Targeting of adenovirus serotype 5 infectivity to EGFRvIII-expressing cells using a bispecific CAR-scFv.

Abstract

A mutant form of the Epidermal Growth Factor Receptor (EGFR), known as EGFR version III, is over-expressed exclusively on various forms of cancer cells. MR1, a single chain antibody variable domain fragment (scFv), is able to bind with high affinity and specificity to EGFRvIII and not to the wild-type receptor. The purpose of this study was to investigate a possible means of developing an adenoviral vector (based on subclass C viruses) that can utilize MR1's targeting ability towards EGFRvIII to infect only cancer cells that express this receptor. Adenovirus infectivity is primarily mediated by binding of the globular carboxyl terminus of its fiber protein, known as the knob domain, to the coxsackievirus and adenovirus receptor (CAR), which is expressed on various cell types. Although we attempted three different approaches to try and target the virus using MR1, including direct incorporation of the scFv into the knob domain of adenovirus, it was the creation of a fusion protein consisting of the extracellular domain of CAR linked to MR1 that suggested increased infectivity to EGFRvIII expressing cells. According to ELISA analysis, the CAR/MR1 fusion protein is able to retain the two independent binding affinities of its composite subunits (bispecificity) as strongly as the two independent proteins do for the knob domain and EGFRvIII, respectively. Additionally, a U87MG EGFRvIII transduced cell line, as opposed to its parental U87MG cell line, showed increased infectivity by adenovirus when incubated with CAR/MR1. These findings suggest that CAR/MR1 is able to bind to and re-direct infection of adenovirus to EGFRvIII expressing cells.