A 24rgd

Elucidation of the fiber knob/CAR and RGD/integrin interactions and the frequent low expression of CAR and high expression of integrins in tumors led to the development of second generation A 24 adenoviruses with improved infectivity in tumors. This development was fueled by the recognition that incorporation of an ACDCRGDCFCG peptide (RGD-4C) motif into the H1 loop of the fiber knob (H1 loop) is capable of facilitating adenoviral entry of tumor cells by bypassing the CAR interaction (51) (see Fig. 9). Curiel and coworkers were the first to demonstrate that this RGD motif could favorably augment the delivery of adenoviral vectors (51). Subsequently, in order to improve the infectivity of A 24, Fueyo et al. reported a modified A 24 that included the RGD motif within the H1 loop, called A 24-RGD (18). In vitro analyses have demonstrated that the presence of this RGD motif increases infection of glioma cells by as much as 6x more than infection with adenoviruses lacking RGD in the H1 loop. Blocking adenoviral infectivity mediated by CAR by incubating tumor cells with fiber knob protein did not significantly inhibit infection of RGD-containing adenovirus. In contrast, incubating them with RGD peptide to block integrin binding significantly reduced adenoviral infectivity. Thus, insertion of the RGD motif into the fiber knob allows for CAR-independent infection of gliomas.

The increased infectivity of A 24-RGD has been shown to translate into increased tumor cell killing (18). Interestingly, in cells with low-CAR expression, A 24-RGD had higher cytopathic effects than A 24 or wild-type adenovirus. However, in cells with high-CAR expression, wild-type virus was more potent than A 24-RGD. Thus, the enhanced killing of A 24-RGD was most evident in tumor cells with low-CAR expression. This enhanced killing by A 24 RGD has been shown most convincingly in an intracranial glioma xenograft model of a low-CAR expressing glioma cell line (U87) (see Fig. 10). Intratumoral injection of A 24-RGD into established intracranial U87 xenografts was associated with a longer survival time (mean 131 d) than intratumoral injection of A 24 (mean 50 d). Furthermore, although long-term survivors were identified with both treatments, 60% of A 24-RGD treated mice, but only 15% of A 24-treated mice, survived for

Fig. 10. Efficacy of A 24 and A 24-RGD in an intracranial glioma xenograft model. U87 glioma cells (low CAR expression) were implanted in the frontal lobes of nude mice, and after 3 d they were treated with the agents shown in the legend. A 24 treatment resulted in a significant increase in survival time relative to controls. Use of A 24-RGD further increased survival time in this model (60% of animals were cured). Treatment with Onyx-O15 had no effect. A 24-RGD was as effective as wild-type adenovirus, indicating that its virulence was not significantly attenuated by the modifications.

Fig. 10. Efficacy of A 24 and A 24-RGD in an intracranial glioma xenograft model. U87 glioma cells (low CAR expression) were implanted in the frontal lobes of nude mice, and after 3 d they were treated with the agents shown in the legend. A 24 treatment resulted in a significant increase in survival time relative to controls. Use of A 24-RGD further increased survival time in this model (60% of animals were cured). Treatment with Onyx-O15 had no effect. A 24-RGD was as effective as wild-type adenovirus, indicating that its virulence was not significantly attenuated by the modifications.

more than 4 mo. Thus, there is substantial evidence that the addition of the RDG motif increased the capacity of A 24 to kill low CAR-expressing tumor cells.

Importantly, and in contrast to the case with tumor cells, the addition of the RGD motif to A 24 does not translate into increased killing of normal human astrocytes. Thus, the selectivity imposed by the mutant E1A is maintained in RGD modified A 24. Specifically, A 24-RGD was shown to be unable to replicate in serum-starved (non-dividing) normal human astrocytes, whereas wild-type adenovirus (not containing mutant E1A) was able to replicate. Indeed, A 24-RGD replication was 1000 to 10,000x greater in glioma cells than it was in normal human astrocytes. Thus, it is expected that A 24-RGD should have a high therapeutic index when used in the treatment of brain tumors. Moreover, recent reports have suggested that most normal cells in the brain have low levels of «^3 or «^5 integrins on their surface in situ, which would further extend the therapeutic index in glioma treatment.

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