Nuclear Medicine and Biology
Volume 39, Issue 1 , Pages 3-13, January 2012

Preparation and preclinical evaluation of 177Lu-nimotuzumab targeting epidermal growth factor receptor overexpressing tumors☆☆☆

Received 6 April 2011; received in revised form 30 June 2011; accepted 3 July 2011. published online 28 September 2011.

Article Outline

Abstract 

Objectives

Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) which recognizes the external domain of the epidermal growth factor receptor (EGFR) with high specificity. It was demonstrated that h-R3 has a unique clinical profile for immunotherapy of adult gliomas and pediatric pontine gliomas. The aim of this work was to evaluate the conjugate 177Lu-h-R3 as a potential radioimmunoconjugate for radioimmunotherapy (RIT) of tumors overexpressing EGFR.

Methods

h-R3 was modified with the macrocylcic ligand S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and the acyclic ligand S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA); the immunoconjugates were labeled with no-carried added 177Lu. Specificity and affinity were tested using radioimmunoassays in a cell line overexpressing EGFR. Biodistribution in mice, healthy or bearing A431 epithelial carcinoma xenografts, was performed for 11 days. Tumor uptake, the influence of the nature of the chelate and the way of administration were studied. Absorbed dose in tumor and selected organs was calculated using the OLINDA/EXM software; the data from the animals was extrapolated to humans.

Results

177Lu-h-R3 conjugates were obtained with specific activity up to 915 MBq/mg without significant loss of immunoreactivity. The binding of 177Lu-h-R3 conjugates to A431 cells showed to be EGFR specific, and the affinity was similar to native h-R3. Tumor uptake reached a maximum value of 22.4±3.1 %ID/g at 72 h and remained ∼20% ID/g over 1 week. Locoregional application showed better tumor/nontumor ratios than intravenous application.

Conclusions

177Lu-h-R3 should be considered for further evaluations as a potential radiopharmaceutical for RIT of tumors overexpressing EGFR.

Keywords: Radioimmunotherapy, Nimotuzumab, 177Lu, Monoclonal antibody

 

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1. Introduction 

Radioimmunotherapy (RIT) is a therapeutic modality that involves the use of monoclonal antibodies (mAb) to deliver the energy of beta- or alpha particles to targeted cells. Even when this technique is approved, it is still in the early stages of development [1]. Relevant results have been obtained in the treatment of refractory non-Hodgkin's lymphoma (NHL) employing ibritumomab tiuxetan (Zevalin) and/or iodine (131I) tositumomab (Bexxar), the first radioimmunoconjugates (RIC) approved by the US Food and Drug Administration (FDA) in 2002 and 2003, respectively. Nevertheless, the treatment of solid tumors using this modality remains to be thoroughly investigated. Discrete advances have been achieved in the treatment of minimal residual disease, locoregional applications, pretargeted RIT and as a combination of therapies [2], [3].

Epidermal growth factor receptor (EGFR) is a target of anticancer therapies due to its overexpression in a variety of malignant epithelial tumors and it is associated with a poor prognosis [4], [5]. In fibroblasts cells, the expression of EGFR ranges from 40,000 to 100,000 receptors per cell [6], [7]. In contrast, EGFR is overexpressed in the majority of solid tumors, including breast and ovarian cancer, colon cancer, head-and-neck cancer and non–small cell lung cancer (NSCLC), with some breast cancers expressing up to 2×106 EGFR receptors per cell [8]. Several approaches have been used to inhibit the EGFR-associated signal transduction cascade. Monoclonal antibodies bound to a specific region of EGFR have been successfully used to inhibit its dimerization and autophosphorylation [5], [9], [10].

Nimotuzumab (TheraCIM, CIMher, Theraloc) is a humanized mAb (IgG1) obtained by transplanting the complementary determining regions of the murine monoclonal antibody ior-egf/r3 to human framework which recognizes the external domain of EGFR with high specificity [11], [12]. Unlike most of the drugs against the EGFR, h-R3 does not show severe adverse effects in the clinic; for example, no serious skin rashes have been reported [13]. h-R3 has demonstrated very encouraging results in pediatric pontine (brainstem) and adult gliomas [14]. It is undergoing clinical trials in non–small cell lung cancer and a Phase II monotherapy trial in Europe in patients with advanced metastatic pancreatic cancer [15]. 188Re-labeled h-R3 showed promising results in a Phase I trial for the radiation treatment of gliomas via an in-dwelling catheter into the postoperative cavity following resection [16].

Beta radiation radionuclide emitters such as 131I (t1/2=8.0 days; β−=0.6 MeV and Eγ=0.364 MeV) and 90Y (t1/2=2.67 days; β−=2.28 MeV) have been successfully used in RIT. Residualizing radionuclides, such as 90Y and 177Lu, are potentially more suitable radionuclides for RIT [17]. 177Lu (t1/2=6.7 days, Eγ=0.208 MeV, β−=0.497 MeV, max range in tissue penetration=2.0 mm) is being strongly considered for RIT since it combines the advantages of both 90Y and 131I and the ability to form stable complexes with macrocyclic and acyclic ligands [18], [19], [20], [21]. Its half-life is appropriate for preparation, transport and successful delivery of therapeutic doses to the tumor by radioimmunoconjugates such as monoclonal antibodies. In addition, 177Lu emits two low-energy γ lines with energy of 113 and 208 keV that are suitable for imaging and assessment of delivered doses.

The aim of this work was to evaluate the RIC 177Lu-nimotuzumab (177Lu-h-R3), in a model of human epithelial carcinoma, as a potential radioimmunoconjugate for RIT of tumors overexpressing EGFR. The effects of the nature of the bifunctional chelate used for 177Lu labeling and the administration approach, intravenous vs. locoregional, were also studied.

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2. Materials and methods 

2.1. Reagents and equipment 

177Lu no-carrier added (nca in 0.05 mol/L HCl), produced indirectly via the 176Yb(n,γ)177Yb→(β−)→177Lu reaction, was generously donated by Isotope Technologies Garching (ITG, Munich, Germany). 90YCl3 (555 MBq in 22 μl of 0.05 M HCl) was purchased from PerkinElmer. Bifunctional chelators, p-SCN-Bn-DOTA and p-SCN-Bn-DTPA were purchased from Macrocyclics (Dallas, TX, USA). Nimotuzumab was purchased in 10-ml vials (5.0 mg/ml in phosphate-buffered saline) from Oncoscience AG (Wedel, Germany). Chelex 100 in sodium form was purchased from Sigma (Steinheim, Germany). All other reagents were purchased from Merck (Darmstadt, Germany) or Fluka (Steinhelm, Germany) with the highest purity available. Ultrapure water (Aquatec Water Systems, California, USA) was used for all procedures. To avoid metal contamination, all the solutions used in conjugation and radiolabeling reactions were passed through a Chelex 100 column (1×10 cm), and all glassware was washed with 2 M HCl and ultrapure water.

Size-exclusion high-performance liquid chromatography (SE-HPLC) was performed at room temperature on an Agilent 1200 series chromatographic system (Agilent, Waldbronn, Germany) equipped with a Rheodyne injector (Milford, USA) and an online γ-ray detector (Raytest, Straubenhardt, Germany). The Gina Start version 4.07 software (Straubenhardt, Germany) was used for data processing. UV measurements were performed on a Spectronic Unicam, Heλios-α spectrophotometer (ThermoSpectronic, Cambridge, UK) using a 1-cm sample cell. Radiation counting of TLC plates was performed with a Cyclone Plus Storage Phosphor System (PerkinElmer, USA) or miniGITA (Raytest). The Vivaspin 6 and 20 centrifugal devices (molecular weight cutoff 30 kDa) were purchased from Sartorius (Stedim Biotech, Goettingen, Germany).

2.2. Conjugation of nimotuzumab with p-SCN-Bn-DOTA 

The original solution of h-R3 was concentrated to 10 mg/ml by ultrafiltration using Vivaspin 20. Concentrations of mAb were determined by UV spectrophotometry at 280 nm. Ten milligrams of humanized mAb h-R3 in 0.01 mol/L phosphate buffer (pH 7.2) was mixed with p-SCN-Bn-DOTA (20- or 50-fold molar excess) or p-SCN-Bn-DTPA (20-fold molar excess) previously dissolved in phosphate buffer (pH 8.5) and final pH was adjusted to 8.5 with NaOH 1 mol/L. Afterwards, the reaction mixture was incubated overnight at 4°C. The immunoconjugates were purified by ultrafiltration on Vivaspin 6 until the absorbance in the ultrafiltrate at 280 nm was nearly zero. Protein concentration was determined by Bradford assay. Conjugates were stored at 4°C for further use. Immunoconjugates were characterized by SE-HPLC. About 20 μl of the conjugate was injected onto a TSK-Gel SW 3000 (7.5×300 mm, 10 μm, TosoHass) column using 0.9% NaCl/0.05% NaN3 as the mobile phase. The flow rate was maintained at 1 ml/min, and the elution was monitored by UV spectrophotometer at 280 nm.

2.3. Determination of the average number of chelates per antibody molecule (L/P) 

The average number of chelates linked to an antibody molecule was determined using 90Y by a radioactive method previously described [22]. Briefly, 49 μg (10 μl) of the conjugates was added to 30–50 μl of NH4OAc 0.5 mol/L (pH 7.0). Afterwards, 10, 20 or 30 μl (3.33×10−4 mol/L) of a standardized YCl3 solution spiked with 90Y was added. The reaction mixture was incubated at 42°C for 3 h, then 1/9 of the reaction volume of DTPA (0.01 mol/L, pH 6.0) was added and the reaction mixture was incubated at room temperature for another 15 min. An aliquot of 2 μl of the reaction mixture was developed on SG-ITLC plates using a 10% (0.4 mol/L) solution of ammonium acetate and methanol (1:1) as the mobile phase. The number of chelates per antibody molecule was calculated from the ratio of counts remaining at the origin to the total number of counts multiplied by the mole ratio of metal (Y3+)/mole of conjugate.

2.4. 177Lu Radiolabeling 

Aliquots of 177LuCl3 (15–185 MBq, 1–25 μl) were added to 25–50 μl of 0.5 mol/L NH4OAc buffer at pH 7.0, followed by 50–100 μl (0.200–0.540 mg) of conjugates. Afterwards, the reaction mixture was incubated at 42°C for 1.5 h. In order to scavenge any free radiometal for further quality control, a solution of DTPA or EDTA (7–17 μl, 0.01 mol/L, pH 6.0) was added to the reaction vial and the mixture was incubated for 15 min at room temperature. In these conditions, the radioimmunoconjugates remain at the origin, while radiometal-DTPA/EDTA complexes migrate to Rf 0.4–0.8. The effect of the number of chelate per antibody molecule and the nature of the chelate on the radiolabeling efficiency and specific activity were studied.

2.5. Quality control 

Radiolabeled conjugates were purified from unbound radiometal, when needed, by size exclusion chromatography on PD-10 columns (GE Healthcare, Buckinghamshire, UK) eluted with phosphate buffered saline at 0.01 mol/L. Radiometal labeling efficiency and radiochemical purity were determined by thin-layer chromatography performed on SG-ITLC plates (Pall Corporation, USA), using 10% (w/v) ammonium acetate/methanol (1:1) as the mobile phase. SE-HPLC was also employed using a TSK-Gel SW 3000 (7.5×300 mm, 10 μm, TosoHass) column, with an isocratic mobile phase of 0.9% NaCl/0.05% NaN3 and a flow rate of 1.0 ml/min.

2.6. Cell culture 

The human squamous carcinoma cell line A431 (CLR 1555; American Type Culture Collection) was used in all cell experiments. The A431 cell line was cultured in DMEM high-glucose medium. Media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin (penicillin 100 IU/ml and streptomycin 100 μg/ml). All media, supplements, antibiotics and buffers used in cell culture were purchased from PAA The Cell Culture Company (Pasching, Austria). During cell culture and cell experiments (unless otherwise stated), cells were grown at 37°C in incubators with humidified air, equilibrated with 5% CO2.

2.7. Saturation binding experiment 

For the saturation binding experiment, A431 cells were cultured ∼2×105cells/well on 12-well plates (BD Falcon, Becton-Dickinson, UK) in 1 ml of medium for 48 h prior to the studies. Cells were treated with solutions of different concentrations (100 μl/well, 3.0–120.0 nmol/L) of 177Lu-DOTA-h-R3 or 177Lu-DTPA-h-R3 and incubated for 3 h at 4°C. The nonspecific background binding was studied by adding a 100 times excess of native nimotuzumab to some wells. Triplicate cell dishes were used for each measuring point. After incubation, the medium was discarded and cells were washed with ice-cold phosphate buffer solution (PBS). Cells were lysed using 0.5 ml of 0.5 M NaOH containing 5% SDS, and the radioactivity was measured in a γ-counter (Wizard 3″, Wallac, Turku, Finland). Dissociation constants (Kd) were estimated from the nonlinear fitted curves using GraphPad Prism software (GraphPad Software, California, USA). For cell counting, a representative parallel sample from the experiments was trypsinized for about 15 min and the cells were counted using a fully automated cell analyzing system (CASY TT, Roche Innovatis, Germany). The mean was used as a cell number for all wells.

2.8. Competitive binding experiment and immunoreactivity 

Biological activities of the 177Lu-h-R3 conjugates were estimated by competitive inhibition experiments against native nimotuzumab. Therefore A431 cells were cultured as described above. The cells were treated with 177Lu-h-R3 conjugates (100 μl/well, 50 ng, ∼75000 cpm/well) premixed with different concentrations of native h-R3 in cell culture medium and incubated for 3 h at 4°C. Afterwards, the cells were treated as described above. Concentration values that caused 50% of inhibition (IC50) of 177Lu-h-R3 to its receptor were estimated from the nonlinear fitted curves using the GraphPad Prism software. The immunoreactive fraction of the 177Lu-h-R3 conjugates was determined using the method described by Konishi et al. [23] based on competitive binding assay using a fixed cell concentration and different dilutions of radioimmunoconjugate. Normalized cell bound radioactivity was plotted as a function of h-R3 concentration, and the immunoreactivity fraction was estimated by fitting the data to the equation previously described [21].

2.9. Animal experiments 

Male Balb/c nu/nu mice, 6 weeks old, purchased from Charles River Laboratories (Sulzfeld, Germany) were used for in vivo experiments. All animal experiments were performed according to the Animals Ethics Committee of the Institute of Nuclear Research, Rez, Czech Republic. For biodistribution studies in a human tumor model overexpressing EGFR, subcutaneous xenografts were established from the A431 human epithelial carcinoma cell line by injection of cell suspension (100 μl, 2–3×106 cells) in the right flank of the mice. The tumors were allowed to grow for 2 weeks before the experiments were performed.

2.10. Biodistribution in healthy mice 

177Lu-h-R3 conjugates (10 μg, 0.1 ml in normal saline solution, 150–250 kBq) were intravenously injected via the tail vein into healthy mice weighing 20–26 g. At 24, 72 and 168 h after injection, the mice were anesthetized, euthanized by cervical dislocation and dissected. Organs of interest were collected, rinsed of residual blood and weighed. Sample activity was measured in an automatic γ-counter (Wizard 3″, Wallac). Tissue activity was calculated in percent of the injected dose per gram of tissue (%ID/g) as an average of three or four animals. The effect of the number of chelates per antibody molecule and the nature of the ligand in the in vivo behavior of the conjugates were studied.

2.11. Biodistribution in a human tumor model overexpressing EGFR 

Mice bearing A431 human epithelial carcinoma xenografts were injected intravenously via the tail vein with approximately 150 to 280 kBq (100 μl, 10 μg) of 177Lu-DOTA-h-R3. Groups of three to four animals were anesthetized, euthanized by cervical dislocation and dissected at 24, 72, 96, 168, 216 and 264 h after injection. In addition, the 177Lu-DOTA-h-R3 conjugate (100 μl, 10 μg, 150–280 kBq) was applied directly into the volume of the tumor tissue for a second group of mice, to study the in vivo behavior of locoregionally applied radioimmunoconjugate. Groups of four animals were anesthetized, euthanized by cervical dislocation and dissected at 24, 72 and 96 h after injection. At the selected time points for each experiment, blood was collected by cardiac puncture and tumors and main organs were collected, weighed and measured in an automatic γ-counter. For the blocking experiment, mice bearing the A431 tumor xenograft were coinjected with 0.15 mg of nimotuzumab. The tails were also measured for radioactive content to determine the accuracy of the injections. The percent of injected dose per gram of tissue was determined using standards representing the injected dose per animal, and tumor-to-nontumor (T/nT) tissue ratios were also calculated.

2.12. Dosimetry 

Dose calculations were done with biodistribution data from A431 xenografts mice using the OLINDA/EXM software [24]. The number of disintegrations that occur in the source region per unit activity administered was calculated from the time–activity curves without correction for radioactive decay. The dose for a particular organ was obtained as exponential interpolation of tabulated data between 0.01 and 2 g for the sphere model. Dose estimations in humans were calculated by extrapolation of the data, from the biodistribution study, in animals to humans using the percent kilogram per gram method [25].

2.13. Statistical analysis 

Statistical analysis was performed to determine the differences in tissue uptakes among the conjugates using analysis of variance (two-way ANOVA test) by the software package GraphPad Prism. The ANOVA test was followed by Bonferroni's multiple comparison test (P<.05). The same approach was used to determine the differences between intravenously and locoregional application.

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3. Results 

3.1. Conjugation and radiolabeling 

The reaction of h-R3 with p-SCN-Bn-DOTA or p-SCN-Bn-DTPA at a molar ratio of 20:1 produced conjugates with an average of 4.1±1.3 (mean±S.D., n=5) chelating groups per protein molecule, while increasing the conjugation ratio to 50:1 for p-SCN-Bn-DOTA gave conjugates with 7.4±1.5 (mean±S.D., n=5) chelators per hR3. The concentration of conjugates ranged from 5.0 to 6.0 mg/ml as determined by Bradford assay. Immunoconjugates were successfully labeled with 177Lu under relatively mild conditions. The radiolabeling yield of conjugates was 99.1±0.5 % (mean±S.D., n=5) and 92.3±4.1% (mean±S.D., n=5) for 177Lu-DOTA-h-R3 and 177Lu-DTPA-h-R3, respectively (Fig. 1). No difference in the radiolabeling yield was observed between the conjugates modified with increasing molar ratios of p-SCN-Bn-DOTA. Specific activities of up to 673 [177Lu-(DOTA)n-h-R3, n=4–5], 915 [177Lu-(DOTA)n-h-R3, n=7–9] and 478 [177Lu-(DTPA)n-h-R3, n=4–5] MBq/mg were obtained. When purification was applied, the radiochemical purity was greater than 98%.

3.2. Saturation and competitive binding experiments 

The immunoreactive fractions were 94.5±3.1% and 88.3±4.6% (mean immunoreactivity±S.E.) for 177Lu-DOTA-h-R3 and 177Lu-DTPA-h-R3, respectively. The results from the binding competitive experiments showed a sigmoid curve. The binding of 177Lu-hR3 conjugates to A431 cell lines showed to be epidermal growth factor receptor specific (Fig. 2). Fifty percent displacements (mean IC50±S.D., n=3) were achieved at 34.9±2.7 and 44.3±5.1 nmol/L of unmodified h-R3 for 177Lu-DOTA-h-R3 and 177Lu-DTPA-h-R3, respectively. The Kd determined by saturation binding experiment of the radiolabeled h-R3 was 13.8±1.9×10−8 and 12.2±1.3×10−8 mol/L for 177Lu-DOTA-h-R3 and 177Lu-DTPA-h-R3, respectively. The number of binding sites per cell was ∼2×106 calculated from the Bmax value (Fig. 3).

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  • Fig. 3. 

    Saturation binding assay of 177Lu-(DOTA)n-h-R3 (n=4–5) and 177Lu-(DTPA)n-h-R3 (n=4–5). The dilutions were assayed in triplicate, and the average binding was calculated and plotted.

3.3. Biodistribution in healthy animals 

The biodistribution study in healthy animals was performed in order to compare the behavior of 177Lu-h-R3 conjugates, mainly in blood, liver, spleen, lung and bone. The results are summarized in Fig. 4. At 24 h, significantly higher concentration in blood of 177Lu-DOTA-h-R3 than that of 177Lu-DTPA-h-R3 (P<.05) was observed. Statistically, no significant differences were observed in blood concentration between 177Lu-DOTA-h-R3 conjugates prepared with increasing conjugation ratios, but the liver uptake was significantly higher for the 177Lu-DOTA-h-R3 conjugate with the higher DOTA/h-R3 ratio (P<.01). At the 72- and 168-h time points, the concentration in blood of 177Lu-(DOTA)n-hR3 (n=4–5) was significantly higher than that of 177Lu-(DTPA)n-hR3 (n=4–5) and 177Lu-(DOTA)n-hR3 (n=7–9) conjugates (72 h, P<.01 and 168 h, P<.001). No significant differences in lungs, kidneys, heart, brain, stomach and bone uptakes among the 177Lu-h-R3 conjugates (P>.05) could be observed in the whole study. Statistical analysis showed that there was no significant difference in tissue-to-blood ratios for 177Lu-(DOTA)n-h-R3 (n=4–5) until 168 h (P>.05). The liver-to-blood ratios increased with time for 177Lu-(DOTA)n-h-R3 (n=7–9, P<.001) and for 177Lu-(DTPA)n-h-R3 (n=4–5, P<.05). For all preparations, bone-to-blood ratios remained constant over time (P>.05).

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  • Fig. 4. 

    Biodistribution of 177Lu-h-R3 radioimmunoconjugates in tissue/organs collected at different time points from healthy mice. Values are expressed as mean % injected dose/g (%ID/g)±S.D. in groups of three to four animals for each time point.

3.4. Biodistribution in xenograft-bearing mice 

The biodistribution studies of 177Lu-(DOTA)n-h-R3 (n=4–5) were conducted in A431 human epidermal carcinoma xenografts in Balb/c nude mice. For intravenous application, the biodistribution study was performed over a period of 264 h and for the locoregional application at 24-, 72- and 96-h time points. Uptakes in the tumor and major organs of interest over time are shown in Fig. 5.Tumor uptake reached a maximum value of 22.4±3.1 %ID/g at 72 h. From 72 to 216 h, the average tumor uptake was 18.3±0.3 %ID/g. Tumor uptake decreased to 12.6±2.8 %ID/g from 216 to 264 h. Normal tissue uptakes were lower than 9 %ID/g in all tissues and they decreased over time, except for bone and spleen whose values remain approximately constant throughout the whole study. Bone uptake was low with a maximum value of 2.7±1.1 %ID/g at 24 h and an average of 1.6±0.5 from 72 to 264 h. At 168 h, T/nT tissue ratios reached maximum values of 2.6, 2.4, 6.1, 6.6, 23.5 and 17.5 for blood, liver, spleen, kidneys, muscle and bone, respectively. When the 177Lu-(DOTA)n-h-R3 (n=4–5) conjugate was applied locoregionally to A431 xenografts in Balc/c nude mice, tumor uptake was significantly higher at 24 and 72 h than that observed for intravenous application (P<.001). Tumor uptake peaked 32.7±2.9 %ID/g at 72 h. There was no statistically significant difference observed in tumor uptake at 96 h (P>.05). Locoregional administration also resulted in significantly lower concentration in blood (P<.001) and uptakes in liver (P<.05), lungs (P<.05) and kidneys (P<.01) than that observed for intravenous application at 24 and 72 h. Locoregional application of the 177Lu-DOTA-h-R3 conjugate showed T/nT ratios 4.6, 9.8, 10.5, 9.8 and 11.7 times higher for blood, liver, spleen, lungs and kidneys, respectively, than those calculated for the intravenous application. At 72 h, the T/nT ratios mentioned above were two times higher for locoregional than for intravenous application. At 96 h, tumor uptake in mice coinjected with nimotuzumab (6.4±1.8 %ID/g) was significantly lower than in mice injected only with radioimmunoconjugate (intravenous: 18.4±3.3 %ID/g; locoregional: 20.3±2.1 %ID/g).

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  • Fig. 5. 

    Biodistribution of the 177Lu-(DOTA)n-h-R3 (n=4–5) radioimmunoconjugate in tissue/organs collected at different time points from A431 tumor-bearing mice. Values are expressed as mean %ID/g±S.D. in groups of three to four animals for each time point.

3.5. Dosimetry 

Absorbed dose to tumor and selected organs for the A431 xenograft-bearing mice calculated with OLINDA/EXM are shown in Table 1. Tumor and liver received the highest doses among the selected organs. Calculations made for spheres of various radii showed high absorbed dose for small tumors. Table 2 shows the organ dose in humans calculated from the extrapolation of the biodistribution in mice to humans. Since the clearance 177Lu-DOTA-h-R3 is through the reticuloendothelial system, those organs involved received the highest doses.

Table 1. Dosimetry of 177Lu-DOTA-h-R3 in BALB/c nu/nu mice bearing EGFR overexpressing A431 xenografts
TissueMass (g)Dose
(Gy/MBq)
Brain0.35±0.030.03±0.00
Heart0.11±0.010.55±0.15
Kidneys0.34±0.050.38±0.01
Liver1.22±0.211.97±0.00
Lungs0.14±0.030.58±0.05
Spleen0.18±0.040.83±0.07
Stomach0.25±0.110.15±0.03
Tumor0.73±0.322.42±0.00

Tumor0.01195.00
0.120.30
0.54.12
12.08
21.05
Table 2. Dosimetry of 177Lu-DOTA-h-R3 extrapolated to human from animal data
Target organEDEED
Brain0.00E0001.11E 05
Breasts5.43E-051.81E-05
Small intestine0.00E0003.30E-06
Stomach wall0.00E0009.03E-05
Heart wall4.52E-040.00E000
Kidneys1.82E-031.51E-04
Liver2.37E-031.98E-03
Lungs3.40E-033.40E-03
Muscle9.11E-047.59E-05
Pancreas0.00E0005.50E-06
Red marrow6.91E-056.91E-05
Skin0.00E0003.36E-06
Spleen1.52E-031.26E-04
Thymus0.00E0003.57E-06
Thyroid1.81E-053.02E-05
Urinary bladder wall0.00E0003.41E-05
Effective dose equivalent (EDE)1.08E-02
Effective dose (ED) 6.25E-03

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4. Discussion 

Even when in the early 2000s, Bexxar and Zevalin were approved by the FDA for RIT of non-Hodgkin's lymphoma with very encouraging results and a high percentage of patients entering long-term remission [26], so far no further radioimmunoconjugate has been approved for RIT of solid tumors. The above-mentioned fact has been due to physical, chemical, biological, clinical, regulatory and financial limitations that have impeded the progress of these drugs [27]. As an example, the doses shown to be effective in hematologic tumors are insufficient in epithelial cancers [3]. Nevertheless, there has been progress in locoregional applications and in the treatment of minimal residual disease [2]. Improving the development of mAbs, radiochemistry, dosimetry, prediction of tumor response, host toxicities and better targeting strategies should be manifested in the progress of therapy of solid tumors in the near future.

Nimotuzumab is a remarkable mAb directed against the EGFR [14]. Nimotuzumab has demonstrated a very good clinical profile. Its clinical benefits are equivalent to or better than other anti-EGFR mAbs: no severe skin, renal, gastrointestinal mucosa and cutaneous effects are shown in several clinic trials [28]. Next to adult gliomas and pediatric pontine, nimotuzumab is under investigation for other potential indications such as nasopharyngeal cancer, head-and-neck cancer, esophageal cancer, breast cancer, prostate cancer, uterine cervical cancer and colorectal cancer [14]. Previous reports combined with the accumulating clinical data on nimotuzumab showed that nimotuzumab is expected to have similar efficacy to cetuximab and other high-affinity antibodies in tumors overexpressing EGFR [29]. Radioimmunoscintigraphy of nodal metastatic disease using [99mTc] h-R3 (DiaCIM) was also performed in a Phase I trial, but little correlation between EGFR expression and positive tumor imaging was observed [12]. Moreover, as stated above, encouraging results were obtained in a Phase I single-dose study of intracavitary-administered h-R3 labeled with 188Re in adult recurrent high-grade glioma [16]. Therefore, the favorable decay properties of 177Lu and its ability to form stable complexes with macrocyclic and acyclic ligands have made 177Lu as an interesting radionuclide for the labeling of h-R3.

Nimotuzumab was conjugated with p-SCN-Bn-DOTA and p-SCN-Bn-DTPA, essentially, as described previously [30], [31] with some modifications. The purification step using the Vivaspin centrifugal device was included or repeated two times more than previously described [30], [31]. The nature of the ligand did not affect the efficiency of conjugation. The radiolabeling yield of conjugates was affected by the nature of the chelate used for modification of h-R3. Milenic et al. [32] reported higher incorporation of 177Lu for the CHX-A″-DTPA derivative than for the C-DOTA and PA-DOTA derivatives. In our study, labeling of the conjugates modified using p-SCN-Bn-DOTA resulted in higher yields and specific activities than labeling of the p-SCN-Bn-DTPA conjugate. In the case of the 177Lu-DOTA-h-R3 radioconjugate, no further purification after the radiolabeling reaction was necessary. The radiolabeling efficiency and specific activities obtained in this study were higher than those previously reported for similar conjugates [33], [34], [35], [36].

Cell studies confirmed that the immunoreactivity of the h-R3 was not considerably compromised by the conjugation and radiolabeling process. The binding of 177Lu-(h-R3) conjugates to A431 cells was receptor specific as estimated by the ability to compete with native h-R3. The IC50 values of the 177Lu-(h-R3) conjugates were similar to those reported for nimotuzumab, when h-R3 competed with 125I-EGF using microsomal fraction of a placental membrane extract [37]. Saturation binding assay showed that the affinity of the 177Lu-h-R3 conjugates was similar to unmodified nimotuzumab [9].

It is well known that antibodies are slowly cleared through the reticuloendothelial organs [38]. Consequently, the in vivo behaviour of the 177Lu-h-R3 conjugates in healthy animals was studied with special regard to blood, liver and spleen. The radiolanthanides that dissociate from the conjugate, in vivo, can form colloids in the blood increasing the liver uptake or can accumulate in bones due to high affinity of lanthanide metal ions to phosphate anion resulting in myelotoxicity [20], [21]. For this reason, bone uptake was also used to evaluate the in vivo stability of the 177Lu-h-R3 conjugates. The increasing amount of DOTA in the conjugate showed increased uptake in liver and faster clearance from blood. The results are in agreement with those reported by Knogler et al. [39]. The uptake in liver, spleen and bones observed in our study was lower than those previously reported for conjugates modified with four to five groups of p-SCN-Bn-DOTA or p-SCN-Bn-DTPA [34]. The release of the radiometal from the conjugate and its metabolism resulted in nonspecific accumulation of the radioactivity in different tissues and gives us an idea of the stability of the conjugate. Tumor/blood ratio calculations are useful for this estimation. Base on liver/blood ratios, 177Lu-(DOTA)4–5-h-R3 showed higher in vivo stability than 177Lu-(DTPA)4–5 and 177Lu-(DOTA)8–9.The bone/blood ratio did not increase with time for all radioimmunoconjugates, suggesting high stability of the metal–ligand complexes.

The 177Lu-(DOTA)n-h-R3 (n=4–5) conjugate was chosen for further evaluation in A431 human epidermal carcinoma xenograft Balb/c nude mice due to the fact that it showed better radiolabeling yields and in vivo stability in healthy mice. The highest uptake over time was observed in the tumor. Notable activity was also observed in liver, spleen, kidneys and lung consistent with that previously reported for the murine version of the h-R3 labeled with 90Y using the DOTA-Ph-Al derivative [40] and with the normal excretion of monoclonal antibodies [41]. The activity in normal tissue was always less than 10 %ID/g and decreased over time. Blocking experiment showed that uptake of the conjugate was specific to the target. In this study, the tumor uptake values and tumor to nontumor ratios are higher than those previously reported for h-R3 and the parental murine mAb labeled with 99mTc and 90Y [35], [37], [42]. The 177Lu-DOTA-h-R3 conjugate showed lower uptake in liver and spleen than other EGFR-targeting antibodies [43], [44], [45].

An interesting phenomenon was observed when the biodistribution results in healthy mice and A431 human epidermal carcinoma xenograft mice were compared. The concentration in blood of the radioimmunoconjugate was 1.4 and 2.9 times higher in healthy mice than in A431 xenograft mice for 72 and 168 h, respectively. At 168 h, spleen, lung and kidney uptakes were significantly higher in healthy mice. The results are in complete agreement with those obtained by Coliva et al. [46] and Zacchetti et al [36]. An explanation to this fact might be that the tumor carrying a high density of the target antigen could take up the conjugate from the blood.

As we mentioned above, promising results using RIT have been achieved in the treatment of minimal residual disease and in locoregional applications. Consequently, the 177Lu-(DOTA)n-h-R3 (n=4–5) conjugate was locoregionally administered in mice bearing A431 human epidermal carcinoma xenografts. The results were compared with those obtained from the biodistribution when the conjugate was intravenously administered with particular attention paid to tumor uptake and tumor to nontumor ratios. Significantly higher tumor uptake and T/nT ratios at 24 and 72 h were observed when the conjugate was locoregionally administered, suggesting that locoregional administration would decrease the nonspecific accumulation of radioactivity in the normal tissue while increasing the tumor uptake.

Dosimetric calculations showed that the 177Lu-DOTA-h-R3 radioimmunoconjugate will deliver the highest dose into the tumor. The smaller the tumor, the higher the dose deposited. The results obtained are in agreement with those previously reported for similar 177Lu conjugates [17], [36]. The results from the extrapolation of animal data to humans in this study showed doses to normal organs lower than those reported by Iznaga-Escobar et al. [47] for 99mTc-h-R3. It is necessary to mention that, in the extrapolation of the animal data obtained in this study to humans, cross-reactivity of nimotuzumab is not taken into account. In this respect, locoregional anti-EGFR RIT might be a more promising option. Biodistribution and internal dosimetry, in patients with malignant gliomas, after locoregional administration of 188Re-nimotuzumab were studied [48]. In this study, the normal organs which received the highest absorbed doses, in decreasing order, were the kidneys, liver and urinary bladder. Even when mean absorbed dose in tumor regions was higher for patients treated with 555 MBq, adverse events were observed. Nevertheless, locoregional single dose of 188Re-nimotuzumab of 370 MBq was safely used in the routine treatment of patients [16], [48]. The demonstrated necessity of high EGFR expression for stable binding of nimotuzumab to the receptor suggests that nimotuzumab would preferentially target tissues overexpressing the target antigen while minimizing toxicity [9], [49], [50], [51]. This fact led us to think that, due to the nuclear properties of 177Lu, the tumor-targeting properties and the stability of 177Lu-nimotuzumab, the use of this radioimmunoconjugate could improve the therapy outcome and the absorbed dose in normal tissue.

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5. Conclusions 

According to the results presented above, 177Lu-DOTA-h-R3 could be obtained with high specific activity without significant loss of immunoreactivity and it specifically accumulated in EGFR overexpressing tumors. Consequently, 177Lu-DOTA-h-R3 seems to have a potential for further evaluations as a radiopharmaceutical for RIT of EGFR overexpressing tumors. The results presented in this report could be of use for future preclinical and clinical studies using this radioimmunoconjugate. Further optimization of the radioimmunoconjugate, such as reduction of its size in order to reduce even more the uptake in normal tissues and the use of 90Y for RIT of larger-size tumors, is ongoing.

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Acknowledgments 

We gratefully acknowledge Dr. Mark Harfensteller and Isotope Technologies Garching (ITG, Munich, Germany), for providing the n.c.a.177Lu. The authors would like to thank Ing. Aliona Frai for her support in the animal study. We thank Lenka Maresova and Ludmila Jandova from the Department of Radiopharmacy, Institute of Nuclear Research, Husinec-Rez, Czech Republic, for outstanding support during the biodistribution studies.

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☆ This research was supported by EUREKA Grant No. E08018 and Project NPVII 2B06165 from the Czech Ministry of Education, Youth and Sports.

☆☆ Conflicts of interest statement: The authors declare that they have no conflicts of interest.

PII: S0969-8051(11)00154-5

doi:10.1016/j.nucmedbio.2011.07.001

Nuclear Medicine and Biology
Volume 39, Issue 1 , Pages 3-13, January 2012

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