144. In Vivo Targeting hTfR and EGFR Using Aptamers

Aptamers which bind cell surface receptors represent an exciting and potentially important class of ligands for the development of diagnostics and therapeutics. However, if aptamers are to be utilized in vivo, and more importantly for the targeted delivery of therapeutic cargoes, the molecular function must be robust, especially with regard to specificity and activity. Because assay formats for determining the binding specificity of aptamers to their cell surface targets vary greatly, and because some molecules have yet to be tested in vivo, it remains difficult to compare the function of these molecules and to identify which candidate molecules could be used for future development as both diagnostics and therapeutics. In order to assess this, we have chemically synthesized 15 different aptamers from the literature which target a range of human cell surface receptors associated with cancers. These include aptamers targeting EGFR, EPCAM, nucleolin, PSMA, PTK7 and hTfR, as well as some aptamers which have been derived from variations on ‘whole cell SELEX’ or similar approaches against cancer cells lines. Using standardized conditions, we assayed each aptamer in vitro using flow cytometry on a panel of different cancer cell lines to compare relative binding affinity and specificity. Molecules which function in vitro were subsequently assessed for tumor targeting capabilities in vivo using near-infrared imaging. Interestingly, some molecules which function robustly in vitro demonstrate little to no activity in vivo. Of the molecules tested,three demonstrate significant activity in vivo. These include an anti-EGFR aptamer, an anti-hTfR aptamer and a molecule identified via an ‘internalization selection method’ that is readily internalized by cancer cells, but shows little uptake by normal tissue. Taken as a whole, our data demonstrate some surprising differences in the apparent affinities and specificities of different aptamers when assayed under these standardized conditions. They also provide us with molecules which we can now think about transitioning for in vivo applications. Data on using these molecules to deliver cytotoxic drugs in vitro and in vivo will be presented.