| ObjectiveThe aim of this study was to radioiodinated AC 133.1 monoclonal antibody?mAb?by two different ways,and comparing their characteristics in vitro and in vivo.Methods1.AC 133.1 mAb preparations were from ascites.2.N-succinimidyl-3-?tri-n-butylstannyl?benzoate?ATE?method:using high performance liquid chromatograph to separate and purify the intermediate product N-succinimidyl-[131]benzoate?131I-SIB?which was acquired through 131I reacted with ATE,coupling it with AC 133.1 mAb at alkaline conditions?pH=8.7?.3.Iodogen method:131I was conjugated to AC133.1 mAb by solid phase oxidation method.4.Measuring the stability of radioiodinated compounds which mixed with proper volume of fetal bovine serum?FBS?,and calculateing its radiochemistry?RCP?at different time points?0,1,3,and 7 d?.5.Determining the equilibrium dissociation constant KD by cell saturated assay.6.Comparing its uptake in tumor and thyroid of the radioiodinated AC 133.1 mAb.7.Statistics analysis:Independment sample's t-test,P<0.05 was considered statistically significant.Results1.The concentration of AC 133.1 mAb was 4?g/?l.2.The retention time of 131I-SIB in UV peak was about 11.0 min,and its value in emission peak was 12.0 min.The radiolabeling yield,RCP of radioiodinated AC 133.1 mAb by ATE method?131I-SIB-AC133.1 mAb?were 70.75 5.59%,91.05 1.53%,respectively;and the corresponding value of AC 133.1 mAb radiolabeled by Iodogen method?131I-AC133.1 mAb?were 71.02 7.00%,92.36 1.97%.3.The RCP of 131I-SIB-AC133.1 mAb at different time points?0,1,3,and 7 d?were 91.05 1.53%,89.52 1.64%,85.31 1.96%,79.64 1.33%,respectively;the corresponding value of 131I-AC133.1 mAb were 92.36 1.97%,90.14 1.36%,86.52 2.68%,81.20 3.75%;statistic analysis showed there was no significant difference between the two groups of RCP at the same time.4.The equilibrium dissociation constant KD of ATE and Iodogen methods were?4.76 0.30? 10-8 M,?4.05 0.19? 10-8 M,respectively;there was no significant difference in the two groups?P=0.254?.5.The%ID/g of 131I-SIB-AC133.1 mAb in tumor at different time points?1,3,5,and 7d?were 4.63 0.07,8.69 0.34,7.25 0.11,6.62 0.11;the corresponding value of 131-AC133.1 mAb were 2.25 0.26,3.75 0.18,5.78 0.47,4.64 0.29,the uptake of 131I-SIB-AC133.1 mAb in tumor was higher than 131I-AC133.1 mAb at the same time point?P=0.001,P<0.001,P=0.002,P<0.001,respectively?.6.The radioactivity uptake?%ID/g?of thyroid with ATE methods at different time points?1,3,5,and 7 d?were 0.99 0.12,082 0.05,0.74 0.21,0.35 0.05,respectively;and the corresponding value of Iodogen method were 2.48 0.18,2.05 0.26,1.76 0.34,1.32 0.13,respectively;the radioactivity uptake of thyroid by Iodogen method was higher than ATE method at the same time point?P<0.001,P=0.001,P=0.005,P<0.001,respectively?.ConclusionRadioiodination of antibody by ATE method had a better stability in vivo.ObjectiveThe purpose of this study was to utilize radioimmunotherapy?RIT?to target CD133?+?colonic cancer stem cells?CSCs?and observe whether it could prevent tumor development in nude mice.Methods1.Assessing the maximum tolerated dose?MTD?in HCT116-tumor bearing nude mice.2.Drawing the tumor growth curve and recording the survival time of nude mice,then calculating the tumor volume doubling time?VDT?and median survival time of four groups from it.3.Analyzing CD133 expression by flow cytometry of four groups.4.Testing protein level of cancer stem-cell related biomarker,these markers include:CD133,ALDH1,Lgr5,E-cadherin,Vimentin,Snaill.5.Testing cell proliferative index of tumors by ki-67 staining.6.Tumor H&E staining.7.Bio-distribution,reflecting the pharmacokinetic characteristics of 1311-SIB-AC133.1 mAb,and calculating the tumor radiation absorbed dose from it.Results1.The MTD of HCT116 tumor-bearing nude mice was 16.65 MBq.2.The VDT and survival time of 131-SIB-AC133.1 mAb group were longer than other three groups?P<0.001?,with a value of 9.36 0.45 d for 131I-SIB-AC133.1 mAb group,and the tumor VDT were 6.29 0.78 d,6.42 0.35 d,6.89 0.30 d for saline,131I-IgG1 and AC 133.1 mAb groups,respectively;there were no significant differences between saline group and 131I-IgG1,AC133.1 mAb groups?P=0.494,P=0.062,respectively?,the same results between 13lI-IgG1 and AC 133.1 mAb groups?P=0.167?.3.The survival time of 131I-SIB-AC133.1mAb group was longer than other three groups?P<0.001?.From the survival curve,we calculated the the median survival time of saline,131I-IgGl,AC133.1 mAb and 131I-AC133.1 mAb groups were 27.8,29.4,32.6 and 44.0 days,repectively.4.CD133 expression in 131I-SIB-AC133.1mAb group was 7.15 0.95%,which was significant lower than the controls?saline group:21.70 1.61%,131I-IgG1 group:20.12 11.46%,AC133.1 mAb group:18.24 2.53%?;there were no significant differences between saline group and 131I-IgG1 AC133.1 mAb groups?P=0.366,P=0.056,respectively?,the same results between 131I-IgG1 and AC133.1 mAb groups?P=0.237?.5.The protein level of cancer stem-like markers?CD 133,ALDH1,Lgr5,Vimentin,Snaill?were lower than other groups,while the protein level of E-cadherin in 131I-AC133.1 mAb group was higher than the controls.6.Ki67 analysis showed reduced proliferative index in the 131I-SIB-AC133.1 mAbgroup?9.52 1.33%?,whereas the groups of 131I-IgG1,AC133.1 mAb and saline groups had significantly higher proliferative indices of 33.46 1.72%,28.60 2.88%,41.56 2.52%,respectively?P<0.001?;the proliferative index of saline group was higher than 131I-IgG1,AC133.1 mAb groups?P=0.014,P=0.001,respectively?,and the proliferative index of131I-IgG1 group was higher than AC133.1 mAb?P=0.043?.7.H&E staining of tumor tissues showed a large necrotic area in the 131I-SIB-AC133.1 mAb group;however,no obvious necrosis was observed in the controls.8.From the biodistribution data,we calculated the mean absorbed radiation dosimetry of tumor in 131I-SIB-AC133.1mAb was 5,966.34 54.90 cGy.ConclusionThe study suggested that targeting CSCs by radioimmnotherapy could inhibit tumor development. |
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