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111In-(Diethylenetriamine pentaacetic acid)n-trastuzumab-(IRDye 800CW)m

111In-(DTPA)n-trastuzumab-(IRDye 800CW)m
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: April 5, 2009.

Chemical name:111In-(Diethylenetriamine pentaacetic acid)n-trastuzumab-(IRDye 800CW)m
Abbreviated name:111In-(DTPA)n-trastuzumab-(IRDye 800CW)m
Agent Category:Antibody
Target:Epidermal growth factor receptor 2 (EGFR2/neu)
Target Category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT) or gamma planar imaging; near infrared fluorescence imaging
Source of signal / contrast:111In and IRDye 800CW
  • Checkbox In vitro
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No structure available.



The human epidermal growth factor receptor 2 (HER2), also known as neu, ErbB-2, or cluster of differentiation 340 (CD340), has a crucial role in the normal development, proliferation, survival, and apoptosis of the cell (1). HER2 is a tyrosine kinase (TK)-dependent receptor and regulates its own expression through the TK signaling pathway. An amplified expression of HER2 has been reported in 20–25% of breast cancer primary tumors and indicates a poor prognosis for the patient (2). Because of its role in the progression of cancer, HER2 or its associated TK are targeted either with antibodies or with small-molecule drugs (or a combination of the two) for the treatment of this ailment (3, 4). One of these antibodies, trastuzumab, which is a humanized monoclonal antibody (MAb) that targets the extracellular domain of the HER2, is approved by the United States Food and Drug Administration for the treatment of breast cancer and is being evaluated in clinical trials for the treatment of several cancer types. Trastuzumab has also been conjugated with different radionuclides or fluorescent compounds and has been used under preclinical conditions for the diagnosis of breast cancer with different imaging techniques (5-7). In addition, this MAb has also been conjugated to gold nanoparticles (8), quantum dots (9), iron oxide (10), and nanocrystals (11) for the detection of cancer.

The use of MAbs for imaging after conjugation to either radionuclides or fluorescent dyes has its limitations. Radionuclide gamma ray emissions can penetrate tissue thereby permitting external detection, but have a physical half-life, and, therefore non-invasive imaging must be performed within a certain time period before the signal becomes difficult to detect or is limiting (12). Though fluorescent compounds do not have half-lives, deep tissue targets in humans may not be visible because of adsorption, but, compared with radionuclides, fluorescent compounds yield higher signal/noise ratios ex vivo that can assist in visualizing cancerous tissue during surgical resection (12). On the basis of these observations, Sampath et al. developed a dual-labeled trastuzumab-based imaging agent for the detection of HER2 expression in breast cancer tissue (12). The investigators synthesized radioactive indium (111In) and IRDye dual-labeled trastuzumab (111In-diethylenetriamine pentaacetic acid [DTPA])n-trastuzumab-(IRDye 800CW)m or 111In-trastuzumab-IRDye 800CW) and used it for in vivo scintigraphic and fluorescent imaging of HER2-positive tumors in athymic nude mice bearing xenograft tumors.



Sampath et al. described the synthesis of 111In-trastuzumab-IRDye 800CW (12). Trastuzumab and IRDye 800CW were obtained from commercial sources. The MAb was conjugated with DTPA as described by Tang et al. (13). The yield of this reaction was typically 98%. As determined with mass spectroscopy, 10 molecules (n) of DTPA were reported to be conjugated to each molecule of trastuzumab to yield (DTPA)n-trastuzumab. The conjugation of (DTPA)n-trastuzumab with IRDye 800CW was performed with a commercially available kit as described by Sampath et al. (12). This reaction had a typical yield of 90%, and a spectrophotometric analysis of the final product from this reaction revealed that 7–10 molecules (m) of IRDye800CW were conjugated to (DTPA)n-trastuzumab to yield (DTPA)n-trastuzumab-(IRDye 800CW)m.

To label (DTPA)n-trastuzumab-(IRDye 800CW)m with 111In, indium chloride in sodium acetate buffer was mixed with (DTPA)n-trastuzumab-(IRDye 800CW)m in phosphate-buffered saline (PBS) for 30 min to yield 111In-trastuzumab-IRDye 800CW (12). The procedure used for purification of the radiopharmaceutical and the radiochemical purity were not reported. Specific activity of the dual-labeled MAb was reported to be 7.4 MBq/nmol (0.2 mCi/nmol).

Stability of the dual-labeled MAb was determined in cell culture medium containing 10% fetal bovine serum for 24 h at 37°C (12). High-performance liquid chromatography (HPLC) was used to detect any degradation products produced from the dual-labeled MAb during the stability study. The column used for the HPLC analysis of the degradation products was not reported. 111In-Trastuzumab-IRDye 800CW was reported to be stable for 14 d at 4°C, but exposure to serum at 37°C resulted in ~6%/d degradation of the radiopharmaceutical as determined with HPLC.

For use as a control, human IgG was dual-labeled to yield 111In-(DTPA)p-IgG-(IRDye 800CW)q as described above (12). The number of DTPA molecules (p) conjugated to the IgG was determined with mass spectroscopy; however, the number of DTPA residues conjugated to each IgG molecule was not reported. The ratio of IRDye 800CW to MAb (q) was calculated with spectrophotometric analysis, and for a single batch of 111In-(DTPA)p-IgG-(IRDye 800CW)q the dye/MAb ratio was reported to be ~3. The yield of this reaction was reported to be 90%. Specific activity and stability of the dual-labeled human IgG was not reported.

In Vitro Studies: Testing in Cells and Tissues


Human breast cancer cell lines SKBr3 (overexpress HER2) and MDA-MB-231 (have negligible expression of HER2) were used to study the in vitro binding characteristics of (DTPA)n-trastuzumab-(IRDye 800CW)m and IRDye 800CW (12). For competitive binding studies the SKBr3 cells were treated with trastuzumab or human IgG for 30 min at 37°C before exposure to (DTPA)n-trastuzumab-(IRDye 800CW)m for 1 h at the same temperature. The cells were then washed twice with PBS and stained with Sytol Green (in ethanol) for 15 min to fix the cells and stain the nuclei. Only the SKBr3 cells were reported to bind (DTPA)n-trastuzumab-(IRDye 800CW)m as confirmed with confocal microscopy. SKBr3 cells pretreated with unlabeled trastuzumab, but not with human IgG, showed no binding of (DTPA)n-trastuzumab-(IRDye 800CW)m. Only a negligible amount of the free IRDye 800CW was reported to bind to the SKBr3 cells.

Animal Studies



For the in vivo imaging studies in nude mice, a derivative of the SKBr3 cells designated SKBr3-luc, which expresses the firefly luciferase (luc) gene, was used to develop xenograft tumors in nude mice (12). The SKBr3-luc cells were used to monitor the growth of xenograft tumors with bioluminescence imaging in the animals. Whole-body fluorescence imaging was performed 24 and 48 h after the respective administration of 111In-trastuzumab-IRDye 800CW (n = 3 animals), 111In-IgG-IRDye 800CW (n = 5 animals), and free IRDye 800CW (n = 5 animals) to the different groups of mice. For the competition studies, five mice were pretreated with 200-fold molar excess of unlabeled trastuzumab for 24 h before administration of 111In-trastuzumab-IRDye 800CW. At 48 h, whole-body fluorescence imaging data produced a tumor/muscle ratio (TMR) for mice injected with 111In-trastuzumab-IRDye 800CW of 2.25 ± 0.2 compared with TMR of 1.35 ± 0.1 for animals administered 111In-IgG-IRDye 800CW. Animals treated with the free IRDye 800CW had a TMR of 1.44 ± 0.18. The TMR of animals pretreated with trastuzumab was significantly lower (P = 0.0048) compared with animals treated with 111In-trastuzumab-IRDye 800CW. Single-photon emission computed tomography (SPECT) was performed on animals 48 h after the treatment with 111In-trastuzumab-IRDye 800CW. The TMR of the tumor region was reported to be 2.66, and whole-body scintigraphy images of the animals also showed a similar trend with a TMR of 2.17.

Dissection of the major organs 48 h after treatment of the animals with the respective imaging agents, except IRDye 800CW, for ex vivo imaging revealed that fluorescence was found primarily in the liver and kidneys of the animals (12). Fluorescence with IRDye 800CW was mainly in the kidneys, and no accumulation of the fluorescent dye was observed in the liver. A comparison of the TMRs between the nuclear and optical modalities showed that the TMRs observed with optical imaging were significantly (P = 0.001) higher than those observed with SPECT. However, a statiscally high (P = 0.0001) correlation, with an R value of 0.9476, was reported between the TMRs determined with the optical and SPECT modalities for the dual-labeled trastuzumab (111In-trastuzumab-IRDye 800CW).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information

NIH Support

Work presented in this chapter was supported by National Institutes of Health Grants P50 CA58183, R01 EB003132, and R01 CA112679.


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