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Experiment Study Of The Two-step Pretargeted Technique For Lymphoma With Biotinyled CD45Monoclonal Antibody And188Re-Avidin

Posted on:2015-06-09Degree:MasterType:Thesis
Country:ChinaCandidate:W L ZhengFull Text:PDF
GTID:2284330431969254Subject:Imaging and nuclear medicine
Abstract/Summary:PDF Full Text Request
Background:Radioimmunotherapy (RIT) is currently one of the most promising cancer treatments, especially in hematological malignants. Its basic principle is that the antibody injected into the patients can be used as a carrier of radioactive nuclide emitting β ray, navigate automatically into the tumor site, so the radioactive nuclide can be selectively transferred into tumor in order to achieve the purpose of selective destruction of tumor. However, because the ratio of target to non-target are not ideal and less radiation dose is selectively transferred to the tumor RIT did not achieve satisfactory clinical desired results, which restricts its widely application. But pretargeted radioimmunotherapy (PRIT) application can overcome the above-mentioned shortcoming, and it use a separate dosing method in which anti-tumor antibodies pre-target the tumor site first, then radioactive nuclides for imaging and treatment are injected in order to combine with antibodies pre-localized in tumor while radioactive nuclides without combining with antibody are excreted quickly from the body. More and more animal experiments and clinical studies have demonstrated that compared to RII and RIT with the radiolabelling antibodies, PRIT is safe and feasible, and improves the effect of tumor imaging and treatment, can improve radioactive dose directly delivered to the malignant cells and simultaneously reduces the nonspecific radiation toxicity to normal tissue cells. Radionuclides used in clinical RIT treatment should have the appropriate range and emitter-βray with a certain energy. Rhenium-188is a more ideal radioactive nuclide with the beta max energy of2.12MeV, the gamma energy of155keV and physical half life of17.0h. Its beta energy is higher which has strong lethality to the tumor cells, and its gamma ray simultaneously emitting is suitable for imaging, which is help to estimate the absorbed dose and undertake clinical pharmacokinetic study, so it can be simultaneously used in imaging and treatment.188Re has good behavior of nuclear physics and biology, as literature researches have shown that it is likely to play a good role in the treatment of tumor with no serious radiation damage to human body because of its biological half-life of less than10h in humans with less perrhenate uptake in various organs and quickly excreting through the urine.This research used CD45monoclonal antibody (McAb) as a transporter of specifically targeted to lymphoma, and according to the principle of pretargeting technique, first biotinylated CD45McAb was located in tumor cells, followed188Re labeled avidin injected after24h. So the ratio of target to non-target can be improved and the background radioactivity can be reduced because of the high affinity of avidin with biotin and the rapid clearance of labeled avidin in vivo. In this paper directly labeled methods of188Re with avidin and anti-human CD45McAb were explored and established, and as a control group with188Re directly labeled CD45McAb, experiment studies on pretargeting radioimmunoimaging in mice bearing lymphoma model were performed, and the corresponding biological distribution was observed in order to provide an experimental basic for further pretargeted radioimmunptherapy for xenograft lymphoma based CD45McAb.Objection:1To investigate the direct-labeling method of CD45McAb with188Re, and observe the stability in vitro and in vivo and the biodistribution of188Re radiolabeled CD45McAb in normal mice.2To evaluate the effect of two-step pretargeting RII in the xenograft lymphoma mice with human Raji cells by biotinyled CD45McAb and188Re-Avidin.3To observe the inhibition effects on Raji cells cultured in vitro by two-step pretargeting with biotinyled CD45McAb and188Re-Avidin.Methods and materials:1The direct-labeling method of CD45McAb with188Re and its biodistribution experimental in normal mice1.1Radiolabeling of CD45McAb with188ReCD45McAb was radiolabeled with188Re through a direct-labeling method. The labeling was conducted at pH5.0with2-mercaptoethanol and stannous chloride used as reductant of antibodies proteins and188Re respectively, and sodium glucoheptonate as chelate agent. Labeling efficiency and radiochemical purity were measured by paper chromatography.1.2The stability experiment of188Re-CD45McAb in vitroA volume of100μL of188Re-CD45monoclonal antibody solution was respectively incubated at37℃with1mL of rat serum and saline solution (NS). Radiochemical stability was determined taking samples of2μL at different times from1h to24h for analysis by paper chromatography.1.3Biodistribution of188Re-CD45McAb in normal mice9normal Kunming mice were injected188Re-CD45McAb via the tail vein and were killed by cervical at3h、6h and12h post-injection, respectively with3mice each time point. Then organ and tissue such as liver, spleen, kidney, lung, stomach, intestine, muscle, bone 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 by radioactive decay correction for observing biodistribution of188Re-CD45McAb in normal mice in vivo.2Two-step pretargeted radioimmunoimaging in xenograft mice with Raji cell2.1Raji cell linesRaji cell lines were kindly provided by the department of hematology of Nanfang hospital, Southern Medical University. The cells were routinely grown at37℃in a5%CO2atmosphere and95%relative humidity in RPMI-1640culture medium supplemented with10%fetal bovine serum.2.2Establishing of experimental animal model of lymphoma1)24Nod-Scid mice (non-obese diabetic severe combined immunodeficient mice)(19-21g) at age of4-to6-week-old (female), which were known as T ’B’ NK cells severe combined immunodeficient animal models, were bred in SPF laboratory animal center of Southern Medical University.2) The Raji cells with logarithm growth period were collected, centrifuged and suspended in serum-free RPMI-1640medium (>1×107mL). Lymphoma was induced by subcutaneous injection of Raji cells into right armpit with0.2mL Raji cells suspension. All mice were bred in pathogen-free (SPF) environment. 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.2.3Preparation of biotinylated CD45McAb1) The conjugation of biotin to antibody was accomplished through the use of biotin activation ester (NHS-biotin) dissolved in anhydrous dimethyl sulfoxide (lmg/lmL). In a typical reaction, CD45McAb was used at a concentration of lmg/ml in0.1M bicarbonate buffer pH8.5. The solution was gently stirred and NHS-biotin in saline added at molar ratio of1to20with respect to CD45McAb. The reaction was allowed to oscillate reaction for1h at room temperature and the product was separated from free biotin by centrifugation two times in a PD-10chromatography column.2) Determination of biotin groups per antibody moleculeThe average number of biotin groups attached to each CD45molecule was determined spectrophotometrically by using HABA/Avidin reagent.0.9mL HABA/avidin reagent was pipeted into a tube and read A500. Then0.1mL 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 at room temperature. Biotin groups per antibody molecule were calculated according to the specification of HABA/Avidin reagent.2.4Determination of immune activity and the avidin-binding activity of biotinylated CD45McAbImmune activity and avidin-binding activity of biotinylated CD45McAb were measured by enzyme-linked immunosorbent assay (ELISA).2.5Labeling of avidin and CD45McAb with188ReSee the section1.1for radiolabeling.2.6Biodistribution studies of two-step pretargeting in mice bearing human lymphoma model6mice bearing human lymphoma (3per time point) were randomly divided into2groups, experimental group is pretargeted group and the control group is188Re-CD45McAb. Mice in pretargeted group were injected50μg/200uL of the biotinyled CD45McAb through the tail vein, then received intraperitoneal injection7.4MBq (50μg/200μL) of the188Re-Avidin after24h. However mice in the control group were injected100μg/100μL of the188Re-CD45McAb through the tail vein. All mice were sacrificed by cervical dislocation at24h post-injection, after which tissues and organs of interest were collected. The tissue and organ samples were weighed, and radioactive counts were determined with an automated γ-counter. Radiation absorbed doses of tumor and normal tissues were expressed as a percentage of injected doses per gram of tissue (%ID/g).2.7Two-step pretargeted radioimmunoimaging in mice bearing human lymphoma model6mice with implanted human lymphoma were randomly divided into two groups (3per group). The first group was the group of pretargeting radioimmunoimaging in which first the biotinyled CD45McAb was injected, following received7.4MBq of the188Re-Avidin by intraperitoneal injection. Another group was the group of radioimmunoimaging received intravenous injection or intratumoral injection of188Re-CD45McAb with the same dose of7.4MBq. All mice were scanned with SPECT with a middle-energy collimator at30min,1h,2h,6h and23h after the radiopharmaceutical was injected. Using regions of interest (ROI) techniques, uptake of radioactivity in the tumor and normal tissues and organs was determined at the different imaging time point, and then calculated T/NT ratio.3The inhibition effects on Raji cells cultured in vitro by two-step pretargeting with biotinyled CD45McAb and188Re-Avidin.3.1Preparation of188Re-CD45McAb and188Re-AvidinSee the section1.1and2.5for radiolabeling.3.2Competitive binding assay in vitro of188Re-CD45McAb with Raji cellsThe Raji cell concentrations were adjusted to6×104,3×105,6×105,3×106,6×106, plus3for each concentration, each tube was added Re-CD45McAb and incubated the cells for1h in the incubator at37℃, the tubes were measured by counting the total radioactivity (T); removed supernatant after centrifugating (2000r/min,5min) at4℃, the solution was washed two times with PBS, and then measured the binding radioactive counts (B) with cells and calculated the binding rate. While non-specific binding experiments of each tube was firstly added50μg of unlabeled CD45McAb, following added20μL188Re-CD45McAb, other steps was the same as above described.3.3The inhibition effects on Raji cells cultured in vitro by different188Re radiolabel treatment.CCK-8assay was used to determine the inhibition effect on Raji cell proliferation in vitro. The Raji cell concentration was adjusted to4×104/well, dispensed100uL of cell suspension seeded in96well plates, were randomly divided into four groups, including pretargeted group,188Re-Avidin,188Re-CD45McAb,188ReO-4, control group (PBS). And setting a blank group (just only the radiolabelling and culture medium without cells). The plate was pre-incubated for24h in an incubator (humidified atmosphere at37℃,5%CO2), then added10μL of concentration of markers into the culture medium in corresponding different groups of plate, incubated the plate for48h in the incubator. The frozen CCK-8was thawed on the bench top or in a water bath at37℃, and10μL of CCK-8solution was added to each well of the plate, incubated for1-2h in the incubator. The plate was measured the absorbance at450nm using a microplate reader absorbance detection to calculate Raji cell viability rate and the inhibition rate.4Statistical analysisSPSS13.0was used to analyses experimental data. Measurement data were calculated by One-Way ANOVA and categorical data were calculated by chi-square test. P<0.05was considered to have significant difference.Results:1The directly radiolabeling of CD45McAb with188Re and its biodistribution experimental in normal mice1.1The labeling ratio and radiochemical purity of188Re radiolabelled CD45McAb.The labeling ratio of188Re-CD45McAb was86%, the radiochemical purity of188Re-CD45McAb was over90%after the separation and purification using PD-10column.1.2The stability in vitro of188Re-CD45McAbRadiochemical purity of188Re-CD45McAb was74%after24h in room temperature. And the radiochemical purity of188Re-CD45McAb was still64%and63%in NS and serum after24h incubating at37℃water bath.1.3Biodistribution of188Re-CD45McAb in normal miceBiodistribution result of188Re-CD45McAb was shown that the kidney radioactive was significantly higher than other organs with long clearance time in body. The percent injected dose per gram of kidney at3h and24h were14.46±2.77and7.46±2.07respectively. The radioactivity depositing in liver was also relatively higher, while radioactivity distribution in other organs rapidly decreased over time.2Two-step pretargeted radioimmunoimaging in xenograft mice with Raji cell2.1Determination of biotin groups per antibody molecule of biotinylated CD45McAb and the biotin-binding activity of188Re-CD45McAbIt was found to give an average of40biotin groups to each antibody molecule when NHS-biotin/antibody molar ratios from1:20. The immune activity of biotinylated CD45McAb was (90.57±8.13)%at average by ELISA. Biotin-binding activity of188Re labeled avidin was (72.37±15.32)%at average by the biotin-sepharose assay.2.2Biodistribution studies in mice bearing human lymphoma with two-step pretargetingThe biodistribution in mice bearing human lymphoma with two-step pretargeting method showed that the%ID/g of tumor, kidney and liver were respectively (1.34±0.52)%,(6.77±2.32)%and (2.81±1.25)%at24h post-injection, and less radioactivity deposited in the other organs, while the uptake in tumor was still higher after24h. And the ratios of tumor to blood and tumor to muscle at24h post-injection reached4.86and8.00.However biodistribution of188Re-CD45McAb in mice with Raji cell xenograft at24h post-injection showed the intensive radioactivity deposited in liver, spleen and kidney. The radioactivity of blood pool was still higher at24h, while fewer radioactivities accumulated in tumor. The ratio of tumor to blood only reached0.63, and the ratio of tumor to muscle was3.21±0.24.2.3Two-step pretargeted radioimmunoimaging in mice with Raji cell xenograftSPECT imaging results of two-step pretargeting method in mice bearing human lymphoma at30min,1h,6h and23h post-injection showed that during the whole imaging radioactive in the blood pool was low, and more radioactive gathered in the liver and spleen was seen; However transplanted tumor was ambiguously imaged at1h post-injection with radioactivity accumulation gradually increasing as time and clearly imaged at1-6h post-injection, and even at23h. The ratios of tumor to muscle at30min,1h and6h post-injection by using of ROI technology reached1.73,2.15and2.53, respectively.However SPECT imaging of188Re-CD45McAb in mice with Raji cell xenograft at1h,6h and20h post-injection showed that the intensive radioactivity was deposited in liver, spleen and kidney and the radioactivity of blood pool was higher at20h with a few radioactivity accumulations in tumor. The ratio of tumor to muscle at20h post-injection was0.98.3The inhibition effects on Raji cells cultured in vitro by two-step pretargeting with biotinyled CD45McAb and188Re-Avidin.3.1Competitive binding assay in vitro of Re-CD45McAb with Raji cellsThe results after repeated measurement showed that the specific cell binding rate of188Re-CD45McAb with Raji cells was (70.9±21.91)%. And in the competition combination experiments the specific cell binding rate of Re-CD45McAb with Raji cells was only (7.96±0.87)%when188Re-CD45McAb was added with CD45McAb at overdosage at the same time.3.2The inhibition effects on Raji cells cultured in vitro by different188Re radiolabel treatment.Raji cell proliferation inhibition assay by CCK-8method showed that Raji cell proliferation was inhibited in all groups with188Re radiolabel treatment, and the inhibition rate was positively correlated with the radioactivity dose. However at the same dose, the inhibition effect in the group of two-step pretargeting method at each time point were all stronger than those groups of Re-CD45McAb188Re-Avidin and188ReO4-, and there was significantly statistical difference between the group of two-step pretargeting method and the other groups (P<0.05). But there was no significantly statistical difference in the inhibitory effect between the group of188Re-Avidin and the group of188ReO4-(P>0.05).Conclusion:1The method of directly labeled CD45McAb with188Re is not only simple, but alsc has high labeling efficiency and radiochemical purity.188Re-CD45McAb has good stability in vivo and in vitro.2188Re-CD45McAb at post-injection in tumor-bearing mice clears more slowly in blood and obviously gathers in the liver, spleen and kidney with a small amount of radioactivity concentration in the tumor site. However after the introduction of two-step pretargeting method it is observed that radioactivity blood removal is accelerated, radioactivity mainly evacuates through the kidneys, and radioactivity in other organs is rapidly reduced with time. But the radioactivity uptake in tumor tissue is increasing with a long time staying.3It is confirmed that there is better specificity and targeting to lymphoma in mice bearing Raji cell xenograft in the group of two-step pretargeting method. Compared with188Re-CD45McAb, two-step pretargeting RII has significantly improved the ratio of tumor to normal tissue and makes tumor imaging clearer.4The Raji cell proliferation inhibition assay in vitro suggests that two-step pretargeting method has obvious inhibitory effect on the Raji cells. In short the research results may provide an experimental evidence for further two-step pretargeting radioimmunptherapy.
Keywords/Search Tags:Two-step pretargeting technique, 188Re, Avidin-biotin system, CD45McAb, Radiolabeling, Lymphoma
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