|Year : 2013 | Volume
| Issue : 2 | Page : 199-204
Optimization of 90 Y-antiCD20 preparation for radioimmunotherapy
Nazila Gholipour1, Ariandokht Vakili2, Edalat Radfar2, Amir Reza Jalilian3, Ali Bahrami-Samani3, Simindokht Shirvani-Arani3, Mohammad Ghannadi-Maragheh3
1 Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Engineering and Technical, Science and Research Branch, Islamic Azad University, Tehran, 14778-93855, Iran
3 Radiopharmaceutical Research and Development Lab (RRDL), Nuclear Science and Technology Research Institute (NSTRI), Tehran, 14155-1339, Iran
|Date of Web Publication||13-Jun-2013|
Amir Reza Jalilian
Radiopharmaceutical Research and Development Lab (RRDL), Nuclear Science and Technology Research Institute (NSTRI), Tehran, 14155-1339
Source of Support: None, Conflict of Interest: None
Context: The advent of monoclonal antibodies such as Rituximab, in recent years, has brought about decisive progress in the treatment of aggressive and indolent non-Hodgkin's lymphoma.
Aims: A further tried and tested improvement to the unmodified antibody has been its coupling to the beta-emitters Y-90. The optimization of 90 Y-antiCD20 radioimmunoconjugate production and quality control methods for future clinical studies in the country was targeted in this work.
Materials and Methods: The antibody was labeled with 90 Y-yttrium chloride (185 MBq) after conjugation with freshly prepared ccDTPA. Y-90 chloride was obtained by thermal neutron flux (4 × 10 13 n/cm 2 /s) of a natural Y 2 O 3 sample, dissolved in acidic media. Radiolabeling was completed in 24 h by the addition of DTPA-Rituximab conjugate at room temperature.
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: Radiochemical purity of 96% was obtained by using ITLC method for the final radioimmunoconjugate (specific activity = 440-480 MBq/mg). The final isotonic 90 Y-Rituximab complex was checked by gel electrophoresis for protein integrity retention. Biodistribution studies in normal rats were carried out to determine the radioimmunoconjugate distribution up to 72 h.
Conclusion: The results showed that 90 Y-DTPA-Rituximab could be considered for further evaluation in animals and possibly in humans as a radiopharmaceutical for use in radioimmunotherapy against non-Hodgkin's lymphomas. Because of the importance of developing anti-lymphoma B agents in nuclear medicine for country use, an optimized radiolabeling method has been introduced.
Keywords: Quality control, Radiopharmaceutical, Rituximab, targetted therapy, yttrium-90
|How to cite this article:|
Gholipour N, Vakili A, Radfar E, Jalilian AR, Bahrami-Samani A, Shirvani-Arani S, Ghannadi-Maragheh M. Optimization of 90 Y-antiCD20 preparation for radioimmunotherapy. J Can Res Ther 2013;9:199-204
|How to cite this URL:|
Gholipour N, Vakili A, Radfar E, Jalilian AR, Bahrami-Samani A, Shirvani-Arani S, Ghannadi-Maragheh M. Optimization of 90 Y-antiCD20 preparation for radioimmunotherapy. J Can Res Ther [serial online] 2013 [cited 2019 Dec 16];9:199-204. Available from: http://www.cancerjournal.net/text.asp?2013/9/2/199/113350
| > Introduction|| |
Many studies have been focused on the development of anti-CD20 radioimmunoconjugates concerning antibody metabolism, CD20 antigen localization and therapeutic response determination. , Because of the technical and economic restrictions in obtaining these radioimmunoconjugates in many developing countries, developing anti-CD20 radioimmunoconjugates containing Y-90 is an essential requirement in many parts of the world; accordingly, we tried to develop quality control and labeling protocols using low-specific activity Y-90 obtained via (n,γ) reaction.
| > Materials and Methods|| |
90 Y was produced at the Tehran Research Reactor (TRR) using nat Y(n,γ) 90 Y nuclear reaction. Yttrium oxide with purity of >99.99% was obtained from Aldrich, Dorset, UK. Sephadex G-50, sodium acetate, phosphate buffer components and methanol were purchased from Sigma-Aldrich Chemical Co, Gillingham, UK. Rituximab was a pharmaceutical sample (Mabthera) purchased from Roche Co, CA, USA, and was used without further purification. Cyclic DTPA dianhydride was freshly synthesized in our lab based on the conventional method. 
Radio-chromatography was performed by counting of Whatman No. 2 using a thin layer chromatography scanner, Bioscan AR 2000, Paris, France. 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 carried out in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, 2 nd ed. All the rats were male NMRI purchased from Pasteur Institute of Iran, weighing 180-200 g. In each group/interval, five rats, before being sacrificed, were kept at routine day/night light program and were kept under common rodent diet pellets. The animal tissue samples were counted using a Wallac 1220 Quantulus, Perkin Elmer,Turku, Finland and ultra low-level liquid scintillation spectrometer, after sample preparation method instructed by the manufacturer.
A portion (0.2 mL) of Y 2 O 3 (10 mg) solution in 3% HNO 3 (1 mL) was poured in a quartz glass tube followed by sealing using flame and sent for irradiation to the TRR at a thermal neutron flux of 4 × 10 13 n/cm 2 /s for 5 days based on the reported procedure.  The product was delivered to the hot cell and then 0.1 M HCl (1 mL) was added. Final 90 YCl 3 solution was diluted to the appropriate volume with ultra pure water to produce a stock solution. The mixture was filtered through a 0.22-μm biological filter and sent for use in the radiolabeling step. The radionuclidic purity of the solution was tested for the presence of other radionuclides using beta scintillation for the detection of various interfering radionuclides. The radiochemical purity of 90 YCl 3 was checked using two solvent systems for ITLC (A: 10 mM DTPA pH.4 and B: ammonium acetate 10%:methanol [1:1]).
The chelator diethylenetriaminepenta-acetic acid dianhydride was conjugated with Rituximab using a small modification of the well-known cyclic anhydride method. Conjugation was performed at a 1:1 molar ratio. In brief, 20 μL of a 1 mg/ml suspension of DTPA anhydride in dry chloroform (Merck, Darmstadt, Germany) was pipetted under ultrasonication and transferred to a glass tube. The chloroform evaporated under a gentle stream of nitrogen. Commercially available Rituximab (5 mg, 0.5 mL, pH 7.5) was subsequently added and gently mixed at room temperature for 60 min. The conjugation mixture was then passed through a Sephadex G-50 column (2 cm × 15 cm, 2 g in 50 mL of Milli-Q® water) separately and 1 mL fractions were collected and checked for the presence of protein using UV absorbance at 280 nm or visible folin-phenol colorimetric assay. The fractions containing the highest concentration of the immunoconjugate were chosen and kept at 4°C, and for radiolabeling.
The antibody conjugate was labeled using an optimization protocol according to the literature.  Typically, a solution of 90 Y-chloride in 0.1 M HCl (400-600 MBq) was added to a conical vial and dried under a flow of nitrogen. To the above vial, acetate buffer (200 μL, pH 4.8) was added and the vial was vortexed for 30s. The protein-containing fraction with the maximum protein content was added in 1 mL of phosphate buffer (0.1 M, pH 8) to the vial and mixed gently for 30s. The resulting solution was incubated at 25°C for 1 h. Following incubation, ITLC and HPLC were performed to determine the radiochemical purity and, in case of low radiochemical purity, the radiolabeled antibody conjugate was purified from free 90 Y by size exclusion chromatography on a Sephadex G-50 column (15-20 mL bed volume) and eluted with PBS. Fractions (1 mL) were collected and the radioactivity of each fraction was measured by the recently calibrated radioisotope dose calibrator (CRC-7; Capintec Instruments, Ramsey, NJ, USA). The protein presence in each fraction was determined using a fast protein assay method by mixing freshly prepared Folin-Colciteau® reagent (5 μL prepared by mixing 25 μL of fresh CuTartrate solution) and 10 μL of the eluted fractions. The fractions containing the proteins (visible blue color by naked eye) with the maximum radioactivity were tested for purity by ITLC using a radio-TLC scanner. Control labeling experiments were also performed using 90 YCl 3 and DTPA with 90 YCl 3 .
For control experiments, 90 Y-DTPA was prepared for R f and retention time studies in chromatographic methods. For preparation, 37 MBq of 90 YCl 3 (in 0.2 M HCl) was added to a conical vial and dried under a flow of nitrogen. To the Y-containing vial, phosphate buffer (0.1 M, 200 μL, pH 8) was added and the vial spun vortex-like for 30 s (final pH 7.5). A solution of DTPA (2.5 mM) in 300 μL of phosphate buffer (0.1 M, pH 8) was added to the first vial and mixed gently for 30 s. The resulting solution was incubated at 25°C for 1 h.
From the final product, 5 μL was applied to a Whatman No. 2 paper strip followed by developing in 10 mM DTPA (pH 3). Radioactivity was determined by a RTLC scanner.
The radioimmunoconjugate was analyzed for integrity by SDS-polyacrylamide gel electrophoresis (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 then 4-20% polyacrylamide were run according to the method of Laemmli. 
Stability of 90 Y-DTPA-Rituximab in PBS was determined by storing the final solution at 4 °C for 2 days and performing frequent ITLC analysis to determine radiochemical purity. The stability of the conjugated DTPA-Rituximab stored at -20 °C for more than 3 months was also investigated. ITLC analysis of the conjugated product was performed to monitor degradation products or other impurities. After subsequent 90 Y-labeling of the stored conjugated product, both labeling efficiency and radiochemical purity were determined.
For stability testing of the radiolabeled compound in the presence of human serum, the radiolabeled antibody (370 MBq) was incubated with freshly prepared human serum from a healthy male volunteer (three-times of the volume) and the mixture was incubated at 37°C in a water bath with gentle stirring. Portions of the mixture content were 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.
To determine its biodistribution, 90 Y-DTPA-Rituximab was administered to normal rats. A volume (50-100 μL, 0.1-0.2 mg/L of the antibody determined by UV spectrophotometric method) of final 90 Y-DTPA-Rituximab solution containing 180 ± 5 mCi radioactivity was injected intravenously to rats through their tail vein. The animals were sacrificed at the exact time intervals (2, 24, 48 and 72 h), and the specific activity of different organs was calculated as percentage of injected dose per gram using a beta-scintillator detector.
| > Results|| |
The radionuclide was prepared in a research reactor according to the regular methods within the range of specific activity, 12-14 mCi/mg, for radiolabeling use, after counting the samples on a beta scintillator detector for 5 min [Figure 1]. 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.
|Figure 1: Beta spectrum for Y-90 prepared by neutron irradiation of natural Y sample|
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The conjugated DTPA-Rituximab fractions containing the maximum protein content were labeled with 90 YCl 3 solution. The samples were checked by ITLC to find the best time scale for labeling. After 1 h, the free 90 Y/conjugated 90 Y ratio in the labeled sample remained unchanged at 4:96. The mixture could then be passed through another Sephadex G-50 size exclusion chromatography column in order to remove unbound 90 Y cation and/or other low-molecular weight impurities.
The eluted fractions were checked by Folin-Colciteau® reagent and for the presence of radioactivity in order to determine the 90 Y-DTPA-Rituximab-containing fractions. The fraction with the highest radioactivity that contained the maximum color absorbance was chosen as the suitable final product with appropriate specific activity for animal tests. Instant thin layer chromatography using various mobile and stationary phases was performed in order to ensure the existence of only the desired radiolabeled antibody. In all ITLC tests, radiolabeled antibody remained at the origin while other species migrated to other R f s depending on the mobile phase used. For 90 Y detection, the best eluent system was 10 mM DTPA aqueous solution in R f of 0.8. It is worth mentioning that, for 90 Y-DTPA detection, ammonium acetate 10%:methanol (1:1) can be used separately to ensure the absence of any free DTPA in the purified conjugated antibody [Figure 2]. Because of the size and charge of the protein (≈150,000 D), 90 Y-DTPA-Rituximab remains at the origin in all systems used.
|Figure 2: Percentage of injected dose per gram (ID/g %) of 90Y-DTPArituximab in normal rat tissues at 1, 2 and 24 h post injection|
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90 Y-DTPA-Rituximab was incubated in freshly prepared human serum for 24 h at 37°C. The aliquots of the resulting mixtures were analyzed to determine the kinetic stability of the radiolabeled conjugate. Ninety-six percent to 98% of the radioactivity was eluted at the same fraction as 90 Y-DTPA-Rituximab using size exclusion chromatography. Thus, there was no evidence for either degradation or transchelation of 90 Y to other serum proteins over a time period consistent with the normal blood clearance time of rituximab. The stability of the radiolabeled compound in the final formulation was also demonstrated [Figure 3].
|Figure 3: ITLC chromatograms of 90YCl3 solution (up) and final 90Y-DTPA-rituximab solution (down)|
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In order to demonstrate the integrity of the protein after conjugation and radiolabeling, gel electrophoresis was performed on the SDS-PAGE gels using 16% bisacrylamide gel. The loaded samples were Rituximab commercial sample, DTPA-Rituximab and radiolabeled protein samples 1 week after the experiment while being kept in the fridge. The three samples comprised a similar pattern of migration in the gel electrophoresis. Interestingly, the SDS-PAGE results were checked with a reported commercial Rituximab sample. The SDS-PAGE patterns for DTPA conjugate in contrast to starting Rituximab sample and radiolabeled immunoconjugates are shown in [Figure 4].
|Figure 4: Radiochemical purities of the labeling mixture vs. time at optimized conditions|
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The animals were sacrificed by ether asphyxiation at the selected times after injection (2, 24, 48 and 72 h). Dissection began by drawing blood from the aorta, followed by collecting the heart, spleen, muscle, brain, sternum, thighbone, kidneys, liver, intestine, stomach, lung, colon and skin samples. The tissue uptakes were calculated as the percent of beta counts read from the scintillation system in contrast to a standard Y-90 sample with known activity per gram of tissue (% ID/g) [Figure 5].
|Figure 5: SDS-PAGE lane patterns for in house-made standard ladder (1) rituximab (2), DTPA-rituximab conjugate (3) and 90Y-DTPArituximab conjugate (4)|
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| > Discussion|| |
Various monoclonal antibodies are radiolabeled with Y-90 and have been used in the development of possible therapeutic agents. Among those, the anti-CD20 radioimmunoconjugate is still the best clinically used agent [Table 1].
|Table 1: Some Y-90 radioimmunoconjugates developed for the treatment of human malignancy|
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The biodistribution of radiometal-labeled anti-CD20 antibody has already been shown by Y-90 antiCD20 conjugate biodistribution studies. Because radiolabeling of each antibody molecule with one radioisotope atom under the optimal theoretical conditions can be satisfactory, we chose the 1:1 molar ratio. The only possible way to check the yield was RTLC analysis of final radiolabeled antibody at various molar ratios.
The activity was rapidly removed from the blood, which was in agreement with the other reported labeled antibodies.  Likewise, the other radiolabeled proteins of the labeled antibody were accumulated in the liver.
The brain did not show any significant uptake over the period of time. This had already been shown by 123 I-antiCD20 conjugate biodistribution studies.  High uptake in the spleen and reticuloendothelial organs was observed, which was due to the final accumulation of B lymphocytes carrying the radioimmunoconjugate on their surface. Increasing uptake of the spleen in the course of time was the direct result of the depletion of circulating B cells occurring rapidly after administration to the mammals. This has been already shown in humans, which is an important sign of therapy in lymphoma patients. 
As a natural reaction to the depletion of the lymphocytes, the reticuloendothelial system, including spleen, will be the final possible reservoir of the depleted lymphocytes. However, a direct resemblance of CD-20 antigen in human beings and rats has not been demonstrated. On the other hand, Rituximab natural binding was found on lymphoid cells in the thymus, the white pulp of the spleen and a majority of B-lymphocytes in peripheral blood and the lymph nodes in human beings.  This has been observed in our studies on the normal rats as well.
Significant accumulation in the lungs was also observed. Interestingly, we found reports of severe pulmonary reactions using anti-CD20 with pulmonary infiltrates or edema in humans. It should be emphasized that acute symptoms appear within 1-2 h of the initiation of the first infusion.  However, it usually fades after 18-24 h, which can be observed in [Figure 5]. Limited bone uptake can be attributed to the stability of the radiolabeled antibody.
For 90 Y-DTPA-Rituximab, the radiochemical purity was 96% and the labeling and quality control took 24 h. The radiolabeled complex was stable in human serum for at least 24 h, and no significant amount of free 90 Y as well as 90 Y-DTPA was observed. The final preparation was administered to wild-type rats, and biodistribution of the radiopharmaceutical was checked 2-72 h later. 90 Y-Rituximab is a potential probe for therapy of lymphomas. This study has offered optimized production and quality control protocol for 90 Y-antiCD20 radioimmunoconjugate for the future clinical studies in the country using generator-produced Y-90.
| > Acknowledgement|| |
The authors wish to thank Mr Mirfallah for performing animal tests. We acknowledge the financial support of the National Radiopharmaceutical Production Project, 2009 granted by Iranian Government.
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