Avidity effects of MinE07, an anti-EGFR aptamer, on binding to A431 cells


Avidity effects of MinE07, an anti-EGFR aptamer, on binding to A431 cells

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Title: Avidity effects of MinE07, an anti-EGFR aptamer, on binding to A431 cells
Author: Avutu, Viswatej
Abstract: Cell growth, differentiation, and proliferation are all carefully regulated processes. Disruptions in these processes are often associated with malignant tumors. The epidermal growth factor receptor (EFGR), part of the ErbB family of receptors, is known to play a pivotal role in regulating numerous cell growth processes including morphology, differentiation, proliferation, and apoptosis in certain cell types. Overexpression or elevated levels of EGFR activity is associated with many different types of cancers. Numerous targeted anti-EGFR therapies have been developed, including monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors. Apatmers provide an attractive alternative to monoclonal antibodies due to their ease of synthesis and lack of immunogenicity. J18 and E07 are two aptamers which were selected for against EGFR. Due to 2’-fluoro pyrimidines modification, E07 was chosen for in vivo applications. E07 was further remodeled to a minimal length construct that still retained binding affinity for EGFR. A cell growth assay using E07 and the anti-EGFR mAb, Cetuximab, revealed that a much larger dose of aptamer was needed to achieve the same level of growth inhibition as Cetuximab. In an effort to improve the efficiency of E07, an experiment was designed to improve the Kd of E07 with avidity effects – supradditive effects observed upon dimerization or multimerization of monomers. Having been observed with peptides, it was hypothesized that nucleic acids might also display such avidity effects. Five dimeric constructs of the minimized E07 (MinE07) aptamer were tested using flow cytometry assays on A431 cells. Two variables were also tested: the orientation of the monomers in the dimeric construct and the distance separating the two monomers. Constructs were assembled in three different schemes. First, DNA organizers containing fluorophores (fluorescein) were used to direct formation. In a second strategy, the extensions added to MinE07 directed the formation of the dimer. Lastly, the dimer was created via transcription off of an ssDNA template. FACS data revealed that none of the constructs significantly produced avidity effects. However, Construct 3 did inconsistently demonstrate slight avidity effects. Depending on the conditions of the A431 cells, the cell surface and subsequent assays can change dramatically. The head-totail orientation proved to be more promising in permitting avidity effects. Because no significant avidity effects were seen, the effect of intra-aptamer distance on binding affinity could not truly be studied.
Department: Biochemistry
Subject: College of Natural Sciences epidermal growth factor receptor aptamers EGFR avidity effects E07 MinE07 A431 cells
URI: http://hdl.handle.net/2152/13407
Date: 2010

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