Three-step Pretargeted Radioimmunoimaging In Nude Mice Bearing Human B Cell Lymphoma By CD45Monoclonal Antibody

[Objective]1. To establish the labeling method of DTPA-biotin and human IgG with99mTc and observe the stability in vitro and in vivo and the distribution study of99mTc radiolabelled DTPA-biotin and hIgG in normal mice.2. To evaluate the value of three-step pretargeting radioimmunoimaging in nude mice bearing human B cell lymphoma by CD45monoclonal antibody.[Materials and methods]1The radiolabeling of DTPA-biotin and hIgG with99mTc and its distribution experimental in normal mice1.1Experimental animal24Kunming mice at age of4-to6-week-old (any male and female) with weight of19-21g were provided by experimental animal center of Southern Medical University.1.2Radiolabeling of DTPA-biotin with99mTcDTPA-biotin was radiolabeled with99mTc by directly labeling method, and its labeling ratio and radiochemical purity were determined by paper chromatography with Xinhua No.1filter paper as stationary phase and acetone and saline as developing agent, respectively. The radiochemical purity was respectively determined at1h、3h、6h and12h after purification.1.3hIgG radiolabelled with99mTchIgG was radiolabeled by reduction method using2-mercaptoethanol, and its labeling ratio and radiochemical purity were determined by paper chromatography with Xinhua No.1filter paper as stationary phase and mixture solution of30%ammonia:ethanol:water (1:2:5) and saline as developing agent, respectively. The radiochemical purity was respectively determined at1h、3h、6h and12h after purification.1.4The stability experiment of99mTc-DTPA-biotin and99mTc-hIgG in vitroA volume of500μl of99mTc-DTPA-biotin and99mTc-hIgG solution was respectively incubated at37℃with1ml of saline solutin (NS). Radiochemical stability was determined taking samples of2ul at different times from1h to12h for analysis by paper chromatography.1.5Biodistribution and imaging of99mTc-DTPA-biotin and99mTc-hIgG in normal mice24normal mice were randomly divided into two groups, each group had12mice. The first group received intravenous injection of99mTc-DTPA-biotin and another group was intravenously injected with99mTc-hIgG. Each group received the dosage of200uCi/0.1ml injected via the tail vein and had3mice undertake SPECT image at1h、3h、6h and12h post-injection, respectively. Then these mice were immediately sacrificed after imaging. The organ and tissue such as liver, spleen, kidney, lung, stomach, intestine, muscle, skeletal and blood were taken and weighed by electronic balance. The radioactive counting was determined by gamma counter. The percent injected dose per gram of tissue (%ID/g) was calculated as radioactive decay correction. 2Three-step pretargeted radioimmunoimaging in nude mice bearing human B cell lymphoma by CD45monoclonal antibody2.1Raji cell linesRaji cell lines were provided by the department of hematology of southern hospital, Southern Medical University. The cells were routinely grown at37℃in a5%CO2atmosphere and95%relative humidity in RPMI1640culture medium supplemented with10%fetal bovine serum.2.2The CD45expression of Raji cells detected by flow cytometryThe Raji cells with logarithm growth period were collected, washed by PBS twice and counted. Cells were transferred to tubes with cell concentration of106cells per tube. The cells were incubated with CD45monoclonal antibody for30min at4℃, and washed with phosphate-buffered saline (PBS). At the same time the isotype of IgG1was as a negative control antibody. The positive cell count expressed CD45was analysised by flow cytometry and the percentage of positive cells was calculated by counting cells with fluorescent marker.2.3Lymphoma experimental animal model(1)24nude mice (18-22g) at age of4-to6-week-old (male and female) were provided by Guangdong Province Experimental Animal Center and the Experimental Animal Center of Southern Medical University.(2) The Raji cells with logarithm growth period were collected, centrifuged and suspended in saline (>1×108/mL). Lymphoma was induced by subcutaneous injection of Raji cells in0.2ml into bilateral hips of each nude mouse. All nude mice were kept in pathogen-free (SPF) environment for raising. The sites of injection were observed at regular intervals for the appearance of tumor formation and progression. And after tumor formation its longest and shortest diameter measured by vernier caliper every two days were recorded. 2.4Coupling of biotin to CD45monoclonal antibodyThe conjugation of biotin to antibody was accomplished through the use of biotin activation ester (NHS-biotin) dissolved in anhydrous dimethyl sulfoxide (1mg/1ml). In a typical reaction, CD45was used at a concentration of10mg/ml in0.1M bicarbonate buffer pH8.5. The solution was gently stirred and NHS-biotin in saline added at molar ratio from30to50with respect to CD45. The reaction was allowed to proceed for1hr at room temperature and the product was separated from free biotin by centrifugation two times in a PD-10chromatography column.2.5Determination of biotin groups per antibody moleculeThe average number of biotin groups attached to each IgG molecule was determined spectrophotometrically by using HABA/Avidin reagent.900μl HABA/avidin reagent was pipeted into a tube and read A500. Then100μl of biotinylated protein was added, mixed by inversion and read A500. The assay was based on the binding of the dye HABA avidin and the ability of biotin to displace the dye in stochiometric proportions. This displacement of dye was accompanied by a change in absorbance at A500. The time of A500determination was set when the color of reaction system changed from orange to yellow as combining of biotin with HABA/avidin reagent for about1minute. Biotin groups per antibody molecule were calculated according to the specification of HABA/Avidin reagent.2.6Labeling of biotin and CD45monoclonal antibody with99mTcSee the section of radiolabeling.2.7Biodistribution studies in nude mice bearing human lymphoma with three-step pretargeting12nude mice bearing hman lymphoma (3per time point) were injected with100μg/100μl of the biotinyled CD45antibody through the tail vein, followed with200μg/100μl of avidin intravenously after48hr and then received intravenously 7.4MBq (20μg/100μl) of the99mTc-DTPA-biotin after another48hr. The mice were sacrificed by cervical distlocation at1,3,6, and12hr after injection, after which tissues and organs of interest were collected. The tissue and organ samples were weighed, and counts were determined with an automated γ-counter. Uptake of radioactivity in the tumor and normal tissues and organs was expressed as a percentage of injected dose per gram of tissue (%ID/g).2.8Pretargeting radioimmunoimaging with three-step in nude mice bearing human lymphoma6nude mice with implanted hman lymphoma were randomly divided into two groups (3per group). The first group was the group of three-step pretargeting radioimmunoimaging in which first100μg/100μl of the biotinyled CD45antibody was injected, followed by200μg/100μl of avidin and finally received7.4MBq (20μg/100μl) of the99mTc-DTPA-biotin. Another group was the group of radioimmunoimaging received intravenous injection of99mTc-CD45antibody with dose of7.4MBq (20μg/100μl). All nude mice wer scanned with SPECT with a low-energy collimator at2,6, and12hr after the radiopharmaceutical was injected in the tail vein and then immediately sacrificed after imaging for biodistribution study. The tissue and organ samples were collect, weighed and counted. Uptake of radioactivity in the tumor and normal tissues and organs was expressed as%ID/g and the ratio of tumor to normal tissue.2.9Statistical analysesAll experimental data were analysed by the statistical software of SPSS13.0. The quantitative parameters (T/B and T/M at12h) were expressed as the mean+SD (X±S).The two samples were compared using independent samples t test, P<0.05was considered as statistical significance.[Results] 1. The radiolabeling of DTPA-biotin and hIgG with99mTc and its distribution experimental in normal mice1.1The labeling ratio and radiochemical purity of99mTc radiolabelled DTPA-biotin and hIgG.The labeling ratio of99mTc-DTPA-biotin is over80%, the labeling ratio of99mTc-hlgG is over70%, the radiochemical purity of99mTc-hIgG is over90%after the separation and purification using PD-10column.1.2The stability in vitro of99mTc radiolabelled DTPA-biotin and hIgG99mTc-DTPA-biotin was relatively more stable in NS, more than75%remaining intact after12hr. And99mTc-hIgG was stable in NS, more than85%remaining intact after12hr.1.3Biodistribution and imaging of99mTc-DTPA-biotin and99mTc-hIgG in normal mice(1) SPECT imaging of99mTc-DTPA-biotin in normal mice post-injection at1hr,3hr,6hr and12hr showed the high renal uptake and less radioactivity distribution in blood and muscle. It indicates that99mTc-DTPA-biotin excrets mainly through the urinary system.(2) SPECT imaging of99mTc-hIgG in normal mice post-injection at1hr,3hr,6hr and12hr showed the intensive radioactivity depositing in liver and spleen and high retention in blood, while less radioactivity depositing in gut, muscle, skeletal.2Three-step pretargeted radioimmunoimaging in nude mice bearing human B cell lymphoma by CD45monoclonal antibody2.1The CD45expression of Raji cell and the establishment of Raji cell xenograft lymphoma in nude miceThe CD45expression of Raji cells was detected by flow cytometry with positive expression rate of78.5%.16of24nude mice had tumor formation and the rate of tumor formation rate is67%.2.2Determination of biotin groups per antibody moleculeIt was found to give an average of12biotin groups to each antibody molecule when NHS-biotin/antibody molar ratios from30:1to50:1.2.3Radioimmunoimaging of99mTc-CD45antibody in nude mice with Raji cell xenograftSPECT imaging of99mTc-CD45antibody in nude mice with Raji cell xenograft at3hr,6hr and12hr post-injection showed the intensive radioactivity depositing in liver, spleen and kidney, and high radioactivity in blood in12hr, while a few radioactivity accumulation in tumor in12hr. The%ID/g of tumor was0.89±0.13at12hr, and the ratios of tumor to blood and tumor to muscle reached1.6and2.5, respectively.2.4Biodistribution and radioimmunoimaging studies in nude mice bearing human lymphoma with three-step pretargetingSPECT imaging results in nude mice bearing human lymphoma with three-step pretargeting at1hr,3hr,6hr,12hr post-injection showed that there were the intensive radioactivity depositing in liver and spleen, and less radioactivity retention in blood during the whole imaging; However for transplant tumor the tumor was ambiguously imaged at lhr post-injection and clearly at3-6hr post-injection. It was also observed that as time continued the accumulation of radioactivity in tumor increased gradually, and even at12hr the tumor still be clearly imaged. The%ID/g of tumor at3,6,12hr post-injection was1.73±0.22,1.24±0.03and0.94±0.07, respectively. And the ratios of tumor to blood at3,6,12hr post-injection reached3.5,4.9and7.8, respectively, while the ratios of tumor to muscle8.2,8.9and10.4, respectively.[Conclusion]1. The labeling rate of DTPA-biotin and hIgG with99mTc was respectively more than80%and over70%, the radiochemical purity of99mTc-hIgG is over90%. It was shown that they had a good stability in vitro and an evident difference in blood clearance and biodistribution in normal mice. All thses may provide an experimental data for the selecting of different components in pretargeting radioimmunoimaging based on avidin-biotin system.2. Compared to CD45antibody radiolabeling with99mTc, Three-step pretargeting RII with99mTc-DTAP-biotin had improved the ratio of tumor to normal tissue. Tumor could be revealed at lhr after injection of radiolabel, and clearly revealed at3hr post-injection and so it may achieve early imaging for tumor. In short, the research results may provide an experimental evidence for the application of three-step pretargeted radioimmunptherapy by CD45antibody in nude mice with Raji cell xenograft.