Avidin-biotin system pretargeting radioimmunoimaging and radioimmunotherapy and its application in mouse model of human colon carcinoma

AIM： To evaluate the multi-step pretargeting radioimmunoimaging （RII） and radioimmunotherapy （RTT） in nude mice bearing human colon carcinoma with avidin-biotin system labeled with 153^Sm. METHODS： Two- and three-step strategies for avidinbiotin system pretargeting techniques were established. In a three-step procedure, human colon carcinoma bearing nude mice were first injected with biotinylated monoclonal antibody （McAb-Bt） followed by cold avidin （Av） 48 h later and then 153^Sm-DB2 24 h thereafter; whereas the twostep procedure consisted of injection of 153^Sm-SA 48 h after pretargeting with biotinylated anti-CEA monoclonal antibody （CEA McAb-Bt）. SPECT imaging and biodistribution were performed at 4, 24, 48, or 72 h after injection of 153^Sm-labeled compounds. Five groups of nude mice subcutaneously grafted with human colon carcinoma were treated 3 d after grafting. One group received the injection with 100 μg CEA McAb-Bt followed by cold avidin （80 μg） after 2 d and 11.1 MBCl I53Sm-DB2 after 1 d. Four control groups were treated respectively with 11.1 MBq 153^Sm- CEA McAb, ii.i MBq 153^Sm-nmIgG, ii.i MBq 153^Sm-DB2, 100 μL normal saline. Toxicity was evaluated by changes of leukocyte count, and the efficacy by variation in tumor volume. Histological analyses of tumors were performed. RESULTS： The three-step procedure allowed faster blood clearance and yielded higher tumor blood ratios （5.76 at 4 h and 12.94 at 24 h） of the 153^Sm-DB2. The tumor was clearly visualized at 4 h in y-imaging after the injection of 153^Sm-DB2, while a significant accumulation of 153^Sm-SA in the tumor was observed only 24 h after the injection and tumor blood ratios at 4 and 24 h were 1.00 and 2.03, respectively, in the two-step procedure. Pretargeting RIT and 153^Sm-CEA McAb had a strong tumor-inhibiting effect.The tumor inhibitory rate was 80.67% and 78.44%, respectively, five weeks after therapy. Histopathological evidence also indicated radioactive damage in tumor tissues as necrosis of tumor cells, while in the other organs such as liver and kidney no radioactive damage was observed. Leukocyte counts showed significant decrease after treatment in groups of 153^Sm-CEA McAb and 153^Sm- nmIgG. CONCLUSION： The two kinds of pretargeting strategies can elevate the target-to-nontarget ratio, decrease the blood background and shorten the imaging time compared to 153^Sm-CEA McAb. Three-step pretargeting RIT is as effident as 153^Sm-CEA McAb, but markedly less toxic. This study provides experimental evidence for the clinical application of pretargeting RII and RIT.

STUDY OF RADIOIMMUNOIMAGING WITH MONOCLONAL ANTIBODY AGAINST THE PATIENTS WITH SCLC

It is an Innovative way to diagnose with cancer with monoclonal antibody (MAb) in vivo. 1-3 After proving MAb 2F7 (IgG2a) had a higher affinity and better specificity to human small cell lung cancer cells in vitro, 4.5 and gave a very clear cut γ- camera pictures for the xenografts of human small cell lung cancer (SCLC) born by nude mice, 6 the possibility of clinical radioimmunoimaging was studied with the MAb 2F7 and its fragment F (ab') 2.

THE SIGNIFICANCE OF RADIOIMMUNOIMAGING IN THE MANAGEMENT OF CANCER PATIENTS

In this article, the significance of radioimmunoimaging(RII)as a mornitoring means for gastric colorectal and lung cancer patients was reported.By using I-131 or Tc-99m labeled C50(antiCEA)and 3H11(anti human gastric cancer), 86 positive lesions were identified in 50 cancer patiens.77 out of the 86 lesions (89.5%)were true positive, 4 false positive by post-surgecal pathology,biopsy or CT scan.The other 5 remained to be unexplained and needed further follow-up.In our clinical practice,RII with antitumor McAbs suggested its unique value in the management of cancer petients.Totally, 14 occult lesions in 9 cancer petients missed by to other examination were identified by this technique.And owing to the RII findings,all of these patients got proper treatment in time.

RADIOIMMUNOIMAGING OF HUMAN BREAST CANCER XENOGRAFTS IN NUDE MICE BY USING HUMAN ANTI-HUMAN BREAST CANCER MONOCLONAL ANTIBODIES

In this study, the human breast cancer-bearing nude mice model has been established and the radioim-munoimaging was carried out by using human anti-human monoclonal antibodies. The results showed that the breast tumor-bearing nude mice received 131I-McAb-CM-l had a clear image of the xenograft during 4 to 6 days after injection and at the same time T/NT all over 1. 0. The highest T/NT reached 7.1. It demonstrates that McAb-CM-1 can specially combine with breast cancer tissues and hopefully it could be used clinically to improve accurate rate of the early breast cancer diagnosis.

Application of labeled radioimmunoimaging tracing in detecting pulmonary embolism in rabbits after bone cement perfusion and relevant treatment effects

Background During the process of bone cement joint replacement, some patients show a series of complications, such as a sudden drop in blood pressure or dyspnea. The cause of the complication is considered to be due to emboli caused by the femur prosthesis insertion. The purpose of the present study was to detect the pulmonary embolism in rabbits after bone cement perfusion by radioimmunoimaging, and to explore its protective measures. Methods Forty rabbits, 2.5-3.0 kg weight, were randomly assigned to four groups, with ten rabbits in each group. Group I （no intervention）： Bone cement perfusion was done after medullary cavity reaming and pressurizing. Group II （epinephrine hydrochlorideintervention）： The medullary cavity was rinsed with a 1：10000 normal saline-diluted epinephrine hydrochloride solution followed by bone cement perfusion after medullary cavity reaming and pressurizing. Group III （fibrin sealant intervention）： The medullary cavity was precoated with fibrin sealant followed by bone cement perfusion after medullary cavity reaming and pressurizing. Group IV （blank control group）： The medullary cavity was not perfused with bone cement after reaming. In each group, the rabbits underwent femoral head resection and medullary cavity reaming. Before bone cement perfusion, 2 ml of developing tracer was injected through the ear vein. Radionuclide imaging was performed at 60, 120, and 180 minutes after bone cement perfusion, and the pulmonary radioactivity in vivo was measured. The rabbits were immediately sacrificed, and the pulmonary tissue was removed and its radioactivity was measured in vitro. Pulmonary tissue was then fixed and the pulmonary embolism and the associated pathological changes were observed. Results The pulmonary radioactivity in vivo was measured at 60, 120, and 180 minutes after bone cement perfusion. The radioactivities of the four groups were 11.67±2.16, 14.59±2.92 and 18.43±4.83 in group I; 8.37±3.05, 10.35±2.24 and 11.48±2.96 in group Ⅱ; 3.91±1.19, 5.53±2.95 and 7.25±1.26 in group Ⅲ; 1.04±0.35, 1.14±0.87 and 1.43±0.97 in group Ⅳ The radioactivities of groups Ⅰ, Ⅱ, Ⅲat 60, 120 and 180 minutes were significantly higher than group Ⅳ（P 〈0.05）. The pulmonary embolism could be detected. Pretreatment with epinephrine hydrochloride and fibrin sealant significantly decreased the pulmonary radioactivity in group Ⅱand group Ⅲ, but it was still higher than in the group Ⅳ. Conclusions Radioimmunoimaging is an alternative method for the dynamic observation of rabbit pulmonary embolism after bone cement perfusion. Radioimmunoimaging is the optional way to evaluate the effect of pretreatment with epinephrine hydrochloride or fibrin sealant on pulmonary embolism after bone cement perfusion.

Preliminary radioimmunoimaging and biodistribution of ^131iodine-labeled single-chain antibody fragment against progastrin-releasing peptide（31-98） in small cell lung cancer xenografts

Background Monoclonal antibodies （mAbs） such as DD3,raised against progastrin-releasing peptide（31-98） （ProGRP（31-98）） antigen,have been used to target small cell lung cancer （SCLC）.However,as an intact mAb,DD3 is cleared slowly from the body,with an optimal radioimmunoimaging time of 72 hours.More recently,a singlechain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research.Thereby,it potentially increases the radioimmunoimaging efficacy.However,there have been few studies with this antibody fragment.The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of 1311I-anti-ProGRP（31-98）scFv in nude mice bearing SCLC xenografts.Methods Anti-ProGRP（31-98） scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry.131I-anti-ProGRP（31-98） scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram （%ID/g） in various organs was calculated.Similarly,the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated.After injection of 131I-anti-ProGRP（31-98） scFv,treated mice were imaged at 1,24,and 30 hours.Then the tumor/base ratios were calculated.Results ProGRP was highly expressed in NCI-H446 cells and xenograft tissue.The metabolism of 131I-anti-ProGRP（31-98） scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes,respectively.The %ID/g of 131I-anti-ProGRP（31-98） scFv in xenografts was much higher than in healthy tissues at 12 hours after injection,reaching a maximum of （5.38±0.92） %ID/g at 24 hours.Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours,with 24 hours proving optimal.Conclusion 131I-anti-ProGRP（31-98）scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance,indicating thatit could be a promising agent for SCLC radioimmunoimaging.

Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment:a radioimmunoconjugate for hepatocellular carcinoma imaging

AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）2 with 99mTc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）2.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat 99mTc-labeled HAb18 F（ab’）2 in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of 99mTc-labeled HAb18 F（ab’）2.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION 99mTc-HAb18 F（ab’）2 conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.

Construction of Human ScFv Phage Display Library against Ovarian Tumor

In order to construct a single chain fragment variable （ScFv） phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes （VH） and light chain variable region genes （VL） were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 10^9 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 μ 10^12 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Localization and Biodistribution of Conjugate ATG－Dex－DNR in Nude Mice as Models for Human Leukemia

I-labelled anti-thymoglobuline (ATG), 131 I-labelled immunoconjugate ATG-Dex-DNR and 131 I-labelled Ts-MoAb as control antibody, respectively.were injected by intraperitoneal (i. p.) administration into nude mice used as models for human T-cell leukemia. SPECT imaging was performed from day 1 to day 8 following i. p. injection. The results showed that radioimmunoimaging of buman tumor xenografts was clearest day 3 after injection in both of ATG and ATG-Dex-DNR groups, whereas it's not the case in Ts-MoAb group. Nude mice were killed 8th day after injection with antibody or conjugate. The tumor, as well as different dissected normal organs including heart, liver, lungs, kidney, femur and intestine, were harvested, weighed precisely, and radioiodine-counted. T/NT ratios in experimental group was greater than 1. 0 (ranged from 1. 246-7. 865). and in control group they were less than 1. 0 (ranged from 0. 263-0. 757, except for tumor/femur ratio). Our results indicated that ATG and ATG-Dex-DNR had specific affinity to cell line of Tcell leukemia CEM.

Clinical application of radioimmunoi maging with (99)~m Tc-BDI-1in the diagnosis of bladder cancer

To evaluate the clinical application of radioimmunoimaging (Rll ) with 99m Tc-BDI-1 in thediagnosis of bladder cancer. Methods 32 pstients with bladder cancer and 5 with normal bladder were studied. Rll was performed 1 hour after intravesical administration of 111-222 MBq 99m Tc-BDI-1 followed by washing out and perfusing bladder with 50 ml PBS. The radioactivity ratio of target over background (C T/C B) was calculated. Results The radiochemical purity of 99m Tc-BDI-1 Wasgreater than 95%. Rll showed radioactive accumulation area for most of bladder c0ancers but no radio-active concentration for normal bladder. The sensitivity and specificity were 91 .7% and 81 .8% respectively with the assignment of the positive criterion of C T/C B≥ 1.40. There was no statistical difference in sensitivity between tumors with diameters ≥ 1 cm (1. 0-4.2 cm) and < 1 cm (0.2-0.8 cm). C T/C B was related to the pathological grade (G 1-G 3) of the tumor. Conclusions Rll by intravesical administration provides a new noninvasive method for the diagnosis of bladder cancer in morphology and in nature of tumor with high sensitivity and specificity. It may be used for the early diagnosis of bladder cancer, follow-up after surgery, and diagnosis of in situ tumor.