Imaging And Therapy Effect Of¹⁸⁸Re Labeled Small-molecular Peptides Derived From VEGF In A549Tumor Bearing Nude Mice

Backgrounds and Objectives：Radionuclide imaging and targeted therapy is a research focus in nuclear medicine.Molecular probes or tumor-targeted drugs deliver different kinds of radioactive nuclide tothe tumor area, and achieve the purpose of tumor imaging and therapy. Peptide-basedradiopharmaceutical for receptor-mediated imaging and therapy develops rapidly due to itsmany peculiar advantages such as high receptor binding affinity, high tumor penetration,favorable pharmacokinetics and few side effects. The prior and the most important step fordeveloping tumor-targeted peptides is to select the appropriate receptor that has a hightumor/non-tumor ratio.Many receptors (over)expressed on tumor cells are widely under investigation fortumor diagnosis and therapy. Among all the receptors, Vascular Endothelial Growth FactorReceptor (VEGFR) is the critical factor in tumor angiogenesis and has been shown to beover-expressed by many kinds of malignant tumor such as lung cancer, breast cancer andneuroglioma. Many of these malignancies have a poor prognosis with advanced disease,current treatments are suboptimal, and therefore there is heightened interest in developingnovel treatments, of which the utilization of the over-expression/ectopic expression ofVEGFR could be one useful approach.VEGF125-136is a dodecapeptide that binds to VEGFR specifically and could block thesignal pathway of VEGFR. In the early stage of our study, we radiolabelled VEGF125-136andproved its tumor targeting ability. We further modified this peptide with bioinformatics andgot two small-molecule peptides (QKRKRKKSRKKH, RKRKRKKSRYIVLS). These twonew peptides showed higher affinities to A549cells than VEGF125-136both in vitro and invivo. In addition these two peptides could inhibit the growth of A549cells with inhibitionrates of40%and70%, respectively. This study intends to radiolabelled these two peptides with radionuclide188Re, toexplore their imaging and therapy effect in A549tumor-bearing nude mice.Methods：1. Preperation of188Re labeled peptides: The two peptides were radiolabelled with188Re by using three bifunctional chelating agents (EC, MAG3and HYNIC), theradiolabeling rates and specific activities were identified, respectively. The stabilities of188Re-EC-peptides were tested both in saline and in human plasma.2. Construction of VEGFR-1over-expression model: Lentivirus was used as a vectorto transport VEGFR-1gene to A549cells. Immunofluorescence was used to identify thetransfection efficiency.RT-PCR and western blot were used to detect the expression ofVEGFR-1both in transcriptional and expressional levels.3. Recepter-mediated binding capacities of188Re labeled peptides: Receptorcompetitive binding assay was used to study the peptides’ binding affinities to A549cells.VEGFR-1receptor saturation binding assay and receptor-mediated internalization wereused to verify the specificity of binding assays.4. Biodistribution and SPECT planar imaging of188Re labeled peptides in A549tumorbearing nude mice: Biodistribution were studied by injecting labeled peptides into A549tumor bearing nude mice through caudal vein. At different time point, tumor and someorgans were collected and weighted. The radioactivity of each sample was measured andpercentage injected dose per gram tissue (%ID/g) and tumor/muscle ratio was calculated.Conventional planar imaging of SPECT was performed to detect the image of xenografted.5. Therapeutic effect of188Re labeled peptides in A549tumor bearing nude mice:60tumor bearing nude mice were divided into treatment groups and controlgroups.Corresponding drugs were injected through caudal vein and survival analysis, tumorgrowth curve, necrosis and apoptosis of tumor cells were used to evaluate the therapyeffect.Results：1. The labeling rates of188Re labeled EC-peptides were (85.34±2.05)%and(90.9±3.45)%respectively, which were significantly higher than the MAG3, HYNICconjugated peptides. The188Re-EC-peptides showed favorable stabilities in saline andhuman plasma. 2. Immunofluorescence showed strong fluorescence signal in Lenti-VEGFR-1A549cells, which indicated high transfection efficiency. RT-PCR and western blot confirmed thatthe VEGFR-1over expression model was constructed successfully.3. Receptor competitive binding assay proved the188Re labeled peptides could bind toA549cells in vitro. VEGFR-1receptor saturation binding assay showed A549cells withVEGFR-1overexpressed bound with more amount of labeled peptides, which suggested thebinding site was mediated by VEGFR-1. Internalization assays showed the peptides couldpass through cell membranes by the internalization of VEGFR-1.4. Xenograft tumors were clear visible in2hours and6hours SPECT imaging. Kidneyand bladder were the major organs of excretion, low accumulation of radioactivity werefound in brain, lung, bone and muscle. The T/NT ratio were (4.67±1.2),(4.91±1.23) at2hand (5.87±1.3),(5.46±0.87) at6h，which were significantly higher than188Re-VEGF125-136.5. Compared with control groups,188Re labeled peptides could significantly prolongthe survival time of the nude mice and inhibit the growth of tumor. There were morenecrosis regions and apoptotic cells in A549xenograft tumors treated with radiolabeledpeptides..Conclusion：1. Ethylene Cysteine proved to be a favorable biofunctional chelating agent for188Reradiolabeling due to its simple radiolabeling condition and high labeling rate.2. Radiolabeled peptides could bind to the VEGFR-1with high affinities and enter intocells by the VEGFR-1mediated internalization.3.188Re labeled peptides accumulated in the tumor site with high tumor/muscle ratio invivo.4.188Re labeled peptides possesses anti-cancer properties with radiation-inducedcytotoxicity in the xenograft model of A549cells.