99mTc-labeled ior-egf/r3 monoclonal antibody against epidermal growth factor receptor

99mTc-ior-egf/r3 MAb

Chopra A.

Publication Details



In vitro Rodents



Overexpression of the epidermal growth factor receptor (EGFR) is a characteristic feature of a variety of cancers such as breast, ovarian, lung, head and neck, bladder, and colon cancers (1). The EGFR is a 170-kDa transmembrane protein that promotes cell proliferation on binding of the autocrine epidermal growth factor (EGF) or the transforming growth factor α (TGFα). The EGFR operates through a receptor-associated tyrosine kinase–mediated signal transduction pathway. The progression of some cancers is attributed to both an increased level, and activation of EGFR by EGF and TGFα or to a constitutive activation of the tyrosine kinase pathway caused by the development of a mutated receptor kinase. In an effort to develop therapy against cancers that develop as a result of increased EGFR activity or a mutated receptor kinase, a variety of EGFR inhibitors have been developed that either compete with EGF and TGFα for receptor binding (e.g., monoclonal antibodies (MAb) that target the receptor) or are small molecules that inhibit activation of the receptor tyrosine kinase signaling pathway (2).

The murine MAb ior egf/r3, which binds to the external domain of the human EGFR, was generated at the Center of Molecular Immunology in Havana, Cuba (3), and has been shown to inhibit the EGF-dependent growth of various cell lines (4). This MAb was also labeled with metastable radioactive technetium (99mTc) to obtain 99mTc-ior-efg/r3 and evaluated in clinical trials for the treatment of patients with epithelial and advanced brain tumors (3, 5-8).

A freeze-dried kit formulation for direct labeling of ior-efg/r3 with 99mTc has also been published (9).



The MAb ior-efg/r3 was secreted by a hybridoma produced by the fusion of mouse myeloma and splenocytes from Balb/c mice immunized with a partially purified fraction of the EGFR from human placenta as described by Fernandez et al. (10). Purification of ior-efg/r3 was not described by the investigators. The radiolabeling of ior-egf/r3 with 99mTc has been detailed by Morales et al. (3) and is described briefly below.

The ior-efg/r3 MAb in phosphate-buffered saline (PBS) was reduced with a molar excess of mercaptoethanol at room temperature for 30 min. The reduced MAb was then purified with a Sephadex-G25M column and eluted in PBS. Integrity of the reduced MAb was compared to the native MAb by nonreducing polyacrylamide gel electrophoresis and size exclusion chromatography on a Superose 12 HR 10/30 column. Ellmans reagent and a micro method were used to determine the number of free sulphydryl groups in the reduced MAb (11, 12).

To radiolabel the reduced MAb, an aliquot of the reduced MAb was mixed with Medronate II bone agent methyl diphosphonate (MDP) kit reconstituted in saline. This solution was then mixed with 37 MBq (1 mCi) pertechnetate (99mTcO4-). Labeling efficiency of the reaction was determined by ascending paper chromatography, and radiocolloids were determined by cellulose nitrate electrophoresis. The labeling efficiency of the procedure was >98%. Specific activity of the product was not provided by the investigators.

Preparation of the freeze-dried kit formulation has been described by Morales et al. (9). For this, an aliquot of the reduced MAb was mixed with MDP containing 1% of mannitol, sorbitol, trehalose, glucose, sucrose, or inositol as lyoprotectants. The mixture was then frozen in liquid nitrogen. The frozen mixture was lyophilized for 24 h, vacuum-sealed, and stored at 4ºC until required. To label the freeze-dried formulation, the lyophilized MAb was mixed with 99mTcO4- as described above. The labeling efficiency, determined as detailed above, was >97% only for the trehalose, glucose, sucrose, and inositol formulations. The labeling efficiency of formulations containing mannitol and sorbitol was <93% and contained approximately 7 to 8% 99mTc-MDP as an impurity.

Unless otherwise specified, all of the animal and human studies described below appear to have been performed with the non–freeze-dried version of the 99mTc-ior-egf/r3 formulation.

In Vitro Studies: Testing in Cells and Tissues


No publications are currently available.

Animal Studies



The biodistribution of 99mTc-ior-efg/r3 was studied in Balb/c mice (3). Radioactivity was observed in the blood, kidneys, heart, lungs, and large intestine of the animals at 4 h after administration. At 24 h after injection, a reduced amount of the label was detected in these organs.

Biodistribution of the freeze-dried formulation of the 99mTc-ior-efg/r3 MAb was investigated in rats (9). The radioactivity accumulated primarily in the liver, kidneys, and lungs of these animals. The liver and lungs lost most of the label by 24 h after administration. The kidneys maintained almost a constant level of accumulated radioactivity in the time after the injection. Although no data was presented on the accumulation of radioactivity in the bladder, the investigators suggested that the accumulation of the label in the kidney indicated that the radiolabel was cleared from the animals primarily through the urinary bladder route.

Other Non-Primate Mammals


No publications are currently available.

Non-Human Primates


No publications are currently available.

Human Studies


The biodistribution of 99mTc-ior-egf/r3 was investigated with the use of immunoscintigraphy in patients with tumors of epithelial origin (7). Significant accumulation of the label was observed in the liver (~48% of total), heart (~3.5% of total), and spleen (~3.1% of total) at 5 min after the bolus infusion. At 24 h after infusion, the amount of radioactivity detected in these organs was significantly lower (liver, ~3.2%; heart, ~0.1%; spleen, ~0.06%). The tumor and liver uptake as well as plasma clearance kinetics of radioactivity in the patients were also monitored during this study. An accumulation of radioactivity was observed in the tumors and liver up to 5 h after injection. The liver subsequently showed a steady loss of radioactivity up to 24 h. During the same period a steady loss of label was observed in the plasma. The tumors showed an increase in label accumulation up to 5 h followed by a steady amount of radioactivity up to 24 h after administration of the labeled MAb.

In another study from the same laboratory, the pharmacokinetics and clearance of the labeled antibody were investigated in patients with tumors of epithelial origin (13). With results obtained from this investigation it was concluded the labeled MAb had an elimination half-life of ~21 h. An analysis of the urine samples from these patients showed that clearance of radioactivity through this route was only ~5% of the injected dose at 24 h after administration, and a fast protein liquid chromatography analysis showed little decomposition (i.e., only 6–7%) of the labeled MAb to smaller molecular weight species of the molecule.

The clinical trial safety and diagnostic efficacy of 99mTc-ior-egf/r3 was also investigated in patients with tumors of epithelial origin (8). The overall sensitivity, specificity, accuracy, and positive and negative predictive values for the immunoscintigraphic images were 84.2%, 100%, 86.5%, 100%, and 52.4%, respectively. From this study it was concluded immunoscintigraphy with 99mTc-ior-egf/r3 could be used for the diagnosis and follow-up of patients with tumors of epithelial origin.


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