The formation of a molecular bridge between Ad and a cell surface receptor constitutes the adapter-based concept of Ad targeting (Fig. 1). Adapter function is performed by bispecific molecules or molecular conjugates that link the Ad to alternative cell surface receptors, often concurrently ablating native CAR tropism. This approach is predicated by the aforementioned two-step entry mechanism of Ad, wherein attachment is distinct from internalization. In this way, alternative means
of cellular attachment do not impede Ad cell entry. Bispecific adapter molecules include bispecific antibodies, chemical conjugates between knob-binding single-chain antibodies (Fab) and cell-selective ligands such as folate, Fab-antibody conjugates using antibodies against target cell receptors [e.g., epidermal growth factor receptor (EGFR)], Fab-peptide ligand conjugates [e.g., basic fibroblast growth factor 2 (FGF2)], and recombinant fusion proteins that incorporate Fabs and peptide ligands.
The first in vitro demonstration of Ad targeting via the adapter modality resulted in CAR-independent, folate receptor-mediated cellular uptake of the virion by cancer cells over-expressing this receptor (39). This was accomplished using a bispecific conjugate consisting of a neutralizing Fab chemically linked to folate. A similar targeting adapter consisting of the same Fab fused to FGF2 was used to target Ad vectors to FGF receptor-positive Kaposi's sarcoma and ovarian cancer substrates (40-42). Upon intraperitoneal injection of Ad-Fab-FGF2 coding for herpes simplex virus type I thymidine kinase (HSV-TK) into tumor-bearing mice, survival was prolonged (43). Importantly, decreased hepatic toxicity was demonstrated (44,45). Other Fab-ligand conjugates have been targeted against epithelial cell adhesion molecule, tumor-associated glycoprotein 72, EGFR, CD-40, and others (32,46-52).
An alternative to the chemical conjugate approach was developed by creating a single recombinant fusion molecule formed by the soluble extracellular form of CAR (sCAR) fused to epidermal growth factor (EGF) (53). Increased reporter gene expression was achieved in several EGFR-overex-pressing cancer cell lines compared with untargeted Ad or EGFR-negative cells in vitro. EGF-directed targeting to EGFR-positive cells was shown to be dependent on cell surface EGFR density. Initial proof-of-concept studies were followed up with construction of adenoviruses coding for the adapter and capable of achieving production and secretion of sCAR-EGF in human cells. Further, the effect of such retarget ing on the oncolytic potency of replication competent agents was tested in vitro and in vivo (54). To improve Ad-sCAR-li-gand complex stability, a trimeric sCAR-fibitin-anti-erbB2 single-chain antibody (sFv) adapter molecule was developed (55). The adapter displayed increased affinity to the Ad fiber knob while augmenting gene transfer up to 17-fold in breast and ovarian cancer cell lines.
The above proof-of-principle studies and others have rationalized testing targeting adapters in vivo. A novel bispecific adapter composed of an antiknob Fab chemically conjugated to a monoclonal antibody (9B9) was constructed. 9B9 binds angiotensin-converting enzyme, a surface molecule expressed preferentially on pulmonary capillary endothelium and up-regulated in various disease states of the lung (48,56). Following peripheral intravenous injection of the Ad/Fab-9B9 complex, reporter transgene expression and viral DNA in the lung was increased 20-fold over untargeted Ad. Importantly, reporter gene expression in the liver was reduced by 83%.
Adapter-based Ad targeting studies provide compelling evidence that Ad tropism modification can be achieved by targeting alternate cellular receptors, and that this modality augments gene delivery to CAR-negative target cells. Although two-component targeting has shown promising results for retargeting adenovirus to new receptors, such delivery systems have more complex pharmacodynamics and -kinetics, and their stability in humans has not yet been studied. Therefore, one-component systems may be more easily applicable to human trials.
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