Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

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Summary Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (a-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUCo-144) in all tissues but there were significant differences (variance ratio test, F=27.95, P<0.001) between the proportional increases in AUCo144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCbl..d by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissuespecific fashion.

Of all the polymers applied to molecule altering structural chemistry, polyethylene glycol (PEG) modification has numerous benefits and relatively few drawbacks. PEG is now increasingly being applied to the problems of tumour targeting, both in the context of the passive targeting of PEG-liposomes and in active targeting strategies using PEGylated anti-tumour antibodies. PEG can also serve as a useful linker molecule between targeting moieties and other agents, including cytotoxic or imaging agents and targeted liposomes. Despite these demonstrated benefits and the level of attention which PEGylation has received, relatively little consideration has been given to two key areas: first, the extent to which the coupling method has an impact on both the functionality of the PEG-adduct and the acquisition of beneficial properties; second, that the impact of PEGylation on biodistribution is complex, thus any attempt to optimise a PEG-peptide or PEG-liposome for a particular task must involve an examination of all the individual facets of the effects of PEGylation. Studies investigating the underlying principles of tumour targeting suggest that current views concerning the optimisation of PEGylated vehicles for tumour localisation need to be re-examined.

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