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Year : 2014  |  Volume : 10  |  Issue : 1  |  Page : 112-120

Preclinical evaluation of [ 111 In]-DOTA-trastuzumab for clinical trials

Radiopharmacy Research Group, Radiation Application Research School, Nuclear Science and Technology Research Institute, 11365-3486, Tehran, Iran

Date of Web Publication23-Apr-2014

Correspondence Address:
Amir Reza Jalilian
Nuclear Science Research School, Nuclear Science and Technology Research Institute, 11365-3486, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.131434

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 > Abstract 

Context: Herceptin and its fragments have been radiolabeled and used in the imaging of human epidermal growth factor receptor 2 (HER2)/neu-positive tumors and development of diagnostic kits is of great importance in radiopharmacy.
Aims: In this study, 111 In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-trastuzumab ( 111 In-DOTA-trastuzumab) was successively prepared and evaluated for ultimate use in the HER2 antigen imaging in oncology.
Settings and Design: The conjugate was prepared, labeled and evaluated using in vitro (radioimmunoassay [RIA], enzyme-linked immunosorbent assay (ELISA), stability, binding, internalization)/in vivo (bio-distribution, single-photon emission computed tomography [SPECT]) experiments.
Materials and Methods: 111 In-DOTA-trastuzumab was prepared followed by determination of radiochemical purity (RCP), integrity of protein, immunoreactivity of radiolabeled antibody with HER2/neu antigen (by SkBr3 cell line binding and RIA methods) were determined followed by stability tests, internalization studies and the tissue bio-distribution determination in wild-type rats as well as SPECT imaging in SkBr3-bearing mice.
Statistical Analysis Used: All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05.
Results: 111 In-DOTA-trastuzumab was prepared (RCP >95 ± 0.5%, S.A. 5.3 μCi/μg) with the average number of chelators per antibody of 6:1 showing significant immune-reactivity retention using ELISA. In vitro stability was >90% in phosphate buffered saline and 80 ± 0.5% in serum over 48 h. Cell binding was significant (>0.79). In vitro internalization reached up to %12-13 in 10 h. Significant tumor uptake was observed.
Conclusions: In vitro and in vivo/SPECT imaging in SkBr3-bearing mice demonstrated that 111 In-DOTA-trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and follow-up of HER2 expression in oncology.

 > Abstract in Chinese 

目的:本研究中,铟111-1,4,7,10-轮环藤宁 -1,4,7,10-四乙酸酸曲妥珠单抗依次准备和评估最终用于肿瘤学中HER2抗原成像。
结果:铟111标记的曲妥珠单抗(RCP >95 ± 0.5%, S.A. 5.3 μCi/μg),每抗体平均螯合剂6:1有明显的免疫反应性(ELISA法)。48小时离体稳定性:在磷酸盐缓冲盐水中>90%,血清中80% ± 0.5%。细胞整合明显:(>0.79)。10小时离体内化率12%~13%。可观察到明显的肿瘤摄取。

Keywords: Bio-distribution, indium-111, production, SkBr3 cells, single-photon emission computed tomography, trastuzumab

How to cite this article:
Alirezapour B, Jalilian AR, Rajabifar S, Mirzaii M, Moradkhani S, Pouladi M, Aslani G. Preclinical evaluation of [ 111 In]-DOTA-trastuzumab for clinical trials. J Can Res Ther 2014;10:112-20

How to cite this URL:
Alirezapour B, Jalilian AR, Rajabifar S, Mirzaii M, Moradkhani S, Pouladi M, Aslani G. Preclinical evaluation of [ 111 In]-DOTA-trastuzumab for clinical trials. J Can Res Ther [serial online] 2014 [cited 2021 Jul 26];10:112-20. Available from: https://www.cancerjournal.net/text.asp?2014/10/1/112/131434

 > Introduction Top

111 Indium-111 (T1/2 = 67 h, major photopeak at 171 keV) is an ideal radionuclide in radio immuno scinti graphy studies due to suitable half-life compared to biological half-life of intact immunoglobulin G (IgG) molecules and have attracted considerable interest in the field of targeted diagnosis. Due to the superior bioconjugation of DOTA bi-functional ligands, N-succinimidyl-1, 4, 7, 10-tetra azacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA-NHS) was used as a bi-functional ligand. [1] Various radio labeled DOTA-immunoconjugates have been developed successfully recently. [2],[3],[4] In order to develop herceptin radio immunoconjugates for using in imaging studies, DOTA-trastuzumab was labeled by In-111 chloride for preliminary bio-distribution studies in rats and tumor-bearing mice.

 > Materials and Methods Top

NHS-DOTA was purchased from Macrocycles (NJ, USA). trastuzumab (Herceptin) was a pharmaceutical sample purchased from Roche Co. Radio-chromatography was performed by using a Bioscan AR-2000 radio thin layer chromatography (TLC) scanner instrument (Bioscan, Paris, France). A high purity germanium (HPGe) detector coupled with a Canberra™ (model GC1020-7500SL) multichannel analyzer and a dose calibrator ISOMED 1010 (Dresden, Germany) were used for counting distributed activity in rat organs. Calculations were based on the 171 keV peak for 111 In. All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05. Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, 2 nd edition.

Production and quality control of 111 In-InCl 3 solution

Cadmium electroplating, over a 50 μm gold-coated copper backing, was performed according to the previously reported method. [5] Indium-111 chloride was prepared by 22 MeV proton bombardment of the enriched cadmium-112 target at a 30 MeV cyclotron, with a current of 100 μA for 48 min (80 μAh). After the dissolution of the irradiated target by conc. HBr, the solution was passed through a cation exchange dowex 50 × 8 resin, pre-conditioned by 25 ml of conc. HBr. The resin was then washed by HBr conc. solution (50 ml). In order to remove the undesired impurities of Cd and Cu, the resin was totally washed with DDH 2 O. Indium-111 was eluted with 1 N HCl (25 ml) as 111 InCl 3 for labeling use. The radiochemical yield was 76 ± 2% and isolated yield of the radionuclide was 81 ± 1%.

Control of radionuclide purity

Gamma spectroscopy of the final sample was carried out counting in a HPGe detector coupled to a Canberra multi-channel analyzer for 1000 s.

Chemical purity control

This step was carried out to ensure that the amounts of cadmium, indium and copper ions resulting from the target material and backing in the final product are acceptable regarding internationally accepted limits. Chemical purity was checked by differential-pulsed anodic stripping polarography. The detection limit of our system was 0.01 ppm for cadmium, indium and copper ions (no carrier added, with specific activity of >1.55 GBq/μg indium, >45-50 mCi/μg indium at the time of calibration, <0.08% In-114 m at calibration time <0.16% In-114 m at expiration time). The pH of the solution is about 1.4.

Conjugation of DOTA-NHS with the trastuzumab

Trastuzumab (Roche) is formulated as a lyophilized powder and each vial is designed to deliver 150 mg trastuzumab. The finished product also includes 3.36 mg L-histidine HCl, 2.16 mg L-histidine, 136.2 mg trehalose dihydrate and 0.6 mg polysorbate-20. In the first step, Vivaspin-2 filters (30 kDa; Sartorius AG; 2 × 10 min at 2.684 g) were used for all ultrafiltration purification steps. In short, trastuzumab was diluted with (1 ml) 0.2 M Na 2 CO 3 (pH 9.2) buffer solution. The antibody concentration was measured using a BioPhotometer (Eppendorf) at OD = 280 nm (3.3 mg/ml). The solution was passed through a Vivaspin 2 (20 min, 2.684 g) 2 times in order to remove the impurities. The antibody then can be removed from the upper part of the filter using the bicarbonate buffer (0.2 M Na 2 CO 3 , pH 9.2). The final concentration was re-measured using biophotometric assay as well as structure integrity test using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then DOTA-NHS (1.3 mg, excess 80 times) dissolved in bicarbonate buffer (400 μl, 0.2 M, pH 9.2) was added to the purified antibody solution (3.3 mg/ml) in a borosilicate vial and mixed gently for 20 times by pipetting. The mixture was gently shaken and incubated at room temperature for 12 h. The mixture was then transferred on a Vivaspin 2 cut-off filter (30 kDa) and centrifuged at 2.684 g for 15 min. In order to terminate the conjugation step and provide the suitable radiolabeling pH, the upper filter fraction is washed through using ammonium acetate buffer (0.2 M, pH 5.5) 3 times in order to remove the excess of DOTA-NHS. In this stage to the upper fraction is added the ammonium acetate buffer (1 ml) to dissolve the immunoconjugate followed by pipetting for 10-20 times. The filter is then centrifuged upside-down at 2.684 g for 5 min. The antibody concentration was measured using a biophotometer (Eppendorf) at OD = 280 nm.

Determination of the average chelate: Antibody ratio

The spectrophotometric method for quantization of micromolar concentrations of bifunctional DOTA-NHS ligand in DOTA-monoclonal antibody (mAb) conjugates performed according to the reported method. [6] Briefly, the optical density of arsenazo yttrium(III) complex (2:1, 1 ml), prepared in 5.0 μM AAIII, 1.6 μM Y(III), 0.15 M sodium acetate buffer, pH 4.0, was measured at 652 nm. A standard curve was then plotted by the addition of multiple (×8) 15 μl DOTA-NHS standard solutions (DOTA-NHS dissolved in 0.15 M sodium acetate buffer, pH 4.00), to the above mixture. In the second step, the optical density of 1:2 yttrium(III) complex of arsenazo (1 ml) was measured at 652 nm in the presence of conjugation product in order to determine DOTA-antibody attachments.

Radiolabeling of the antibody conjugate with 111 In

In general, 37 MBq of 111 In-InCl 3 (in 0.2M HCl) was added to a conical vial and dried under a flow of nitrogen. To the indium-containing vial were added acetate buffer (700 μl, pH. 5.5) and the vial vortexed for 10 min. The conjugate containing fraction (500 μg) in acetate buffer with the measured protein content was added to the vial and mixed gently for 5 min using pipetting (×10-20). The mixture is then incubated at 40°C for 90 min followed by testing the radiochemical purity (RCP) by instant thin layer chromatography (ITLC) using a radio TLC scanner (Whatman No. 1, 1 mM DTPA). Finally ethylene di-amine tetra acetic acid (ETDA) solution (10 μl, 10 mM) is added to the labeling mixture and incubated for 10 min in order to scavenge the unlabeled In cation. The mixture is then passed through the disposable PD10 De-salting column (Amersham) in order to further increase the RCP of the mixture. The final solution is then passed through a 0.22 μm on the biological filter for animal studies.


The radioimmunoconjugate was analyzed for integrity by SDS-PAGE. The radiolabeled mAb was evaluated with and without reduction by 2-mercaptoethanol. Approximately, 200,000 cpm of each preparation was applied per lane and the 4-20% polyacrylamide were run according to the method of Laemmli. [7]

Stability testing of the radiolabeled compound in the final formulation

Stability of 111 In-DOTA-trastuzumab in phosphate buffered saline (PBS) was determined by storing the final solution at 4°C for 14 days (almost 5 times of physical half-life) and performing frequent ITLC analysis to determine RCP. The stability of the conjugated DOTA-trastuzumab stored at − 20°C for more than 1 month was also investigated. ITLC analysis of the conjugated product was performed to monitor for degradation products or other impurities. After subsequent 111 In-labeling of the stored conjugated product, both labeling efficiency and RCP were determined. ITLC was performed by sampling the radiolabeled complex on a Whatman paper followed by developing in 1 mM DTPA aqueous solution.

Measurement of transchelation to EDTA

A volume of 0.1 ml aliquot of the 111 In-labeled product was mixed with 0.1 ml of 3 mM EDTA (pH = 7.4) and allowed to incubate for 30 min at room temperature. The entire solution was used for gel column scanning. For a control, another 0.1 ml aliquot was mixed with 0.1 ml of distilled water and also allowed to incubate for 30 min at room temperature prior to gel column scanning. The protein labeling percentage was calculated using the following equation according to the reported method. [8]

Stability testing of the radiolabeled compound in the presence of human serum

Radiolabel stability was assessed by size exclusion chromatography on a Sepharose column (1 cm × 30 cm). The column was equilibrated with PBS and eluted at a flow rate of 0.5 ml/min at room temperature; 1 ml fractions were collected.

Reactivity of trastuzumab towards human epidermal growth factor receptor 2 /neu by the enzyme-linked immunosorbent assay

The native cancerous antigen (HER2/neu) was purified from ascetic fluid of a patient with aggressive small-cell lung carcinoma and metastasis to the peritoneum by an antibody-sepharose affinity (immunoaffinity) column, which is explained elsewhere. [9] Purified HER2/neu was coated onto the wells of microtiter plates (0.6 μg/well) at 37°C overnight. Bovine serum albumin was used as a negative control. The contents of the wells were emptied, washed and blocked with a 4% solution of skimmed milk in PBS (10 mM, pH 7.2) for 1 h at 37°C. At the end of incubation time, wells were washed and added with dilutions of trastuzumab and was incubated at 37°C for 2 h. The content of the wells were emptied, wells were washed added with proper dilution of rabbit anti-mouse conjugated to horseradish peroxidase and incubated at 37°C for 1 h. Finally wells were washed, added with 50 μl of substrate tetramethylbenzidine (Roche Diagnostics) and incubated for 10 min. The reaction was stopped by adding 50 μl of 1 M H 2 SO 4 per well. The plates were read at 450 nm in the ELISA autoreader.

Reactivity of radiolabeled trastuzumab towards HER2/neu antigen by radioimmunoassay

After labeling of mAb with 111 In, the reactivity of radiolabeled mAb trastuzumab toward HER2/neu was considered by RIA. Purified HER2/neu antigen was coated onto the wells of microtiter plates (0.2 μg/well) at 37°C overnight; same concentration of bovine serum albumin (BSA) was used as a negative control. The contents of the wells were emptied, washed and blocked with a 4% solution of skim milk for 1 h at 37°C. Finally, wells were washed and added with 30000 cpm of 111 In-DOTA-trastuzumab and was incubated at 37°C for 2 h. The content of the wells were emptied, wells were washed and the radioactivity of wells were measured by γ-counter.

Cell culture

A HER2/neu positive cell line, SkBr3, was obtained commercially and was grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 μg/ml streptomycin and 100 IU/ml penicillin.

Immunoreactivity of radiolabeled trastuzumab towards SkBr3 cell line

Immunoreactivity of the radiolabeled mAb trastuzumab ( 111 In-DOTA-trastuzumab) towards SkBr3 cell line was determined using a double-inverse of the binding data which may be considered a modification of the Lineweaver-Burk plot as follows. Different numbers (5 × 10 6 , 2.5 × 10 6 , 1.25 × 10 6 , 0.62 × 10 6 , 0.31 × 10 6 and 0.15 × 10 6 ) of SkBr3 cell was located in tubes; 30000 cpm of the radioimmunoconjugate ( 111 In-DOTA-trastuzumab) was added and incubated for 3 h at 4°C. After incubation, the radioactivity of tubes was read by γ-counter. Then tubes were centrifuged in 3000 g and the radioactivities of cells (the pellet, not the supernatant) were read by γ-counter. The immunoreactivity was determined using a Lineweaver-Burk plot and data were analyzed by the Lindmo method. [10]

Internalization assays

Internalization studies were performed using acid dissociation of 111 In-DOTA-trastuzumab. [11] SkBr3 cells were harvested and resuspended at 1 × 10 5 cells/200 μl in medium and cultured in 24 well plates. After 24 h, 30000 cpm of 111 In-DOTA-trastuzumab were added to each well and incubated at 37°C. At various times (4, 8, 12, 16, 24, 30 and 36 h), RPMI medium in wells were removed and the cells, half of wells, were then exposed to 1 ml solution of 0.1 M NaOH and half of wells were exposed to 1 ml solution of 0.1 M acetic acid pH 4.0. After 15 min, the radioactivity content of the supernatant was measured by γ-counter. NaOH solution destroyed the whole SkBr3 cells and this solution was considered as total activity (internalized, bound and dissociated 111 In-DOTA-trastuzumab). Acetic acid was used to remove the interaction between 111 In-DOTA-trastuzumab and HER2/neu at the SkBr3 cell surface and the radioactivity of the solution was considered as bound and dissociated 111 In-DOTA-trastuzumab. Differences between the two activities were considered as internalized 111 In-DOTA-trastuzumab fraction.

Biodistribution of 111 In-DOTA-trastuzumab in normal Sprague-Dawley rats

For biodistribution studies, 111 In-DOTA-trastuzumab and 111 InCl 3 were administered to normal Sprague Dawley rats separately. A volume (50-100 μl) of final radioactive solution containing 90 ± 5.5 μCi radioactivity and 25 ± 5 μg of trastuzumab was injected intravenously to rats through their tail vein. The total amount of radioactivity injected into each rat was measured by counting the 1 ml syringe before and after injection in a dose calibrator with a fixed geometry. Bio-distribution studies were performed using groups of 3 rats sacrificed at 24, 48, 72 and 96 h after injection of each radiolabeled trastuzumab. At each time point, rats were sacrificed using CO 2 gas and normal organs (lungs, stomach, small and large intestine, spleen, blood, heart, muscle, brain, kidneys, bone, skin and liver) were harvested. Furthermore urine and feces samples were collected and counted. Organs were weighed and gamma counted with a HPGe detector counting the area under the curve of the 171 keV peak. The percentage of injected dose of radioisotope per gram (% ID/g) organ was calculated (after correcting for radioactive decay using an aliquot of the injected at each time point). Mean values and standard errors for each tissue and time point were plotted.

Development of SkBr3-Xenograft mice for imaging studies

The biodistribution studies were performed when the tumor volume reached 7-8 mm 3 . All the animal experiments were approved by the local Animal Care Committee.

Imaging of 111 In-DOTA-trastuzumab in xenograft-bearing mice

Images were taken 18, 48 and 72 h after administration of the radiopharmaceutical by a dual-head single-photon emission computed tomography (SPECT) system (SMV-GE, DST-XL). The distance of animal to high energy septa was 12 cm. Images were taken from both normal and tumor bearing mice. The useful field of view was 3.5 mm.

 > Results Top

Radionuclide production

Indium-111, in form of InCl 3 , was prepared by 22 MeV proton bombardment of the enriched 112 Cd target at Cyclone-30 on a regular basis. The target was bombarded with a current intensity of 170 μA and a charge of 1400 μAh. The chemical separation process was based on a no-carrier-added method. (No carrier added, with specific activity of >1.55 GBq/μg indium, >45-50 mCi/μg indium at the time of calibration, <0.08% In-114 m at calibration time <0.16% In-114 m at expiration time). The pH of the solution is about 1.4.

Radiochemical separation was performed by a two-step ion exchange chromatography method with a yield of higher than 95%. Quality control of the product was performed in two steps. Radionuclidic control showed the presence of 171 and 245 keV gamma energies, all originating from 111 In and showed a radionuclidic purity higher than 99% (E.O.S.). The concentrations of cadmium (from target material) and copper (from target support) were determined using polarography and shown to be below the internationally accepted levels, i.e. 0.1 ppm for Cd and Cu. [12],[13]

The radioisotope was dissolved in acidic media as a starting sample and was further diluted and evaporated for obtaining the desired pH and volume followed by sterile filtering. The RCP of the 111 In solution was checked in two solvent systems, in 1 mM DTPA, free In 3 + cation is converted to more lipophilic 111 In-DTPA form and migrates to higher Rf (0.8) while any small radioactive fraction remaining at the origin could be related to other In ionic species, not forming In-DTPA complex, such as InCl 4 -, etc., and/or colloids (not observed).

On the other hand, 10% ammonium acetate: Methanol mixture was also used to determine RCP. The fast eluting species was possibly the ionic In-111 cations other than In 3+ (not observed) and the remaining fraction at Rf . 0 was a possible mixture of In 3 + and/or colloids [Table 1] and [Figure 1].
Figure 1: Instant thin layer chromatography chromatograms of 111InCl3 solution in DTPA solution (pH. 5) (left) and 10% ammonium acetate:methanol (1:1) solution (right) using Whatman no. 2

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Table 1: The Rf values of chemical impurities and DOTA-conjugates

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The difference in values of impurity in two solvent systems is possibly due to the presence of colloidal impurity in the sample, considering the purities of both chromatograms indium cation is the only radiochemical species present.

Conjugation of trastuzumab with DOTA-NHS and radiolabeling of trastuzumab with 111 In

In order to overcome the effect of excipients and producing appropriate acidity for conjugation step the pharmaceutical sample was purified by ultra-filtration using the cut-off filters followed by determination of the antibody concentration using spectrophotometry. In order to improve the conjugation step alkaline pH is necessary, thus bicarbonate buffer was used to reconstitute the antibody. In each step, biophotometric assay as well as structure integrity test using SDS-PAGE performed in order to guarantee the quantity and the quality of the antibody.

The use of polymer tubes and other synthetic materials in the conjugation and labeling step interfered with the conjugation reaction, while borosilicate vials were the appropriate vessels. In order to remove the leftover of DOTA-NHS in the reaction and concentrate the antibody, the cut-off filter was used once more (30 kDa).

At this stage, a pH 5.0 buffer was used to recover the antibody in order to terminate the conjugation step and provide the suitable radiolabeling pH and for final fraction the quantity of the antibody was measured at OD = 280 nm.

In order to estimate the number of DOTA prosthetic group on each antibody molecule, the arsenazo yttrium complex (Y[AAIII] 2 ) method was used. The absorbance of Y(AAIII) 2 at 652 nm decreases upon the addition of DOTA-trastuzumab while the corresponding absorbance of AAIII at 538 nm increases. The Y(AAIII) 2 and arsenazo III are the only absorbing species in solution; neither DOTA-trastuzumab nor its Y(III) complex have any absorbance in this wavelength region. The isosbestic point observed at 585 nm is consistent with only two absorbing species for reaction. The data demonstrated the DOTA: Antibody ratio of 6:1 [Figure 2].
Figure 2: Instant thin layer chromatography chromatograms of 111In- DOTA-trastuzumab in DTPA solution (pH. 5) before (right) and after (left) cut-off purification using Whatman no. 2

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Kukis et al. observed that coupling of too many ligands to antibody leads to impairment of immunoreactivity as described for the Lym-1 antibody. [14] Zimmermann et al. also found that unfavorable biodistribution were a more sensitive marker for over-substitution of the mAb than in vitro immunoreactivity. Therefore, substituting 18 ligands per antibody molecule lead to an about 25% lower in vitro immunoreactivity, in vivo tumor uptake was decreased by 80%. At the same time accumulation of radioactivity in the liver was increased about 40%. A c/a ratio up to 11 seemed to be the limit for substituting mAbs without damage to immunoreactivity and effects on biodistribution. [15] Thus, in this study, we optimized conjugation procedure to obtain a chelate-to-mAb molar ratio less than 11 (6:1 for instance), in order to minimize any immunoreactivity loss and unwanted liver uptake.

The conjugated DOTA-trastuzumab fraction was mixed with 111 InCl 3 solution at the appropriate acidity in acetate buffer at 40°C for 60 min followed by testing the RCP by ITLC. By the addition of ETDA to the solution for 5 min, the radiolabeling reaction was terminated as well as production of In-EDTA complex, which can be better removed by size exclusion method. The EDTA scavenging time was shown to be critical in order to maintain the appropriate RCP, increased EDTA incubation time led to the decomposition of radioimmunoconjugate and the reduction of RCP.

RCP determination

ITLC using various mobile and stationary phases was performed in order to ensure the existence of only the desired radiolabeled antibody. Two different solvent systems with two stationary phases were tested. In all tests, radiolabeled antibody stayed at the origin while other species migrated to other R f s depending on the mobile phase used. The R f s of the possible occurring chemical species in chromatography of the reaction steps are summarized in [Table 1] (n = 5).

As shown in the [Table 1], for 111 In 3 + detection, the best eluent systems is chromatographic protocol 2 resulting in Rf 0.9. For 111 In-DOTA detection, system 1 can be used (Rf 0.3). 111 In-DOTA-trastuzumab remains at the origin in all systems used due to the size and charge of the protein (≈150,000 Da). The mixture can be further purified using de-salting column to reach at least 95 ± 0.5% RCP. [Figure 3], demonstrates the ITLC chromatograms of 111 In-DOTA-trastuzumab solution before and after filter purification. The DOTA-trastuzumab conjugate was labeled with 111 In at specific activity as high as 5.3 μCi/μg and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity.
Figure 3: The absorption of arsenazo (III) and yttrium (III) complex against the concentration of DOTA-NHS (error bars represent standard error of the mean)

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Protein integrity test using SDS-PAGE

In order to demonstrate the integrity of the protein after residulation and radiolabeling gel electrophoresis was performed on the SDS-PAGE gels using 16% bisacrylamide gel. The loaded samples were trastuzumab commercial sample, DOTA-trastuzumab and radiolabeled antibody samples 4 weeks after the experiment while kept in the fridge. Gels were stained with Coomassie Blue. The samples were showed to have a similar pattern of migration in the gel electrophoresis reported previously [16] [Figure 4].
Figure 4: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified trastuzumab (Lane 2), conjugated DOTA-trastuzumab (Lane 3) and 111In-DOTA-trastuzumab (Lane 4) monoclonal antibodies. Lanes 1 and 5, molecular mass protein markers

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Stability of radiolabeled protein in the presence of human serum in vitro

[Figure 5] presents the in vitro stability of radiolabeled mAb by using size exclusion chromatography using G-25 Sephadex at 37°C after 0, 6, 12, 24, 48, 72 and 96 h of incubation (in the buffer and human serum). The results showed that after incubation of 111 In-DOTA-trastuzumab (300 μCi) with freshly prepared human serum at 37°C for up to 48 h, more than 82% of total radioactivity eluted in the same position as 111 In-DOTA-trastuzumab. Cole et al. reported that decreases of stability in human serum resulted from mild transchelation of 111 In to serum components like albumin, [17] while Cooper et al. reported the high stability of macrocyclic immunoconjugates such as DOTA-mAb, DO3A-mAb and NOTA-mAb in serum over 48 h [18] which is in agreement with our data. More than 94% of total radioactivity was stable in the final formulation after 48 h, this value is higher than the value for serum due to lack of transchelation activity. The results demonstrated the stability of the radiolabeled antibody and its suitability for in vivo imaging.
Figure 5: Stability of 111In-DOTA-trastuzumab in phosphate buffered saline buffer and human serum up to 96 h

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Reactivity of mAb trastuzumab toward HER2/neu by ELISA

[Figure 6] shows the reactivity of trastuzumab toward the native cancerous HER2/neu purified from metastasis ascites fluid, following the ELISA procedure represented here. Trastuzumab showed high immunoreactivity toward HER2/neu protein. The highest optical density difference from that of the non-specific (reactivity of anti HER2/neu with corresponding concentration of BSA) was found at an antibody concentration of 10 μg. From these data, the antibody was suitable for radiopharmaceutical studies. It was previously found that IgG antibodies of reasonably high affinity (1 × 10 8 /M) are optimal for both imaging and therapy. [19] For example, the anti MUC1 monoclonal antibodies, BC2 and hCTMO1, is reported to have a high affinity toward MUC1 (1.36 × 10 7 /M and 2.6 × 10 7 /M respectively).
Figure 6: Reactivity of purified trastuzumab (2.5, 5 and 10 ìg/well) towards human epidermal growth factor receptor 2 (HER2)/neu (0.6 ìg/well) and bovine serum albumin (0.6 ìg/well) as control and HER2 as specific antigen for trastuzumab at 450 nm OD (different concentrations)

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The two mentioned antibodies are in a clinical trial for breast cancer therapy. [20] In this way, trastuzumab, which exhibited high affinity (5 × 10 9 /M) toward HER2/neu antigen and HER2/neu positive cell line such as SkBr3 cell line may be suitable for in vivo tumor targeting, imaging and therapy. [21]

Reactivity of radiolabeled trastuzumab towards HER2/neu antigen by RIA

Reactivity of radiolabeled mAb trastuzumab towards the HER2/neu antigen (0.2 μg/well) and BSA (0.2 μg/well) is shown in [Figure 7].
Figure 7: Reactivity of radiolabeled trastuzumab (5 μg/well) towards human epidermal growth factor receptor 2 (HER2)/neu (0.2 μg/well) and bovine serum albumin (0.2 ìg/well) as control and HER2 as specific antigen for trastuzumab

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All assays performed in duplicate. Error bars represent standard error of the mean. The 111 In-DOTA-trastuzumab showed high immunoreactivity toward HER2/neu antigen. This result also showed that the chemical binding of 111 In to mAb had no adverse effects on the immunoreactivity of antibody towards HER2/neu antigen.

Immunoreactivity of radiolabeled trastuzumab towards SkBr3 cell line and internalization assays

Zimmermann et al. reported that moderate increase of chelator to mAb ratio leads to a slight decrease in immunoreactivity. Lewis et al. also reported this slight decrease in immunoreactivity is likely due to non-specific conjugation of chelators to mAb, which possibly attaches a chelator in the region of the antigen-binding site. [22] The Immunoreactivity of the radiolabeled trastuzumab ( 111 In-DOTA-trastuzumab) towards SkBr3 cell line was determined under conditions of antigen excess in HER2/neu antigen-expressing SkBr3 human breast carcinoma cells by using a Lineweaver-Burk plot. Under these conditions, the immunoreactivity of radioimmunoconjugate was found to be 0.79 which is suitable for further imaging studies in animal models and possibly human trials. It seems that exact optimization of c/a ratio has led to such suitable immunoreactivity. Thus, conjugation and complexation procedures did not affect the affinity of mAb toward its antigen in this work [Figure 8].
Figure 8: Immunoreactivity of 111In-DOTA-trastuzumab using SkBr3 cell-binding assay (n = 5)

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Internalization assays were performed with the HER2/neu expressing SkBr3 cell line to determine internalized activity following in vitro incubation at 37°C as a function of time. As shown in [Figure 9], internalized activity for radiolabeled trastuzumab reached to the maximum significantly in 8-12 h incubation at 37°C. After 36 h the internalized activity was decreased to 3% in 36 h. [23] The decline in cell-associated activity can be explained by proteolytic degradation of the v in lysosomes, leading to metal transchelation to the internal metal containing biomolecules [Figure 9].
Figure 9: Internalization assay of 111In-DOTA-trastuzumab in different time on SkBr3 cell line (n = 5)

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111 InCl 3 biodistribution in wild-type rat tissues

For better comparison, biodistribution study was performed for free In 3+ . Indium cation almost mimics the ferric cation behavior and is rapidly removed from the circulation and is accumulated in the liver, which is comparable to many other radio-metals accumulation. About %10-11 of the activity accumulates in the liver after 24 h. Furthermore, a major fraction is excreted through the urine as a water-soluble cation. The transferrin-metal complex uptake and final liver delivery looks the possible route of accumulation as reported previously. Spleen and lung also demonstrate significant accumulations [Figure 10].
Figure 10: Biodistribution of 111In-InCl3 (1.85 MBq, 50 μCi) in wild-type rats 2, 4 and 24 h after intravenous injection via tail vein (n = 3)

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111 In-DOTA-trastuzumab biodistribution in normal rat tissues

Kokai et al. showed that during late gestation (Embryonic day 18) and during the early postnatal period (PND-1 and PND-5), two types of tissue expressed HER2/neu antigen (p185) in normal rat. Strong immunoreactivity was detected on epithelial cells of the intestinal villi, the skin, pulmonary bronchioles and proximal renal tubules. In adult animals, immunoreactive p185 was limited to epithelial tissues, including the basal layer of skin, the mucosal epithelium of intestine, bronchiolar epithelium of lung and proximal tubular epithelium of kidney. [24]

As shown in [Figure 11], bio-distribution studies in adult normal Sprague-Dawley rats after injection of 111 In-DOTA-trastuzumab demonstrated that uptake in intestine, skin and lung organs significantly increased during the study time also high uptake in kidney organ during the time study were observed, which is due to the presence of HER2/neu antigens in mentioned tissues also leading to high colon and feces activity content, which is in agreement with the findings of Kokai and co-workers. This has been already shown by other groups, working with 125 I-anti-HER2 probes. [25]
Figure 11: Percentage of injected dose per gram (% ID/g) of 111In- DOTA-trastuzumab in normal Sprague-Dawley rat tissues at 24, 48 and 72 h post-injection (n = 3)

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Due to rapid metal-scavenging property of ceruloplasmin and other metalloproteins in the serum and ultimate liver transfer of the metal, the radioindium cation is not observed in the circulation after 24 h (less than 1%). Although in case of radiolabeled mAb trastuzumab, the circulating content is still significant up to 72 h as observed for other radiolabeled antibodies. Lung, intestines, colon and skin like the expected HER2 neu expression profile demonstrated significant uptake at all-time intervals.

Imaging study in SkBr3-bearing mice

111 In-DOTA-bevacizumab imaging in the tumor-bearing mice showed a distinct accumulation of the radiotracer in the chest region all the time after injection. Distinct tumor uptake was observed at all-time intervals. However, the best tumor uptake was shown at 48 h post injection, leading to the best target: Non target ratio. This uptake is comparable to the biological half-life of the IGs esp. IgG [Figure 12].
Figure 12: Single-photon emission computed tomography images of 111In-DOTA-trastuzumab (1.85 MBq, 50 ìCi) in SkBr3-bearing mice model 18 h (A), 48 h (B) and 72 h (C) post injection; 1: Tumor sites, 2: Injection site

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 > Discussion Top

The HER2/neu antigen is a transmembrane receptor [26] over expressed in 25-30% of breast cancers. [27] The over expression has been implicated in the carcinogenesis of breast cancer and is an independent prognostic indicator of survival in patients. [28] Trastuzumab is a humanized IgG1 mAb recognizing an epitope in the extracellular domain of the receptor and is used for immunotherapy for HER2/neu-positive tumors. [29]

Breast cancer radioimmunoscintigraphy targeting HER2/neu expression has been proposed by different research groups and could allow direct assessment of the receptor status of primary and metastatic lesions suggesting the effectiveness of Herceptin therapy. Herceptin and its fragments have been radiolabeled and used in the imaging of HER2/neu-positive tumors using In-111, [30] Y-90, [31] Y-86, [32] Br-76 [33] and Zr-89. [34]

In-DOTA-trastuzumab was prepared with suitable RCP (>95 ± 0.5%) specific activity (΋5.3 μCi/μg). Significant immunoreactivity retention obtained (5 × 10 9 /M) using ELISA method. In vitro stability of the labeled product was determined up to 48 h (>90% in PBS and >80 ± 0.5% in human serum). The radioimmunoconjugate SkBr3 cell binding was significant (>0.79). Internalization studies was %12-13 after 10 h. RIA studies using radiolabeled antibody demonstrated significant specific antigen binding (7 times of BSA). The accumulation of the radiolabeled mAb in organs of wild type rats was consistent to HER2 distribution. SPECT imaging in SkBr3-bearing mice demonstrated highest tumor uptake in 48 h.

These data validate this radiopharmaceutical for further clinical testing. Therefore 111 In-DOTA-Trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and treatment studies and follow-up of HER2 expression in oncology.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]

  [Table 1]


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