and C.T. antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin 7, respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody. Adenoviruses (Ad) are nonenveloped viruses with a DNA genome of about 36 kb. Recombinant Ad have been widely used as gene transfer vehicles in preclinical and clinical studies (14). Infection with Ad vectors requires expression of separate cell receptors for attachment and entry. While the attachment of the virus to the cell is mediated by high-affinity binding of the knob domain of the Ad fiber to the 46-kDa coxsackie- and Ad receptor (CAR) (2, 48), internalization of the virus in clathrin-coated vesicles occurs through endocytosis upon interaction of the penton base protein with v integrins (28, 54). In spite of a wide tissue distribution, CAR expression is low or absent in many cell types and tissues which are of interest for experimental or therapeutic gene transfer, including skeletal muscle, endothelium, hematopoietic cells, and tumor cells. Therefore, considerable effort has recently been directed Nortadalafil to the retargeting of Ad vectors toward those cell types. Genetic modification of the Ad fiber protein through incorporation of small peptide motifs into the HI loop (12, 24), a flexible, protruding region in the globular knob domain, through the addition of short peptide sequences at the C terminus of the fiber protein (6, 55), or through more radically reengineering knobless fiber molecules (30), improved the Ad-mediated transduction of cell types expressing ligand binding cell surface receptors. For example, incorporation of an RGD motif into the HI loop of first-generation Ad vectors (12) and high-capacity Ad vectors (4, 23) has been shown to enhance the transduction of CAR-negative integrin-expressing target cells. Similarly, the hexon protein has been modified by incorporation of an RGD peptide (49). Due to structural constraints of the capsid proteins, however, this approach seems to be restricted to small peptide CCND3 ligands. In an alternative approach, bispecific adaptor molecules composed of chemically cross-linked monoclonal antibodies (MAbs) (53) or fusion proteins containing a peptide ligand and a capsid-specific single-chain antibody or a soluble CAR domain (11, 50) have been employed to bridge Ad vector capsid proteins to cell surface receptor molecules. This strategy of tropism modification has also proved to be successful in vivo (40). However, it requires recombinant overexpression or chemical synthesis and modification, as well as extensive purification steps for the adaptor molecule, which may be time-consuming, costly, and difficult to scale up. Therefore, it was highly desirable to design a system based on the binding of unmodified MAbs to Ad vector particles, rendering the adaptor concept considerably more versatile and easy to apply. A stable variant of the immunoglobulin (Ig)-binding B domain of the staphylococcal protein A (46), the so-called Z domain, has been described as a three-helix, 59-amino-acid (aa)-residue module that binds the Fc portion of IgGs with high affinity (9, 36). The entire Z domain or derivatives thereof have been genetically incorporated into envelope proteins of baculovirus (34, 38) and Sindbis virus (21, 37) and into the capsid of adeno-associated virus type 2 (41) and have been shown to retain IgG-binding activity (33, Nortadalafil 37, 41). In this study, we describe the construction of an Ad vector displaying a short modified version of the Z domain, Z33 (7), in the HI loop of the fiber knob and the application of this vector in targeting experiments with specific MAbs directed against Nortadalafil cell surface antigens. The Nortadalafil Z33-modified Ad vector could be very efficiently targeted to epidermal growth factor receptor (EGFR)-expressing tumor cells, as well as to skeletal muscle cells, by complexation with cell-type-specific MAbs. MATERIALS AND METHODS Primary cells and cell lines. A431.
