| Objective:Monoclonal antibody D-D3that against pro-gastrin-releasing peptide (ProGRP) wasradiolabeled with99Tcmby directly labeling method and pretargeted method, respectively.To evaluate the distribution and imaging of the product from these two different methodsin healthy nude mice and nude mice bearing human small cell lung cancer (SCLC). Tofurther study whether the imaging of anti-ProGRP monoclonal antibody labeled bypretargeted method can be used as the best way to early diagnosis of small cell lung cancer.Methods:1. The preparation of biotinylated monoclonal antibody D-D3The anti-ProGRP(31-98)monoclonal antibody D-D3was dissolved in sodiumbicarbonate solution, and biotin activated ester was dissolved in dimethyl sulfoxide(DMSO). Then two compounds were mixed at room temperature for3~4hours in thecondition that maximum ratio of biotin activated ester: antibody was8:1, then was purifiedwith SephadexG50chromatography column; The biotinylated degree and the titer andactivity of biotinylated antibody was measured by HABA/Avidin kit and ELISA method,respectively; and the expression of ProGRP protein in target tissues was detected byimmunohistochemistry using biotinylated antibody as the first Antibody.2. DTPA-biotin and monoclonal antibody D-D3labeled with99Tcm2.1Radiolabeling of DTPA-biotin with99Tcm: The DTPA-biotin was dissolved using0.05mol/L PBS, then added SnCl2solution prepared in hydrochloric acid, and99TcmO4-,then shaked evenly at room temperature. The labeled volume and amounts of SnCl2werechanged to explore the optimal condition, The labeling ratio under these differentconditions was detected by paper chromatography.99Tcm-DTPA-biotin was incubated respectively with NS and healthy serum at37℃and radiochemical purity was measured atdifferent time points to determine the stability of labelled product.2.2D-D3radiolabelled with99Tcmby directly labeling method:99Tcm-D-D3wasradiolabeled by reduction method using2-mercaptoethanol, and99Tcm-D-D3was purifiedby gel column separation method; Labelling efficiency and radiochemical purity weremeasured by paper chromatography and the stability of labelled product was measured.Immunocompetence of labelled product was determined with cell conjugation assay.3. Biodistribution studies of labeled antibodies in normal nude mice and nude micebearing tumorNormal nude mice were administrated by three-step pretargeting technology. First,200μg of biotinylated antibody was injected from the tail vein followed by200μg avidinbiotin after one day, and99Tcm-DTPA-biotin0.148MBq (0.004mCi) at the third day.Normal nude mice were sacrificed with cervical dislocation at designated time points ingroup. The blood and major organs were removed, weighed and counted in a gammascintillation counter to determine the%ID/g (percentage of injected dose related to theorgan weight) for each radioactive substance. Establishment of nude mice model bearingSCLC was divided into two groups (pretargeting group and directly labeling group). Thepretargeting group included Group A: first,200μgof biotinylated antibody was injectedfrom the tail vein followed by200μg avidin biotin after one day, and99Tcm-DTPA-biotin0.148MBq (0.004mCi) at the third day. Group B: antibody without biotin was injected thefirst day, the others were same to group A. Group C: NS was injected the first two days, theothers were same to group A. The directly labeling group was Group D:99Tcm-D-D30.148MBq (0.004mCi) was injected directly. The%ID/g and tumor/non-tumor (T/NT) ratio weremeasured through the same way.4. Imaging studies in normal nude mice and nude mice bearing SCLCAfter injection of the radiolabeled products, the sequential SPECT static imaging ofthe healthy nude mice and the nude mice bearing SCLC were performed to observe theimaging efficacy of labeled product using two different methods. The contour of nude micetransplantation tumor in the different imaging phase was delineated by ROI technology,and then tumor/base (T/B) ratios were calculated. Results:1. The average moral ratio of biotin and antibody in biotinylated monoclonal antibodywas2.5under the condition that active biotin ester and antibody in a quality ratio of8:1,and the immunocompetent of biotinylated antibody was about90.92%.2. The labelling rate of99Tcm-DTPA-bition was88.50±1.04%and the radiochemicalpurity was89.45±0.24%. After incubation with NS at37C for8h, the radiochemical puritywas65.46±1.86%and the radiochemical purity was58.54±0.25%at12h. Meanwhile, afterincubation with healthy human serum for8h, the radiochemical purity was70.36±1.46%and the radiochemical purity was60.81±1.01%at12h. The labelling rate of99Tcm-D-D3was78.69±3.11%and the radiochemical purity was92.14±3.55%. After incubation withNS at37C for8h, the radiochemical purity was64.73±0.34%and the radiochemicalpurity was54.45±0.95%at12h. Meanwhile, after incubation with healthy human serumfor8h, the radiochemical purity was65.49±0.45%and the radiochemical purity was52.92±0.84%at12h. The immunobinding rates of NCI-H446cell was82.1±2.45%.3. The vivo distribution of99Tcm-DTPA-biotin in the normal nude mice which wereadministered three-step pretargeting technology was complied with a two-comparmentmodel of plasma dynamics, which showed that the metabolism of99Tcm-DTPA-biotindepended on liver and kidney with rapid elimination in blood, while the brain and muscledid not uptake much. Group A nude mice bearing SCLC had rapid uptake of99Tcm-DTPA-biotin and got a high tumor/blood ratio. Group B without antibodypretargeting showed the rapid elimination in blood, while less radioactivity in tumor.Group C with direct injection of99Tcm-DTPA-biotin had the tumor uptake same withbackground, which has the same value of blood and tumor, owing to no biotin-avidinpre-positioning and amplification. Group D which used the directly labeling method tooklong time to achieve high tumor/blood ratio.4. Radioimmunoimaging results: There were no positive imaging findings in normalnude mice and nude mice with B, C groups. Clear tumor image of Group A was obtainedat4h after injection with99Tcm-DTPA-bition. The radioactivity in tumor site of Group Dwas accumulated gradually with time and a visible tumor image was acquired at8h. Conclusion:1. Monoclonal antibody can maintain a good immunocompetence after beingcombined with a certain amount of biotin molecules.2. The key factor affecting the rate of biotin-labeled was SnCl2content, while themark volume and PH also affected. The optimal labeling conditions included the totallabeled volume100uL, SnCl25ug and PH7.4.99Tcm-DTPA-biotin and99Tcm-D-D3havecertain stability in vivo and vitro.3. Pretargeting technology has specific enhancement and signal amplification effecton tumor, which can increase the target tumor uptake of99Tcmand superior to the directlylabeling method.It provides a new approach to immune molecule imaging. |
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