Abstract
1487
Objectives: The internalization of antibody is pivotal for anti-cancer immunoconjugates to deliver the drug inside tumor cells. In our previous studies, anti-EGFR antibody LA22 conjugated mitomycin C (LA22-MMC) showed significant in vitro cytotoxicity and in vivo tumor growth inhibition. LA22-MMC killed cells more efficiently than the naked LA22 and Erbitux-MMC in both A431 and A549 cells. We supposed that it was caused by the rapid internalization of LA22. In this study, we evaluate the internalization and tumor uptake of LA22. Methods: LA22 was radiolabeled with 125I. The immunoreactivity of LA22 after iodination, the Kd value of LA22 to EGFR (epidermal growth factor receptor), and the internalization rate of 125I-LA22 were determined in A549 cells. Internalization of unlabeled LA22 was also investigated by an indirect immunofluorescence assay using a confocal microscope. The in vivo tumor uptake of 125I-LA22 was evaluated by both γ-imaging and biodistribution studies in nude mice bearing A549 human lung cancer xenografts. To analyze in vivo internalization of 125I-LA22, tumor tissues were excised and disaggregated, and tumor cell suspensions were obtained. The cell surface bound and internalized activities were measured in a γ-counter. Results: The immunoreactivity of 125I-LA22 was 83%; LA22 binds to EGFR with high affinity (Kd=0.69±0.13nM). 125I-LA22 internalized rapidly into A549 cells (maximum of 65.8% at 20min) and stayed saturated until 60 min. The internalized antibody accumulated in the cytoplasm with coarse punctates by confocal imaging. The clear γ images of xenografted tumors were obtained at 72 h postinjection for 125I-LA22. The biodistribution studies revealed that the tumor uptake of 125I-LA22 was 8.00±0.61 %ID/g at 72 h postinjection (control mIgG was 2.19±0.37 %ID/g). The quantitative uptake (%ID/g tumor) of 125I-LA22 for ex vivo tumor cells, cell surfaces and the intracellular were 1.23±0.08, 0.46±0.05 and 0.78±0.08, respectively, and the in vivo internalization ratio was calculated to be 64.06%. Conclusions: This study demonstrates the in vitro internalization property and in vivo tumor uptake of LA22. The rapid internalization of LA22 makes it a promising vehicle for cancer-targeted drugs delivery.
- Society of Nuclear Medicine, Inc.