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111In-Diethylenetriamine pentaacetic acid-human epidermal growth factor

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894, vog.hin.mln.ibcn@dacim

Created: ; Last Update: January 22, 2008.

Chemical name:111In-Diethylenetriamine pentaacetic acid-human epidermal growth factor
Abbreviated name:111In-DTPA-hEGF
Agent Category:Growth factor
Target:Epidermal growth factor receptor (EGFR)
Target Category:Receptor-ligand binding
Method of detection:Single-photon emission computed tomography (SPECT) or gamma planar imaging
Source of signal:111In
  • Checkbox In vitro
  • Checkbox Rodents

Click here for the protein and mRNA sequence of EGFR.



Radioimmunotherapy is an attractive choice for the treatment of lymphomas because antibodies directed against specific tumor cell targets can be linked to radionuclides such as iodine (131I), yttrium (90Y), etc., for the lethal irradiation of cancer cells (1). The main advantage of these agents is the ability of the emitted β-particles, because of their long path lengths, to provide therapeutic doses that are evenly distributed to cells in the tumor and affect even the surrounding cancerous cells that do not bind the radiolabeled antibody (2). Radioimmunotherapy with β-emitters has shown some success in the treatment of lymphomas, but the long circulation time and high dose of the labeled antibodies resulted in the development of dose-limiting, nonspecific myelotoxicity in some patients (3). In contrast, the short-range, Auger electron–emitting radionuclides such as iodine (125I) and indium (111In) are radiotoxic to cells only after internalization because they cause cellular DNA fragmentation that leads to cell death (4). Reilly et al. envisioned that Auger electron emitters could be used to target cancerous cells after conjugation to internalized antibodies, and the radiopharmaceutical would potentially show little or no toxicity to the bone marrow cells (5).

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that mediates biological activity through an intracellular tyrosine kinase–signaling pathway. It is known to be overexpressed in a variety of human malignancies, and the degree of EGFR expression often indicates the clinical prognosis for the patient because individuals with higher levels of EGFR were shown to have a poor survival rate (6). Blocking EGFR activity appears to be an effective approach for the treatment of cancers, and a variety of agents, including monoclonal antibodies (MAb), have been developed for this purpose (7). The EGFR is particularly a target for the treatment of breast cancer because it is present in most estrogen receptor-negative and hormone-resistant forms of the neoplastic condition (8). In an effort to develop an alternate treatment, a radiopharmaceutical was developed by derivatizing human epidermal growth factor (hEGF) with diethylenetriamine pentaacetic acid (DTPA) and conjugating it with 111In to obtain 111In-DTPA-hEGF (9-16). The labeled hEGF was then evaluated in vitro and in vivo for imaging and possible treatment of hormone-resistant breast cancer in a mouse model (9-16).



The synthesis of 111In-DTPA-hEGF was as described by Reilly and Gariepy (17). Recombinant hEGF as a peptide of either 51 or 53 amino acids (the intact hEGF has 53 amino acids with a molecular mass of 6.2 kDa), available from various commercial sources, was reacted with bicyclic anhydride DTPA (cDTPAA). The hEGF and cDTPAA were mixed in a molar ratio of 1:5 for 30 min in bicarbonate buffer containing sodium chloride (pH 7.5). Excessive DTPA was removed by size-exclusion chromatography on a P-2 mini-column. The average substitution level of DTPA was estimated to be 1.5 μM/μM hEGF. The labeled hEGF was filter-sterilized before use (9, 10).

To label DTPA-hEGF with 111In, the radionuclide (as 111In-chloride) was mixed with acetate buffer (pH 6.0), and the labeled growth factor was purified from free 111In by size-exclusion chromatography on a P-2 column (17). Radiochemical purity of the product was determined to be between 95 and 98% by silica gel instant thin-layer chromatography. Specific activity of the radiolabeled product was reported to be 600–1,200 mCi/μmol (22,200–44,400 MBq/μmol).

A kit for the preparation of 111In-DTPA-hEGF under good manufacturing practice regulations was designed and evaluated by Reilly et al. (18). The kit was prepared by dispensing a filter-sterilized single dose of ~4 nM (250 μg) DTPA-EGF in acetate buffer (pH 6.0) into glass vials, which were capped and stored at 4°C. The kit was then tested for several quality parameters, including stability, as described by the investigators. The kit was determined to be stable under the storage conditions up to 90 days after manufacture. Labeling of the conjugated hEGF was achieved by adding 111In-chloride directly into the vial and incubating it as described above. The labeling efficiency and biological activity of the kit-labeled product was determined to be similar to the product described above (17, 18). The 111In-DTPA-hEGF prepared with the kit was reported to have a stability of >24 h at 4°C. Stability of labeled hEGF was also determined in mouse and human plasma at 37°C. Under these conditions, 27–33% of the 111In was observed to be transchelated to transferrin over a 72-h period (12). The investigators concluded that the kit formulation was suitable to initiate a clinical study of patients with advanced EGFR-positive breast cancer (18).

In Vitro Studies: Testing in Cells and Tissues


The in vitro binding of 111In-DTPA-hEGF was investigated in MDA-MB-468 cells, a human breast cancer cell line that express high levels of EGFR (13). The affinity constant of 111In-DTPA-hEGF for these cells was determined to be 7.5 ± 3.8 × 108 L/mol for 1.3 ± 0.3 × 108 binding sites/cell. The antiproliferative effect of 111In-DTPA-hEGF was also investigated in the MDA-MB-468 cells (10). Compared to DTPA-hEGF, which had a 50% inhibitory concentration (IC50) of 500 pM for these cells, the IC50 of 111In-DTPA-hEGF was <70 pM. In the same study, the antiproliferative effect of 111In-DTPA-hEGF was observed to be stronger than a select group of chemotherapeutic agents also investigated for comparison (10).

The intracellular localization of 111In-DTPA-hEGF was investigated in MDA-MB-468 cells, and internalization of the labeled growth factor was observed to increase from ~70% of the treated dose at 15 min to ~80% at 4 h (5). After 30 min of exposure to the radioactivity, ~7% of the label was located in the cell nucleus and ~2.5% was bound to the chromatin fraction. At 24 h, >15% of the radioactivity was located in the cell nucleus and ~10% was observed in the chromatin. In the same study, the radiotoxicity of 111In-DTPA-hEGF was evaluated in MDA-MB-468 and MCF-7 cells using a clonogenic assay (5). The MCF-7 cells were reported to express ~100-fold fewer EGFR (1.5 × 104 receptors/cell) compared to the overexpressing MDA-MB-468 cells (1.3 × 106 receptors/cell) (5). The investigators observed that 111In-DTPA-hEGF reduced the growth and cloning efficiency of only the MDA-MB-468 cells and did not affect the MCF-7 cells. This indicated that the labeled hEGF affected only the overexpressing EGFR cells.

In another study with MDA-MB-468 cells, the tyrosine kinase function of the EGFR was inhibited with gefitinib and the cells were exposed to 111In-DTPA-hEGF (16). Under these conditions, cells treated with gefitinib were shown to internalize significantly higher amounts (26.0 ± 5.5%) of the label compared to the control cells (14.6 ± 4.0%; P < 0.05) treated with 111In-DTPA-hEGF alone. In addition, the gefitinib-treated cells showed a higher number of DNA strand breaks compared to controls, and a significant (P < 0.01) reduction in survival was observed in clonogenic assays. With results obtained from this study, the investigators concluded that the use of 111In-DTPA-hEGF in combination with tyrosine kinase inhibitors could be a therapeutic option in the clinic (16).

Using several different breast cancer cell lines, Hu et al. showed the nuclear importation and cytotoxicity of 111In-DTPA-hEGF correlated with the EGFR density on the respective cell lines used in the study (11).

Animal Studies



Biodistribution and imaging studies were performed using either athymic mice bearing MCF-7, MDA-MB-231 (a human breast cancer cell line), or MDA-MB-468 cell xenografts or severe combined immunodeficiency mice implanted with metastasized breast cancer JW-97 cells (13). In the same study, the uptake and biodistribution of 111In-DTPA-hEGF in mice with the various tumors was compared to the uptake and biodistribution of the EGFR monoclonal antibody (MAb) 528, which was also labeled with 111In under the same conditions as hEGF (13). Both radiopharmaceuticals showed an uptake in the liver and kidneys of the animals. At 72 h after administration, <0.2% of the injected dose of 111In-DTPA-hEGF was detected in the blood, indicating that it was rapidly eliminated from blood circulation. During the same period, 3% of the injected dose of the labeled MAb 528 was detected in the blood, suggesting that it had slow clearance from the circulation. The tumor/blood ratios were higher for the labeled growth factor compared to the MAb (12.1 versus 6.1, respectively), but all of the tumor/normal tissue ratios were higher for the MAb. Although the MDA-MB-468 and JW-97 tumors could be visualized with both radiopharmaceuticals, the MAb was reported to be a better choice for imaging (13).

In another study, mice bearing xenograft tumors derived from MDA-MB-468 and MCF-7 cells were administered five weekly doses of 111In-DTPA-hEGF at 6 weeks after tumor cell implantation (established tumors) or 1 week after implantation (non-established tumors) (9). The investigators reported that, among these animals, highest tumor localization, radiation absorption, and tumor growth inhibition were observed in the animals that had small, non-established tumors. In a similar study, the investigators showed that, compared to uptake by MCF-7 xenograft tumors in mice, the MDA-MB-468 xenograft tumors in the animals had a higher incorporation of the radioactivity, and the uptake increased after the animals were pre-injected with a 100-fold excess of unlabeled DTPA-hEGF (11). They also noted that, compared to the tumors, a higher amount of radioactivity was incorporated in the liver, kidney, and spleen cells of the animals.

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



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