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66Ga-Labeled NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody

[66Ga]-NOTA-TRC105
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: June 21, 2012.

Chemical name:66Ga-Labeled NOTA-conjugated anti-CD105 (endoglin) chimeric monoclonal antibody
Abbreviated name:[66Ga]-NOTA-TRC105
Synonym:
Agent Category:Antibody
Target:CD105 (endoglin) antigen
Target Category:Antigen
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:66Ga
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

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). 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 in detail by Seon et al. (4). For translation to the clinic, a human/mouse chimeric anti-CD105 mAb (designated c-SNj6 or TRC105) has been 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 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 was visualized with NIRF imaging (8). TRC105 has been conjugated with 64Cu and IRDye 800CW to develop a dual-modality (PET/NIRF) imaging agent that has been used to detect murine breast cancer 4T1 cell tumors in mice (9). Recently it has become possible to produce 66Ga (t1/2 = 9.3 h, E0 = 4.15 MeV), which has a very high specific activity and reacts with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), a bifunctional chelating agent, to yield a product ([66Ga]-NOTA) that has a specific activity of >70 GBq/μmol (>2 Ci/μmol), a value that is several-fold higher than those published in the literature (<4.6 GBq/μmol (~125 mCi/μmol)) (10). The investigators suggested that the high specific activity of the NOTA-conjugated radionuclide could be utilized to label peptides and biomolecules such as proteins and mAbs for PET imaging. In an ongoing effort to develop an immuno-PET agent that would be suitable for the noninvasive imaging of angiogenesis in tumor, TRC105 was conjugated with NOTA, labeled with 66Ga ([66Ga]-NOTA-TRC105), and evaluated for the PET visualization of CD105 expression in 4T1 cell tumors in mice (11).

Synthesis

[PubMed]

The 66Ga preparation used to perform this work was reported to contain <2 ppb nonradioactive Ga and <20 ppb Fe as determined with inductively coupled plasma mass spectroscopy (11).

TRC105 was obtained from a commercial source and conjugated with NOTA as described by Engle et al. (11). On average, ~5 NOTA molecules were conjugated to each of the mAbs as determined with ultraviolet spectrophotometric analysis (9). NOTA-TRC105 was labeled with 66Ga and purified with size-exclusion chromatography using phosphate-buffered saline containing 0.25 M ammonium acetate (pH 7.2) as the mobile phase (11). The final radioimmunoconjugate preparation was 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 [66Ga]-NOTA-cetuximab as described above (11). 66Ga-Labeled cetuximab was used as a control in some studies.

The total time to prepare the two purified 66Ga-labeled mAbs was reported to be 70 ± 10 min (n = 7 preparations), and both labeled mAbs had a radiochemical yield and radiochemical purity of 80.2 ± 4.1% and >95%, respectively (11). The specific activity of the two 66Ga-labeled mAbs was ~1.2 GBq (32.4 mCi)/6.6 nmol.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

A fluorescence-assisted cell-sorting analysis of HUVEC cells (human umbilical vein endothelial cells that have a high expression of CD105) and MCF-7 cells (human breast mammary gland cells that do not express CD105) showed that both TRC105 and NOTA-TRC105 had similar affinities for the CD105 antigen on the HUVEC cells (11). Neither TRC105 nor NOTA-TRC105 showed any binding to the MCF-7 cells. This indicated that the conjugation of NOTA to TRC105 did not alter its affinity or specificity for the CD105 antigen expressed on the HUVEC cell surface (11). No blocking studies were reported.

Animal Studies

Rodents

[PubMed]

The biodistribution of [66Ga]-NOTA-TRC105 was investigated in mice bearing murine breast cancer 4T1 cell tumors as described by Engle at al (11). The animals (n = 4 mice) were given an intravenous injection of the tracer and euthanized at 36 h postinjection (p.i.) to retrieve all the major organs, including the tumors, 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). Maximum radioactivity was detected in the blood (~12.5% ID/g), followed by the tumors (~10% ID/g), lungs (~6% ID/g), and the liver, kidney, and spleen (each of these organs showed an accumulation of ~5% ID/g). All other organs had an uptake of <2.5% ID/g. The tumor/muscle uptake ratio (T/M ratio) was calculated to be 10.1 ± 1.1 at 36 h p.i. When the mice (n = 4 animals) were given a blocking dose of TRC105 (13.3 nmol) 2 h before injecting the radioimmunoconjugate, the T/M ratio decreased significantly (~6.0; P < 0.01), and a simultaneous increase in accumulation of label was observed in the liver and lungs. Pretreatment of the mice with escalating doses of NOTA-TRC105 (ranging from 3.33 pmol to 666 pmol) did not change the amount of radioactivity accumulated in the tumors (8.8 ± 1.2% ID/g, 9.9 ± 1.2% ID/g, 8.5 ± 0.5% ID/g, and 8.0 ± 0.5% ID/g at 3.33, 16.65, 83.5, and 666 pmol NOTA-TRC105, respectively). These studies indicated that [66Ga]-NOTA-TRC105 had a binding specificity for the CD105 antigen.

The uptake of [66Ga]-NOTA-TRC105 was compared to that of [66Ga]-NOTA-cetuximab in the different tissues of mice bearing 4T1 cell tumors (n = 4 mice/group) (11). Except for the liver and the tumors, all other tissues from the animals showed a similar uptake of radioactivity from both 66Ga-labeled mAbs. The tumors showed a significantly higher (P < 0.01) uptake of label with 66Ga-NOTA-TRC105 (~10% ID/g) than with 66Ga-NOTA-cetuximab (~3.5% ID/g). However, the liver showed higher accumulation of the tracer with 66Ga-NOTA-cetuximab (~6.0% ID/g) than with 66Ga-NOTA-TRC105 (~4.0% ID/g). This suggested that the CD105 antigen was targeted specifically by the 66Ga-labeled TRC105 mAb.

For PET imaging of mice bearing 4T1 cell tumors (n = 4 animals), the rodents were given an intravenous injection of [66Ga]-NOTA-TRC105 (dose not mentioned), and whole-body images of the animals were acquired at 4, 20, and 36 h p.i (11). Quantitative data were obtained from the images with region-of-interest analysis as described by Engle et al. (11). The initial time point (4 h p.i.) showed a high level of radioactivity in the blood pool, and this level decreased gradually at the subsequent time points. The tumors were visible at 4 h p.i. and had an uptake of 5.9 ± 1.6% ID/g. The amount of tracer in these lesions increased to 8.5 ± 0.6 at 20 h p.i. and remained constant thereafter (9.0 ± 0.6% ID/g at 36 h p.i.). The liver showed a decrease in accumulation of radioactivity over time (10.6 ± 1.8% ID/g, 8.2 ± 0.4% ID/g, and 6.8 ± 0.5% ID/g at 4, 20, and 36 h p.i. respectively); however, the blood showed a relatively constant level of the tracer at all the time points (10.3 ± 1.9% ID/g, 9.4 ± 0.9% ID/g, and 9.0 ± 0.6% ID/g at 4, 20, and 36 h p.i. respectively). To determine the in vivo target-binding specificity of [66Ga]-NOTA-TRC105, the animals (n = 4 mice) were injected with 13.3 nmol nonradioactive TRC105 2 h before administration of the 66Ga-labeled TRC105 (11). PET images of the animals were acquired as before. The uptake of label in the tumors was 4.4 ± 1.2% ID/g, 5.8 ± 0.6% ID/g, and 7.2 ± 0.6% ID/g at 4, 20, and 36 h p.i., respectively, which was significantly lower (P < 0.05 at 20 h p.i. and 36 h p.i.) than the accumulation observed in the lesions of animals injected with [66Ga]-NOTA-TRC105 alone (for uptake values, see above). The accumulation of radioactivity in the blood and liver of animals given the blocking dose was similar to that of animals injected with [66Ga]-NOTA-TRC105 alone at all the time points. Therefore, this study confirmed the in vivo CD105 binding specificity of the radioimmunoconjugate.

To further confirm the in vivo specificity of [66Ga]-NOTA-TRC105, isotype-matched [66Ga]-NOTA-cetuximab was injected as a control in mice bearing 4T1 cell tumors (n = 4 animals), and PET images were acquired from the animals at the same time points mentioned above (11). From the images, it was calculated that the amount of label in the tumors was 2.7 ± 0.5% ID/g, 4.2 ± 1.2% ID/g, and 4.1 ± 0.8% ID/g at 4, 20, and 36 h p.i., respectively, which was significantly lower than the accumulation of tracer observed with [66Ga]-NOTA-TRC105 at all examined time points (P < 0.05 at 4 h p.i.; P < 0.01 at 20 h p.i. and 36 h p.i.). Results from this study showed that [66Ga]-NOTA-TRC105 bound specifically to the CD105 antigen.

From these studies, the investigators concluded that [66Ga]-NOTA-TRC105 is a suitable agent to detect tumors that expressed the CD105 antigen in rodents (11).

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

NIH Support

This work was supported in part by grant 9U54TR000021 from the National Center for Advancing Translational Sciences (NCATS) and the National Institutes of Health through the UW Radiological Sciences Training Program 5 T32 CA009206-32.

References

1.
Nassiri F., Cusimano M.D., Scheithauer B.W., Rotondo F., Fazio A., Yousef G.M., Syro L.V., Kovacs K., Lloyd R.V. Endoglin (CD105): a review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Res. 2011;31(6):2283–90. [PubMed: 21737653]
2.
Perez-Gomez E., Del Castillo G., Juan Francisco S., Lopez-Novoa J.M., Bernabeu C., Quintanilla M. The role of the TGF-beta coreceptor endoglin in cancer. ScientificWorldJournal. 2010;10:2367–84. [PubMed: 21170488]
3.
Fonsatti E., Nicolay H.J., Altomonte M., Covre A., Maio M. Targeting cancer vasculature via endoglin/CD105: a novel antibody-based diagnostic and therapeutic strategy in solid tumours. Cardiovasc Res. 2010;86(1):12–9. [PubMed: 19812043]
4.
Seon B.K., Haba A., Matsuno F., Takahashi N., Tsujie M., She X., Harada N., Uneda S., Tsujie T., Toi H., Tsai H., Haruta Y. Endoglin-targeted cancer therapy. Curr Drug Deliv. 2011;8(1):135–43. [PMC free article: PMC4353483] [PubMed: 21034418]
5.
Shiozaki K., Harada N., Greco W.R., Haba A., Uneda S., Tsai H., Seon B.K. Antiangiogenic chimeric anti-endoglin (CD105) antibody: pharmacokinetics and immunogenicity in nonhuman primates and effects of doxorubicin. Cancer Immunol Immunother. 2006;55(2):140–50. [PubMed: 15856228]
6.
Hong H., Yang Y., Zhang Y., Engle J.W., Barnhart T.E., Nickles R.J., Leigh B.R., Cai W. Positron emission tomography imaging of CD105 expression during tumor angiogenesis. Eur J Nucl Med Mol Imaging. 2011;38(7):1335–43. [PMC free article: PMC3105181] [PubMed: 21373764]
7.
Hong H., Severin G.W., Yang Y., Engle J.W., Zhang Y., Barnhart T.E., Liu G., Leigh B.R., Nickles R.J., Cai W. Positron emission tomography imaging of CD105 expression with 89Zr-Df-TRC105. Eur J Nucl Med Mol Imaging. 2012;39(1):138–48. [PMC free article: PMC3228902] [PubMed: 21909753]
8.
Yang Y., Zhang Y., Hong H., Liu G., Leigh B.R., Cai W. In vivo near-infrared fluorescence imaging of CD105 expression during tumor angiogenesis. Eur J Nucl Med Mol Imaging. 2011;38(11):2066–76. [PMC free article: PMC3189267] [PubMed: 21814852]
9.
Zhang Y., Hong H., Engle J.W., Yang Y., Theuer C.P., Barnhart T.E., Cai W. Positron Emission Tomography and Optical Imaging of Tumor CD105 Expression with a Dual-Labeled Monoclonal Antibody. Mol Pharm. 2012;9(3):645–53. [PMC free article: PMC3295892] [PubMed: 22292418]
10.
Engle, J.W., V. Lopez-Rodriguez, R.E. Gaspar-Carcamo, H.F. Valdovinos, M. Valle-Gonzalez, F. Trejo-Ballado, G.W. Severin, T.E. Barnhart, R.J. Nickles, and M.A. Avila-Rodriguez, Very high specific activity (66/68)Ga from zinc targets for PET. Appl Radiat Isot, 2012.
11.
Engle J.W., Hong H., Zhang Y., Valdovinos H.F., Myklejord D.V., Barnhart T.E., Theuer C.P., Nickles R.J., Cai W. Positron emission tomography imaging of tumor angiogenesis with a (66)Ga-labeled monoclonal antibody. Mol Pharm. 2012;9(5):1441–8. [PMC free article: PMC3375902] [PubMed: 22519890]
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