Molecular Imaging Application Of Radioiodinated Anti-egfr Human Fab On Egfr-overexpressing Tumor Xenografts

The epidermal growth factor receptor (EGFR; ErbB1) belongs to the group of the tyrosine kinase receptor. Numerous studies have shown a correlation between EGFR overexpression and tumor growth, angiogenesis, invasion, metastasis, and apoptosis inhibition. Clinical data have also confirmed that increased EGFR expression is associated with more aggressive disease, resistance to chemotherapy and radiotherapy, poor prognosis and survival. The role of EGFR in carcinogenesis and its localization on the cell surface as a transmembrane protein make it an ideal target for cancer immunotherapy.Currently, the most commonly used method for detecting EGFR levels is staining by immunohistochemistry (IHC), which has been applied in a majority of published studies. However, there are considerable variations between laboratories in the execution of this method in terms of application of reagents and even differences in the definitions used for high expression or overexpression. Molecular imaging with single-photon emission-computed tomography (SPECT) has been considered one of the most promising ways to show a molecule's expression more directly and exactly than traditional IHC can do.We previously generated a fully human anti-EGFR Fab which affinity is 30 nM. And in this study, we radioiodinated this Fab and evaluated its ability to identify tumors having different EGFR expression levels in xenograft models as a potential molecular imaging reagent for non-invasive diagnosis. The study was designed as following:1. To generate and purify large quantities of anti-EGFR fully human Fab fragments.2. To select human malignant tumor cell lines expressing different EGFR levels, and set up the corresponding xenografts in nude mice. 3. To radioiodinated Fab with 125I and determine 125I-Fab's immunoactivity.4. To evaluate 125I-Fab's diagnostic capability in tumor xenografts.Methods:1. Large scale of Fab was expressed in culture supernate and E.coli's periplasm. The pure Fab was achieved by two-step purification, which was His-tag affinity chromatography and ion-exchange chromatography, respectively.2. The human malignant cell lines A431, U118, U87, M14, MCF-7 and DBTRG were cultured and their total RNA and protein were collected. In order to select the cell lines expressing high, moderate or low EGFR, the cells were compared by RT-PCR and Western blot.3. To determine whether the Fab was able to recognize and bind the EGFR in native conformation on cell surfaces, we performed immunoprecipitation and FACS assay.4. The Fab was radioiodinated with 125I by classic Chloramine-T method, and the immunoactivity of conjugated Fab was confirmed by ELISA and?counting assays.5. The cells expressing different EGFR levels were injected into the right forelimbs of nude mice subcutaneously to set up the corresponding xenografts.?6. About 300?Ci 125I-Fab was injected into mice by tail vein and the whole-body images of each mouse were acquired at different time points post-injection within 3 days by SPECT.7. Computer-assisted region-of-interest (ROI) analysis were drawn around the tumor and in the contralateral region located in the left forelimb (background) to achieve the corresponding values.Result:1. About 20 mg anti-EGFR fully human Fab protein was achieved with the purity over 95 %.2. Cell lines A431, U118 and M14 were selected representing high, moderate and low EGFR expressing levels. The corresponding xenografts were set up successfully. 3. The Fab was radioiodinated and the labeling efficiency was about 60 %and the radiochemical purity was above 97.82%. The resultant 125I-Fab was still able to recognize EGFR specifically.4. The 125I-Fab accumulated only in tumors with high or moderate EGFR expression as shown by SPECT. Moreover it could also discriminate EGFR levels in vivo by ROI assay.Conclusion:We characterized six human tumor cell lines having different levels of EGFR expression, and we selected A431, U118, and M14 for a molecular imaging study with fully human anti-EGFR 125I-Fab. The 125I-Fab had reasonable antigen binding capability and accumulated only in tumors with high or moderate EGFR expression. Moreover, the acquired scintigrams could measure EGFR's density on the tumors, making it a potential reagent for imaging diagnosis of EGFR overexpressing tumors.