The Preliminary Study Of^99mTc-EGFR Nanobodies For Tumor Radioimmunoimaging

ObjectiveIn this study, epidermal growth factor receptor (EGFR) Nanobodies (monomer and multivalent polymers), genetically engineered antibodies, were labeled with the radionuclide99mTc, and prepared as new molecular probes for tumor diagnosis with specific targeting of cancer cell membranes EGFR. The binding of99mTc-EGFR Nanobodies in different cancer cells in vitro, and SPECT imaging characteristics of99mTc-EGFR Nanobodies (monomer, tetramer, pentamer and heptamer) in different xenografted tumor models in vivo were observed. More importantly is the choice of the appropriate one from the4types of99mTc-EGFR Nanobodies for radioimmunoimaging, to analyze possible mechanisms and to provide an experimental basis for clinical applications of this kind of molecular imaging agents. MethodsHuman epidermal carcinoma A431cells, lung adenocarcinoma cell line A549, large cell cancer of the lung NCI-H460cells, human ovarian cancer SKOV3cells, breast cancer MDA-MB-453cells, prostate cancer DU-145cells, choroidal melanoma OCM-1cells were cultured in vitro. The expressions of EGFR on cancer cells membrane were detected by flow cytometry, immunocytochemistry, immunofluorescence cytochemistry. The semi-quantitative analysis of EGFR protein in different cancer cells were detected by western blot.EGFR Nanobodies were labeled through their C-terminal hexahistidine-tag conjugated with technetium-99m tricarbonyl intermediate. The labeled compounds were purified by ultra centrifugal filter methods. The labeling efficiency was determined by thin layer chromatography (TLC), which using silica gel TLC strips up expanded with two mobile phase of0.9%sodium chloride solution and acetone. Four kinds of cancer cells (A431, A549, NCI-H460and OCM-1) were seeded in12-well plates and cultured to adhere overnight. The cells were added to100μL mixtures of0.5%HEPES and0.5%BSA for blocking the nonspecific binding. The groups were divided into99mTc-EGFR Nanobody monomer,99mTc-EGFR Nanobody tetramer,99mTc-EGFR Nanobody pentamer and99mTc-EGFR Nanobody heptamer and the subgroup containing competitive inhibitor, which was pre-incubated with2μg corresponding unlabeled EGFR Nanobody (10μL) for1h at37℃. Subsequently,1μCi (37KBq)99mTc-EGFR Nanobody (1μg,10μL) were added and incubated for1h at37℃in incubator.99mTcO4-was set as negative control group. Each group had four parallel wells. The cells and the supernatant fluid of each well were separated and collected, respectively. The binding rate of99mTc-EGFR Nanobodies were calculated through radioactive counts per minutes (CPM) of above components.The fifty-five male BLAB/C-nu mice xenograft model were prepared by subcutaneous injection of0.5-1.0×107cancer cells (A431, A549, SKOV3and OCM-1) in left upper limb near the armpit. The imaging experiments were performed until the tumor was growing to0.8-1.0cm of diameter. Targeting of99mTc-EGFR Nanobodies in xenografted tumors were tested by SPECT with a pinhole collimator at different time points (30,60,90,120and180min) after injection of7.4-18.5MBq (approximately10μg). The characteristics of radioactive distribution were observed, and the ratios of tumor and non-tumor were analyzed through drawing regions of interest (ROT). The EGFR expression in tumor tissues were detected by immunochemistry (IHC) after imaging finished. ResultsIn vitro, there were different expressions of EGFR in7kinds of cancer cells, which were in good condition, different shapes, and rapid proliferation. A431, A549, NCI-H460and SKOV3cells have higher EGFR expression, MDA-MB-453and DU145cells have lower and OCM-1cells have negative.The method of99mTc labeled EGFR Nanobodies is simple and feasible. The radiochemical purity was greater than95%after ultra filtration and stable in vitro within6h. The binding rate of99mTc-EGFR Nanobodies to A431, A549and NCI-H460cancer cells was significantly higher than99mTcO4-(P<0.05). It can be decreased in varying degrees by using corresponding unlabeled EGFR Nanobodies, part of the differences have statistically significant (P<0.05). EGFR negative expression OCM-1cells have different degrees of binding rate of99mTc-EGFR Nanobodies.The A431xenografts tumor tissues were better displayed after postinjection of99mTc-EGFR Nanobody pentamer, and the maximum T/NT was about2.9at90min after injection. A549, SKOV3and OCM-1xenografts tumor tissues were not clearly displayed at different timepoint after postinjection of99mTc-EGFR Nanobody monomer, tetramer and heptamer, respectively."mTc-EGFR Nanobody monomer was rapidly accumulated and clearanced by kidney and bladder at1h after intravenously injected.99mTc-EGFR Nanobody tetramer and heptamer were mainly accumulated in the liver and partly in the kidneys.ConclusionsThere were some mismatch in vitro and in vivo experiments. The binding of four kinds of99mTc-EGFR Nanobodies to EGFR overexpression cancer cells were higher, and specificity was found by competition inhibitor experiments. In vivo SPECT imaging, only99mTc-EGFR Nanobody pentamer had more accumulation in over expressed EGFR tumor tissues, especially in A431xenografts tumor tissues.99mTc-EGFR Nanobody monomer was rapidly excreted by the kidneys due to its lower molecular weight.99mTc-EGFR Nanobody tetramer and heptamer mainly accumulated in liver, the possible mechanisms are larger molecular weight and lower tumor penetration. Therefore,99mTc-EGFR Nanobody monomer,99mTc-EGFR Nanobody tetramer and heptamer are not appropriate for radioimmunoimaging, and99mTc-EGFR Nanobody pentame has the potential value for the further study.