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Author
Garcia Torrado, Alejandro
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Abstract
Glioblastoma (GBM) is the most aggressive primary brain tumor, and life expectancy is 15-20 months after diagnosis. The narrow therapeutic index of chemotherapies, the difficulty to cross the blood-brain barrier in the tumor margins and the heterogeneity of the cells in tumor mass, decrease the effectiveness of current treatments. This master’s research work is part of a European project that aims to develop a targeted gene delivery system as a novel treatment for GBM.
Herein we generate targeting ligands to direct the gene delivery system to tumor cells overexpressing the epidermal growth factor receptor (EGFR) with maximal efficiency. For this purpose, we select two antibody analogs, a nanobody and a Designed Ankyrin Repeat Protein (DARPin), and we devise a strategy to conjugate them to the nanoparticle in an oriented manner. This strategy consists in incorporating reactive non-canonical amino acids (ncAAs) or cysteine residues at selected sites that should enable the conjugation without affecting antigen binding.
Although we managed to clone the amber stop codons necessary to encode the ncAAs in the nanobody, this protein was not expressed under any of the wide range of conditions attempted. Conversely, we succeeded in expressing and purifying the anti-EGFR DARPin. We then assessed the binding capacity of the DARPin on cells expressing different levels of EGFR. We found that antibody aparent Kd ranged from 0.9 nM to 35 nM in good agreement with expected EGFR expression. Competition assays showed 75% decrease in binding of the labelled DARPin when pre-incubating cells with the non-labeled protein. These results confirmed that the protein was correctly folded and that interaction was dependent on EGFR. Moreover, for these binding experiments we used the DARPin fused to glutathione S-trasnferase (GST), which indicated that the DARPin has the capacity to target large cargoes to EGFR+ cells.
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