| BackgroundRadioimmunoimagine (RII) plays an important role in early tumor diagnosis.To date,there were only five RII drugs approved by FDA in clinical studies. However, the use of RII can be limited by the poor diagnostic efficiency and high false negative results. Therefore, it is a cornerstone for RII to futher develop a novel antibody probe targeting tumor. Neuropilin-1(NRP-1), a co-receptor for Semaphorin3A (Sema3A) and vascular endothelial growth factor (VEGF), plays an important role in neoplastic processes of angiogenesis, growth and metastatic spread. Overexpression of NRP-1has been frequently detected in a wide range of human tumors, for example, lung cancer, gastric cancer, colon cancer, pancreas cnacer, gliomas, etc. Futhermore, increasing evidence has demonstrated that there is a correlation between NRP-1overexpression and poor prognosis, and short survival for some cancer types. Therefore, NRP-1has become an attractive target for cancer molecular imaging and therapy. In the study, to further develop an anti-NRP-1antibody-based probe for future clinical translation, an monoclonal antibody--A6targeting to NRP-1was labeled with131I by Iodogen method, The resulting probe,131I-A6-MAb, was then evaluated in tomor models bearing U87MG cell to study whether the SPECT probe can be used for imaging of NRP-1positive tuomor in vivo.Methods(1) An anti-NRP-1mAb-A6was obtained by rProtein A column purification from the ascites, and its affinity was identified by ELISA methods.(2) A6was labeled with131I by Iodogen method under the optimum labeling conditions, then the labeling efficiency and the stability were detected in vivo by TLC.(3) The bioactivity of13II-A6in U87MG cells was measured by cell uptake assay, binding test, internalization assay.(4) The nude mice bearing human U87MG glioma cells were randomly divided into4groups with4in each group. The nude mice were sacrificed by cervical dislocation and dissected at24,48,72,96,120h respectively after intravenous injection of7.4MBq131I-A6. The biodistribution of the agent was measures as%ID/g..(5) SPECT/CT imaging was performed in8mice including unblocking groud (n=4) and blocking group (n=4) at different time (12.24.48.72.96.120h) after injection of7.4MBq131I-A6MAb with or without a100-fold (700μg) unlabeled A6MAb.Results(1) The purity of purified anti-NRP-1mAb-A6was more than95%, concentration was4mg/mL.(2) The Paper chromatography showed that the labeling efficiency and the radiochemical purity of131I-labeled A6were98%. The stability of131I-A6-MAb in PBS buffer solution were maintained over85%in96h;(3)131I-A6-MAb had rapid accumulation in U87MG cells and reached the highest value of15.80±1.30%at1h, and it bound to NRP-1with low nanomolar affinity (KD=1.67±0.14nM) in U87MG cells. The internalization rate of131I-A6-MAb reached highest25.7±3.0%at8h(4) The in-vivo biodistribution study showed that131I-A6-MAb displayed relatively high levels of radioactivity accumulation in U87MG tumors. The value of tumor uptake for131I-A6-MAb were6.0±1.2.8.8±1.7.11.4±2.0.8.2±1.4ID%/g at24h in unblocking and blocking (2.5.5、10mg/kg) groud, respectively. Furthermore, in5mg/kg blocking groud, the tumor uptake of31I-A6-MAb was the most highest, however in lOmg/kg blocking groud, the tumor uptake decreased. The liver uptake were7.6±1.5.6.4±0.9.5.8±0.7.6±0.8ID%/g respectively. As time progressed, the radioactivity in the tumor and other normal tissue (except thyroid) was decreased,but a faster clearance of131I-MAb was observed in the blood. At120h p.i., the radioactivity in the blood were0.8±0.1.1.2±0.3、2.0±0.3.6.0±1.3ID%/g, and the tumor:blood ratios (T/B) were1.8±0.5.2.3±0.7.2.4±0.6.0.9±0.2; (5) The SPECT imaging showed the tumor uptake of31I-A6-MAb increased from12h to24h gradually after injection. Co-injection of an excess (700μg) of unlabeled A6antibody resulted in significant reduction in tumor uptake.Conclusion(1) We developed a anti-NRP-1antibody A6with high affinity and purity a with an affinity chromatography purification method(2)131I-A6-MAb can be easily synthesized by Iodogen method with high radiochemical purity. The specific tumor uptake of131I-A6-MAb, which correlates with NRP-1expression in gliomas, makes it a new promising tumor targeted radiotracer.(3) Saturation the uptake of131I-A6-MAb in normal tissue could increase the tumor uptake, and5mg/kg may be the optimal dose. |
PDF Full Text Request