86 × 104 counts/s/ mm2, 1.56 × 105 ± 1.54 × 104 counts/s/mm2, and 1.19 × 105 ± 2.41 × 104 counts/s/mm2 at 4, 24, and 48 h p.i., respectively. The fluorescence intensity of tumors from animals pretreated with unlabeled TRC105 was significantly reduced (4.04 × 104 ± 1.20 × 104 counts/s/mm2, 8.97 × 104 ± 0.66 × 104 counts/s/mm2, and 7.75 × 104 ± 1.36 × 104 counts/s/mm2 at 4, 24, and 48 h p.i., respectively; P < 0.05 at 24 h and 48 h p.i. compared to animals injected with [64Cu]-NOTA-TRC105-800CW alone). These results were similar to those obtained from the PET study and indicated that [64Cu]-NOTA-TRC105-800CW had an in vivo binding specificity for the CD105 antigen.

Results obtained from ex vivo PET and NIRF images of the major organs harvested from the animals were similar to those obtained with whole-body PET and NIRF imaging (11). The ex vivo images showed that tumors from mice injected with [64Cu]-NOTA-TRC105-800CW had a very high contrast compared with the contrast of tumors from animals injected with a blocking dose of either unlabeled TRC105 or [64Cu]-NOTA-cetuximab-800CW. The NIRF signal intensities of the in vivo and the ex vivo tumors were similar at 48 h p.i. (1.19 × 105 ± 2.41 × 104 counts/s/mm2versus 1.31 × 105 ± 1.58 × 104 counts/s/mm2) and had a linear correlation R2 value of 0.74.

Biodistribution data obtained at 48 h p.i. from different tissues of the mice showed that the tumors had the maximum accumulation of radioactivity from [64Cu]-NOTA-TRC105-800CW (~12.5% ID/g) compared with ~10.0%ID/g, ~6.0% ID/g, and ~6.5% ID/g in the liver, lungs, and kidneys, respectively (11). In addition, a significantly decreased (P < 0.05) uptake of radioactivity was observed in the tumors of mice pretreated with a blocking dose of unlabeled TRC105 (~4.0% ID/g) or in tumors of mice injected with [64Cu]-NOTA-cetuximab-800CW (~4.5% ID/g).

Frozen tumor sections were treated with TRC105 and an anti-CD31 antibody (the CD31antigen is an endothelial biomarker) followed by exposure to appropriate secondary antibodies to visualize the expression of CD105 and CD31 in the lesions (11). Fluorescence microscopy of the immunostained sections showed that there was co-localization of the CD105 and CD31 stains in the frozen sections, indicating that TRC105 bound only to the endothelial cells. In addition, CD105 was expressed only on actively proliferating endothelial cells of the tumor, which are located toward the periphery of the tumor (compared with the center of the lesion) where angiogenesis is most active. Mature blood vessels showed a very low expression of CD105.

From these studies, the investigators concluded that PET/NIRF agents can be used for the detection of tumors in rodents (11).

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 the National Institutes of Health through the UW Radiological Sciences Training Program 5 T32 CA009206-32.


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