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177Lu-Labeled h-R3 (nimotuzumab), a humanized monoclonal antibody targeting the external domain of the epidermal growth factor receptor

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

Created: ; Last Update: February 2, 2012.

Chemical name:177Lu-Labeled h-R3 (nimotuzumab), a humanized monoclonal antibody targeting the external domain of the epidermal growth factor receptor
Abbreviated name:[177Lu]-h-R3
Agent Category:Antibody
Target:Epidermal growth factor receptor
Target Category:Receptor
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of signal / contrast:177Lu
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



The epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane protein that promotes cell proliferation through a receptor-associated tyrosine kinase (TK)–mediated signal transduction pathway and is known to participate in the development, survival, and migration of normal cells and a variety of cancerous cells (1). In addition, overexpression of the EGFR in malignant tumors appears to correlate with a poor prognostic outcome for the patient (1). In an effort to develop therapies against cancer, a variety of anti-EGFR monoclonal antibodies (mAbs) that inhibit activation of the receptor (see Di Fede et al. (2) for details) or small molecules that interfere with stimulation of the receptor TK pathway (for details, see Kumar et al. (3)) have been developed and approved by the United States Food and Drug Administration for use in the clinic.

Vallis et al. developed ior egf/r3, a murine mAb with a high affinity for the extracellular domain of the EGFR, labeled it with 99m-technetium, and used it for the molecular imaging of the receptor (4). However, because of immunogenicity, the 99mTc-labeled mAb was considered to have limited clinical application, particularly for patients who required repeated imaging investigations. To alleviate this problem, a humanized ior egf/r3 was developed by combining the complementary determining regions of the murine antibody with a human immunoglobulin-1 framework, and this new agent was designated as h-R3 or nimotuzumab (5). Subsequently, 99mTc-labeled h-R3 was investigated for its tissue distribution and imaging properties in clinical trials using single-photon emission computed tomography (SPECT) (4, 5). From these studies, the investigators concluded that 99mTc-h-R3 can be used for the detection of tumors that overexpress EGFR, but it showed a high accumulation in the liver and kidneys of the patients. In another clinical study, the biodistribution of 188Re-labeled h-R3 was investigated with SPECT in patients with high-grade gliomas after locoregional administration; it was shown that this mode of administration of 188Re-h-R3 was safe and that the radiolabeled mAb could be used for the treatment of this malignancy (6). In a continued effort to develop a radioimmunotherapeutic agent that can be used in the clinic for the radioimmunotherapy of cancerous tumors that overexpress the EGFR, Beckford Vera et al. produced 177Lu-labeled h-R3 and investigated its biodistribution in healthy mice and mice bearing A431 human cell epithelial carcinoma xenograft tumors (7).



h-R3 was purchased from a commercial source and conjugated with either S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) or the acyclic ligand S-2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA) for conjugation with 177Lu as described by Beckford Vera et al. (7). When a 20:1 ratio of p-SCN-Bn-DOTA or p-SCN-Bn-DTPA was used to produce the conjugates, the average number of chelating groups per molecule of h-R3 was determined to be 4.1 ± 1.3 ([177Lu]-(DOTA)4–5-h-R3 and [177Lu]-(DTPA)4–5-h-R3, respectively) (n = 5 experiments). When the conjugation ratio of p-SCN-Bn-DOTA was increased to 50:1, the chelate:h-R3 ratio increased to 7.4 ± 1.5 ( [177Lu]-(DOTA)7–9-h-R3) (n = 5 experiments).

The radiolabeling yields of 177Lu-DOTA-h-R3 and 177Lu-DTPA-h-R3 were 99.1 ± 0.5% and 92.3 ± 4.1% (n = 5 determinations for each conjugate), respectively. The radiochemical purities of the purified 177Lu-labeled preparations of all the different conjugates were reported to be >98%. The specific activities of [177Lu]-(DOTA)4–5-h-R3, [177Lu]-(DOTA)7–9-h-R3, and [177Lu]-(DTPA)4–5-h-R3 were 673 MBq/6.6 pmol (18.2 mCi/6.6 pmol), 915 MBq/6.6 pmol (24.7 mCi/6.6 pmol), and 478 MBq/6.6 pmol (12.9 mCi/6.6 pmol), respectively.

In Vitro Studies: Testing in Cells and Tissues


Using a competitive binding assay with a fixed number of A431 cells and different dilutions of the radioimmunoconjugates, the immunoreactivities of [177Lu]-DOTA-h-R3 conjugates and [177Lu]-DTPA4–5-h-R3 were determined to be 94.5 ± 3.1% and 88.3 ± 4.6%, respectively (7).

Competitive binding studies in the presence of non-radiolabeled h-R3 revealed that the IC50 concentrations for [177Lu]-DOTA-h-R3 conjugates and [177Lu]-DTPA4–5-h-R3 were 34.9 ± 2.7 nmol/L and 44.3 ± 5.1 nmol/L, respectively (7).

From saturation binding experiments, the Kd values of [177Lu]-(DOTA)4-5-h-R3 and [177Lu]-DTPA4–5-h-R3 were determined to be 13.8 ± 1.9 × 10−8 mol/L and 12.2 ± 1.3 × 10−8 mol/L, respectively (7). The number of binding sites per A431 cell was calculated to be ~2 × 106 from the Bmax value.

Animal Studies



The biodistribution of the various [177Lu]-h-R3 conjugates was studied in normal Balb/c nu/nu mice (n = 3–4 animals/time point) as described by Beckford Vera et al. (7). Little difference in the accumulation of radioactivity from the two 177Lu-DOTA-h-R3 conjugates was observed in the blood, but at all the time points a significantly higher amount of label (P < 0.001–0.05) from [177Lu]-(DOTA)4–5-h-R3 was observed in circulation compared with that from either [177Lu]-(DOTA)7–9-h-R3 or [177Lu]-(DTPA)45-h-R3. At all time points (24, 72, 96, 168, 216 and 264 h postinjection (p.i.)), the liver showed a significantly higher (P < 0.01) uptake of radioactivity from [177Lu]-(DOTA)7-9-h-R3 compared with the other two conjugates. Other organs (brain, heart, lungs, kidneys, bone, and stomach) showed almost no difference in uptake of the tracer from all the [177Lu]-h-R3 conjugates. Because a higher yield of [177Lu]-(DOTA)4–5-h-R3 compared to that with [177Lu]-(DTPA)4–5-h-R3 was obtained during synthesis (>99% versus ~92%) all subsequent studies were performed only with the former 177Lu-labeled h-R3 conjugate.

The biodistribution of [177Lu]-(DOTA)4–5-h-R3 was investigated in mice bearing A431 cell xenograft tumors (7). The animals (n = 4–5 mice/time point) were injected with 150–280 kBq (4.4–7.6 μCi; 66.6 pmol) of the labeled conjugate through the tail vein and euthanized at various time points ranging from 24 h to 264 h p.i. to harvest all the major organs and the tumors. All organs showed a similar trend in uptake and retention of radioactivity from the radiolabeled mAb; maximum uptake was observed at 24 h p.i., and it decreased gradually up to 264 h p.i. The tumor showed a maximum uptake of 22.4 ± 3.1% injected dose per gram tissue (ID/g) at 72 h p.i., which decreased to 18.3 ± 0.3% ID/g by 216 h p.i. and to 12.6 ± 2.8% ID/g at 264 h p.i. The tumor/non-tumor tissue (T/nT) ratios were the highest at 168 h p.i., with reported values of 2.6, 2.4, 6.1, 6.6, 23.5, and 17.5 for blood, liver, spleen, kidneys, muscle, and bone, respectively. See below for results from blocking studies.

In another study, the biodistribution of [177Lu]-(DOTA)4–5-h-R3 was studied after a locoregional injection of the labeled mAb (i.e., the radiolabeled mAb was injected at the tumor site on the animals) was given to mice (n = 4 animals/time point) bearing A431 cell xenografts (7). The animals were euthanized at different time points ranging from 24 h to 96 h after the injection, and all the major organs, including the tumors, were collected to determine the uptake of radioactivity by the various tissues. In this study, significantly higher uptake of label by the tumors was observed at 24 h and 72 h p.i. compared with the intravenous injection (i.v.) (P < 0.001). The uptake in the lesion peaked to 32.7 ± 2.9% ID/g at 72 h p.i. (compared with 22.4 ± 3.1% ID/g in animals given the i.v. treatment); however, at 96 h p.i. no significant difference in uptake of the label by the tumor (between the i.v. and the locoregional injection) was observed (P > 0.05). At 24 h and 72 h p.i. with the locoregional injection, a significantly lower amount of the tracer was observed in the blood (P < 0.001), liver (P < 0.05), lungs (P < 0.05), and kidneys (P < 0.01) compared with that after the i.v. treatment. After the locoregional injection of [177Lu]-(DOTA)4–5-h-R3, the T/nT ratios for the blood, liver, spleen, lungs, and kidneys were 4.6, 9.8, 10.5, 9.8, and 11.7 times higher, respectively, than those calculated for the i.v. application. At 72 h p.i., the T/nT ratios mentioned above were two times higher for the locoregional injection than after the i.v. treatment.

In a blocking study, mice bearing A431 cell tumors were injected with [177Lu]-(DOTA)4–5-h-R3 in the presence of excess non-radiolabeled h-R3 (~67 pmol) (7). At 96 h p.i., the uptake of label by the tumor was significantly lower (6.4 ± 1.8% ID/g) than in mice injected only with the radioimmunoconjugate (18.4 ± 3.3% ID/g and 20.3 ± 2.1% ID/g after i.v. and locoregional injections, respectively).

From these studies, the investigators concluded that [177Lu]-(DOTA)4–5-h-R3 can be used to target cancerous tumors that overexpress the EGFR; however, further evaluation of the radiolabeled mAb is necessary before it can be used in the clinic (7).

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.


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Torres L.A., Coca M.A., Batista J.F., Casaco A., Lopez G., Garcia I., Perera A., Pena Y., Hernandez A., Sanchez Y., Romero S., Leyva R., Prats A., Fernandez R. Biodistribution and internal dosimetry of the 188Re-labelled humanized monoclonal antibody anti-epidemal growth factor receptor, nimotuzumab, in the locoregional treatment of malignant gliomas. Nucl Med Commun. 2008;29(1):66–75. [PubMed: 18049099]
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