Adenovirus type 5 uptake by lung adenocarcinoma cells in culture correlates with Ad5 fibre binding is mediated by ?v?1 integrin and can be modulated by changes in ?1 integrin function

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Abstract
Background Recombinant adenoviruses (Ad) have been employed as vectors for a wide variety of gene therapy applications, but their use has been hindered by problems relating to efficacy and safety. The efficiency of Ad‐mediated gene transfer depends on the interaction of the fibre and penton base proteins with their corresponding cell receptors. Ad infection is initiated by the formation of a high affinity complex between the fibre protein and a host cell protein that for most Ad serotypes is CAR (the coxsackie B virus and Ad receptor). A second molecule, the MHC class I, may also be involved in Ad type 2 and Ad type 5 uptake. Ad internalization results from the interaction of the penton base protein with cell surface integrins αvβ3 and αvβ5. In this study, we addressed the interaction between Ad type 5 (Ad5) and its receptors on lung derived adenocarcinoma cells in culture. Methods Using flow cytometry, we determined the level of expression of attachment and internalization receptors that are expressed on the cell surface of A549, H322 and H441 lung‐derived adenocarcinoma cells in culture. The level of αvβ1 cell surface integrin was assessed by immunoprecipitation. Measuring the level of luciferase gene expression at different viral titres quantitated Ad5 uptake by these cells. The kinetics of binding of Ad5 fibre knobs to A549, H322 and H441 cells was assessed in direct binding studies using 125I labelling of purified recombinant Ad5 fibre‐knob domains. In order to assess the functionality of integrins, adhesion assays were performed in the presence or absence of activators of integrin function. In competition experiments, prior to exposure to the virus, the cells were pre‐incubated with purified recombinant Ad5 fibre‐knob domains, function blocking anti‐integrin antibodies, or integrin activating agents, prior to the introduction of luciferase expressing Ad5. Results We found that Ad5‐mediated gene transfer in A549, H322 and H441 adenocarcinoma cells in culture is highly variable and that this variation correlates with specific binding of Ad5 fibre‐knob domain binding to the cell surface. We also found, for the first time, that Ad5 infection is mediated by integrin αvβ1 and that functional activation of β1 integrin by means of the specific anti‐β1 monoclonal antibody, TS2/16, induced increased A549 cell adhesion to fibronectin and vitronectin and also enhanced Ad5 uptake by these cells. Conclusions Our studies demonstrate that the Ad5 fibre‐knob domain interaction with CAR represents a major determinant of Ad5‐mediated gene transfer to lung‐derived adenocarcinoma cells in culture. The finding that integrin αvβ1 is involved in Ad5 infection has implications for the use of recombinant Ad5 vectors for cancer gene therapy, since αvβ1 is expressed at high levels and acts as an alternative vitronectin receptor in many epithelial and some melanoma tumours which express no αvβ3 and constant low levels of αvβ5. The fact that the β1 integrin‐activating antibody TS2/16 can enhance αvβ1‐mediated Ad5 infection suggests that the efficacy of Ad5‐mediated gene transfer might be influenced not only by the level of cell surface expression of integrins but also by their state of activation. Copyright © 2001 John Wiley & Sons, Ltd.