1. Background
Radioimmunotherapy (RIT) is a targeted therapy with antibodies to concentrate therapeutic radioactive substances in tumors and cancer cells, by decreasing the amount of drug the side effects will be reduced (1).
The main goal of RIT is transferring a lethal dose of radiation to tumor cells. Therapeutic efficiency differs based on immunological of antibody, tumor penetration, antibody affinity, and the radioactive properties of the conjugated radionuclide. Generally, it takes several days for antibodies to penetrate solid tumors (2).
In RIT, the half-life must be long enough. A 3 - 8-day half-life is appropriate for this purpose. (1).
Non-Hodgkin lymphoma (NHL) is a cancer affecting lymphocytes. NHL is classified into various types: Fast-growing and slow-growing (3). NHL is an ideal case for treatment with radiopharmaceuticals (4).
Rituximab is a monoclonal chimeric antibody against antigen CD20 for treating NHL or chronic lymphocytic leukemia (5).
NHL has a higher sensitivity to medium-dose-rate compared to high-dose-rate radiation, and normal tissues tolerate LDR radiation effects better than HDR ones (4). Medium-energy beta-emmiters such as 131I(0/5 Mev) and 177Lu(0/8 Mev) up to 1 mm can penetrate tumor tissue and kill hundreds of tumor cells (4).
In the present study, rituximab was labeled with 131I and 177Lu which are two medium-energy beta-radiators, and results of the radiolabeling efficiency, stability, immunoreactivity, and biological distribution were compared.
2. Methods
2.1. Materials
The anti-CD20 antibody was purchased from Sobhan Oncology in the form of vials containing 100 mg in 10 mL, and purified by using borate buffer (pH = 8 - 8.5) and 13000-Dalton dialysis tubes. The Lu-177 radionuclide was prepared by 176LuCl3 bombardment under neutron bombardment and the (n, γ) reaction in Tehran Research Reactor (TRR) at the flux of 2.6 × 1013 n.Cm-2.S-1 for 14 days and dissolving it in 1M HCl. The DOTA-NHS chelator was purchased from Macrocyclice. Other chemicals were used without further purification.
2.2. Preparation of 177Lu-Rituximab
One mg of DOTA-NHS was added to 5 mg of Ritiximab in borate buffer (pH = 8-8.5), and was shaked for 24 hours at 2°C - 8°C. It will be purified by using 0.25 M Ammonium Acetate Buffer in 13KD dialysis tubes for 24 hours at 2°C - 8°C (6, 7). For determining the number of DOTA conjugated to the antibody, 50 mL of the DOTA-Ab complex was added to 950 μL of the Arsenazo reagent and incubated for 20 min at room temperature in a dark place. The UV absorbance was determined at 590 nm against the Arsenazo as standard solution, then the number of conjugated DOTA chelators per mole of antibody was calculated (8, 9).
For radiolabeling 300 μg of the DOTA-Ab complex was incubated with 6 mCi of 177LuCl3 in 0.1 N HCl for 3 hours at 37°C. For purification of radiolabeled antibody, Sephadex G-25 column and for measuring the immunoreactivity of fractions NaI well type gamma counter device was utilized.
2.3. Stability of 177Lu-Rituximab
The stability of 177Lu-Rituximab was determined by paper chromatography on Whatman 3MM and normal saline as solvent at the intervals of 1, 3, 8, 24, and 48 hours. 177Lu-Rituximab was diluted in PBS (1 mCi/mL of phosphate buffer saline) in a refrigerator at 4°C (10). Biodistribution was examined in normal male mice weighing approximately 25 - 32 g. they were kept in standard separate cages with a 12:12 dark/light cycle at 23°C ± 3°C.
After transfer from Pasteur Institute, the animals were kept at the Animal House of the laboratory. The mice had free access to food and water and were fed with mice and rat food. After one week of treatment, the mice were used for tests. The test was performed on series of three mice. 100 μL of 177Lu-Rituxiamb (1 mCi) was administered through vein tail. The animals were sacrificed under CO2 atmosphere 4, 24, and 48 hours post injection and The percentage of the dose per gram of tissue was calculated (%ID/g) (11).
2.4. Immunoreactivity of 177Lu-Rituximab
The immunoreactivity was assessed using Lindmo method (12). 6 × 107 Raji cells in 1 mL of cell culture medium in 2 mL microtubes were poured in another five tubes (each tube contained half the number of cells in the previous one) and the 177Lu-rituximab was added. After 2 hours they were centrifuged and washed multiple times so that only the cell-conjugated activity would remain. The curve of cell-conjugated activity against 1/ number of cells was drawn. The slope of this curve indicates the percentage of intact antibody after radiolabeling process. Each measurement was done in triplicate, and the mean was reported.
3. Results
On average, 3.5 DOTA were conjugated to each antibody molecule. Labeling was performed with 6 mCi of 177LuCl3 per 300 μg of antibody, and the radiolabeling yield was 92%.
After 48 hours at 4°C 82% of 177Lu- Rituximab was stable (Figure 1).
Figure 1.
Stability of 177Lu- Rituximab 1, 3, 8, 24 and 48 hours after labeling at 4°C
Results of biodistribution (Figure 2) indicated the acceptable stability of 177Lu- Rituximab Significant amounts of it after 48 hours remained in blood pool of normal mice.
Figure 2.
%ID/g of 177Lu- Rituximab in normal mice
The immunoreactivity test (Figure 3) shows 85% ± 3% of radiolabelled Antibodies after labeling with Lu-177 were intact and they ca bind to their receptors on the surface of B lymphocytes (8).
Figure 3.
Immunoreactivity of 177Lu- Rituximab
In previous study, Rituximab was radiolabeled with NaI131 the quality control and biodistribution tests were performed for it based on methods explained above (13), with results presented below:
For radiolabeling chloramine-T method was applied. The efficiency was 86% (Figure 4).
Figure 4.
Efficiency of radiolabeling of Rituximab with 1, 5, 7 mCi of NaI131 by chloramine-T method
The stability was examined at 1, 3, 24 after labeling by paper chromatography after 24 hours 70% of 131I-Rituximab was stable (Figure 5).
Figure 5.
Stability 131I-Rituximab by chloramine-T method 1, 3 and 24 hours after labeling at 4°C
The %ID/g of 131I-Rituximab showed acceptable amount of it 48 hours post injection in blood pool (Figure 6). The trace amount of activity in thyroid approved the stability of radiopharmaceutical.
Figure 6.
%ID/g 131I-Rituximab by chloramine-T method in normal mice
4. Discussion
Radiolabeled antibodies are appropriate candidates for targeted therapies. Precise antibody, radionuclide and chelator selection are very important (1-4).
Macrocyclic chelators such as DOTA provide stable binding to the radiometal. In this study 3.53.5 DOTA molecules were conjugated to each antibody (in previous studies have reported 4 molecules). However, the conjugating of 10 or more DOTA, reduces the immunoreactivity by 50% (8, 14, 15).
177Lu is a suitable radionuclide for RIT because of low-energy beta emition and gamma ray (113 KeV 6.5%; 208KeV 11%) for scintigraphy. As the target/non-target rate is higher in low-energy beta-radiating radionuclides than high-energy ones, 177Lu is preferred for scintigraphy in NHL patients (2, 6, 7).
Radiolabeling was performed with 6 mCi 177LuCl3 and 300 μg of antibody with the radiolabeling efficiency 92%. The 177Lu-Rituximab was 82% stable 48 post labeling. The constant amount of radiopharmaceutical in bone confirmed its stability (7).
For in vivo targeting evaluation, immunoreactivity assessment was performed by Lindmo method (12). The result shows 85% ± 3% radiolabeled antibody was intact. The high percentage of antibody in the blood circulation compared to other organs from 4 to 48 hours post injection indicates the high target/non-target ratio.
The 131I decays by beta (0.61 Mev) and gamma (360 Kev). As its half-life (8 days) is similar to the half-life of anti-mouse antibody in human, it is an appropriate radionuclide for RIT. Other advantages of 131I include its easy accessibility, low cost, and known chemistry (13).
Comparing 131I and 177Lu radionuclides (9), every 1 mg of the antibody in the chloramine-T method tolerates only 5 mCi of 131I (radiolabeling efficiency of 86%). At higher activities of 131I, we observed decomposition of antibody, whereas in radiolabeling with 177Lu, every 300 mg of antibody was radiolabeled with 6 mCi (radiolabeling efficiency of 92%) (13).
177Lu-Rituximab was more stable (82% up to 48 hours) but only 70% of 131I-Rituximab after 24 hours at 4°C was stable (13).
Biodistribution was similar in two methods. The stability and retention of radiopharmaceuticals in blood pool of normal mice is a positive factor. The result of cell conjugation and examining The immunoreactivity was 58% ± 3% in 131I-Rituximab by chloramine-T method, and 85% ± 3% in 177Lu-Rituximab which indicats that an acceptable percentage of the antibody shows resistance against radiolabeling.
4.1. Conclusion
Based on results, it can be concluded that radiolabeling with 177LuCl3 has a higher efficiency compared to Na131I, and the resulting radiopharmaceutical has higher stability and immunoreactivity. Biodistribution results were acceptable in the case of both compounds.
