One method of adenoviral retargeting employs antibody conjugates whereby an antibody (Ab) component binds to adenovirus and is cross-linking to a targeting moiety facilitating the binding to an alternative receptor (27). An Ab directed against the fiber-knob region has been used because it not only binds to the virus but also eliminates the undesired adenovirus native tropism by masking the region required for CAR affinity. A conjugate consisting of an anti-knob antibody combined with folate enabled retargeting of adenovirus to folate receptor positive malignancies (28). Along the same lines, adenovirus with a FLAG epitope insertion in the penton base region was retargeted to av^5 integrin of endothelial and smooth muscle cells by using a bispecific conjugate
with anti-avP5 integrin Ab and anti-FLAG Ab (29)29. Further Ab conjugate-based retargeting strategies using Abs of receptor ligands combined with an anti-knob Ab have been reported for various cell surface targets. These Abs targeted the fibroblast growth factor (FGF) receptor (30), epidermal growth factor (EGF) receptor (31), and angiotensin-converting enzyme (ACE) (32). Interestingly, the anti-ACE Ab achieved suitable in vivo targeting for the pulmonary vasculature when combined with transcrip-tional targeting (32). These data indicate that antibody-based targeting can confer CAR-independent infection, infectivity enhancement, and targeting.
Although these approaches validated the concept of adenoviral retargeting using large ligands, antibody-based conjugates encounter production problem. It is rather difficult to maintain structural and functional consistency of the crosslinked bispecific conjugate, making mass-production as a drug nonfeasible. To overcome these problems, fusion proteins created by genetic means are developed as an alternative to chemical crosslinking with the advantage of easier mass-production. The initial report in this field applied a fusion protein of anti-knob single-chain antibody (scFv) and EGF (33). This fusion protein successfully enhanced gene delivery to EGF-receptor positive malignancies. A slightly different fusion protein based system for bridging a targeting moiety to adenovirus uses the extracellular domain of CAR, which binds to the Ad5 fiber-knob region. Combined with the EGF ligand for target recognition, this fusion protein expressed in a Baculovirus system successfully retargeted Ad to EGFR positive cells (34). In contrast with these two methods, which employ extrinsic bridging molecules, genetically modified viruses based on alteration of the viral genome offer several distinct benefits. The most obvious advantage is that production of genetically modified viruses is much simpler relative to targeting conjugates because all components are encoded in the viral genome. So far, several targeting moieties have been successfully incorporated into adenoviruses. An integrin-binding motif and polylysine were successfully incorporated in the carboxyl terminus of fiber (35), whereas the FLAG octapep-tide (36) and RGD-4C integrin-binding peptide (37) were placed in the HI-loop of fiber knob region, yielding clear targeting function. On the other hand, there have been many failed attempts in genetic incorporation of targeting motif into Ad. For example, whereas the HI-loop tolerated the vasoactive intestinal peptide (VIP) sequence, the infectivity of this virus did not show augmented transduction in receptor positive cells (38). This may indicate that successful incorporation does not always translate into successful targeting.
Attempts have also been made to genetically incorporate onto the Ad capsid Ab and scFv, which have clear benefits as target moieties as a result of their high specificity. With the range of current genetic approaches, incorporating these targeting moieties while retaining functionality still remains difficult. For example, although a scFv was successfully incorporated into the Ad capsid as a fusion protein with pIX, it did not show its original binding ability (unpublished data). The reason for this discrepancy has not been clearly elucidated although possible explanations include scFv accessibility, alteration of capsid trafficking, and scFv processing. Instead of using either conjugate-based or genetic strategies above, the combination of both strategies may complement their respective shortcomings. An example of such combination was pursued by incorporating the Fc binding domain from protein A or G into adenovirus (39-41). Because these motifs effectively bind to antibodies, this vector structure enables easy incorporation of various kinds of Abs for targeting. Theoretically, this method has large potential benefit since established antibodies with high affinity and selectivity can be easily exploited to achieve the desired targeting.
A completely different strategy for targeting is to complex Ad with polyethylene gly-col (PEG). Because the Ad capsid would not be exposed after PEGylation, this method creates a stealth-like As vector to neutralization in the blood and also eliminate undesired binding to non-target tissues/cells (42,43). In addition, PEGylation permits incorporation of extrinsic targeting ligand on the surface of the PEG capsule. For example, RGD pep-tide and anti-E-selectin Ab incorporated on the surface of PEG successfully allowed selective transduction of av integrin and E-selectin positive cells, respectively (44).
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