Combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids

Adenoviral-mediated gene transfer is suboptimal in human glioma and limits in vivo gene therapy approaches. There is a need for targeted vectors able to enhance gene transfer into the tumor as well as to lower the viral load in the surrounding normal tissues. We evaluated primary human tumor samples by immunohistochemistry and fluorescence-activated cell sorter for express ion of the Coxsackie-adenovirus receptor and other antigens with potential utility to redirect adenoviruses (Ads) to gliomas, In the majority of the s amples, Coxsackie-adenovirus receptor expression was low. This correlated w ith inefficient gene transfer in vitro, Epidermal growth factor receptor (E GFR) and alpha (v)beta (5) integrins were often highly, but heterogeneously , expressed. We hypothesized that these receptors, overexpressed in tumor b ut not in normal brain, could serve as independent binding sites for altern ative pathways of infection with targeted Ads, We examined this, using Ads that expressed the luciferase reporter gene under the cytomegalovirus promo ter, Targeting to the EGFR was performed with a single-chain bispecific ant ibody directed against the human EGFR and against the fiber knob of the Ad. Targeting to the (alpha (v) integrins was performed by insertion of an int egrin-binding sequence, RGD-4C, in the HI-loop of the Ad. Increased lucifer ase gene transfer in primary glioma cells was observed in 8 of 13 samples w ith EGFR-targeting (2-11 times enhancement; median, 6) and in all of the sa mples with RGD-targeting (2-42 times enhancement; median, 12), Combining th e two targeting motifs further enhanced the gene transfer in primary glioma cells in an additive manner (3-56 times; median, 20), The double-targeted Ads also strongly augmented gene transfer into organotypic glioma spheroids . Conversely, gene transfer into normal brain explants was reduced dramatic ally using Ads targeted to the tumor, Our findings demonstrate the feasibil ity and benefit of binding multiple ligands to the adenoviral fiber knob, T hese vectors have a great potential for clinical use in the context of tumo rs that are usually heterogeneous for target antigen expression at the sing le-cell level.