126. Zc-Ad Vector: A Novel Immunotargeting Vector with a Modified Fc-Binding Domain
Authors: Sam C. Noureddini, Nikolay Korokhov, Abner Mhashilkar, Joanne T. Douglas, David T. Curiel
Journal: Molecular Therapy (2004)

Adenovirus serotype 5 (Ad5) has shown potential as a gene delivery vehicle for numerous gene therapy applications. Because of the paucity of Ad5's primary receptor, coxsackie-adenovirus receptor (CAR), on cancer cells, and its widespread distribution on other cell types, vector targeting to specific receptors on cancer cells has become a mandate for efficient gene transfer. In one targeting scheme, the receptor-selective affinity of immunoglobin G (IgG) molecules has been employed to retarget Ad5 to specific cell types. This system provides for a flexible, modular approach to targeting cancer cells, because of the vector's ability to utilize any of the multiple IgG molecules with characterized receptor affinity. In this targeting schema, the Ad5 fiber capsid protein has been engineered to contain a specific domain (Cd) of protein A from S. aureus, exhibiting binding affinity for the conserved Fc domain common to all IgG molecules. Attempts to fully develop this technology, however, have been confounded by high degrees of viral aggregation, observed after viral-IgG complexes have been formed ex vivo, thus hindering the vector's ability to transfer genes. The proposed cause of this aggregation is the Cd domain's ability to bind the Fab regions of the IgG molecule, in addition to the Fc domain, yielding complexes of multiple virions crosslinked via IgG molecules. To circumvent this problem, we have engineered a novel IgG binding ligand, the Zc domain; a modification of the previous Fc-binding domain employed for this targeting schema. Based on the literature of well-known non-Fab-binding, Fc-binding domains, we have abolished the Zc domain's ability to bind the Fab regions of IgG molecules, via site-directed mutagenesis of a single glycine to alanine substitution. With this structural modification, we hypothesize that the degree of aggregation displayed in Ad-Ig complexes will be lessened, thus, enhancing the vector's ability to transfer genes in situ. Given the promise shown for modifications of this nature at the C-terminus, we have constructed an E1-deleted Ad5 vector containing a C-terminal linker with the Zc domain, and enhanced green fluorescent protein (GFP) as a reporter gene. The Zc-Ad vectors were propagated and incubated with either a monoclonal anti-CD40 IgG, or an Fc anti-CD40 single chain antibody (scFv) fusion protein to provide for CD40 targeted Ad5 vectors. Gene transfer analyses, comparing the targeting efficacy of the Zc vs. Cd viruses, complexed with the IgG and scFv fusion, were carried out with 293 cells expressing CD40. Preliminary data for these experiments demonstrate that the Zc-Ad vector is equally effective as our previous Ig-binding vector system, as displayed by CAR-independent, CD40-mediated gene transfer in vitro. Because aggregation has apparently undermined the potential utility of targeting by Ig-binding Ads hitherto, we propose that the Zc ligand will mitigate aggregation, and optimize these vectors for efficient gene transfer.