A prime objective in the development of AdV for gene therapy is cell type-specific targeting through vector tropism modification. Targeting may augment gene delivery to tissues of interest that do not express sufficient native AdV receptor and may diminish immunogenicity, toxicity, and in vivo sequestration of the vector. Toward this goal, the native AdV tropism needs to be abolished completely, and a new binding ligand with specificity for the target tissue must be added to the AdV. The present study provides such targeted AdV, where the native interaction with primary and secondary AdV receptors is eliminated through genetic means and new binding affinities are introduced through bispecific adapter molecules. These vectors obviate an important drawback of previous two-component vector systems where abolition of native tropism depended on neutralization by the adapter molecule. Standardized production of the latter type of targeted vector with completely blocked native tropism is not trivial. In addition, the stability of such targeting complexes might be insufficient in vivo to retain full neutralization. The best results after systemic vascular delivery of two-component targeted vectors reported so far were obtained using AdV with bispecific antibodies that block the CAR binding domain in the fiber knob. Although this approach considerably reduced non-target organ transduction (13
), more than 90% of gene transfer still occurred in normal tissues (32
). Thus, these studies clearly indicated that more stringent methods for abolition of native tropism were warranted.
By combining specific adenovirus capsid mutations that eliminate CAR and αv
integrin interaction (11
) with bispecific scFv fusion proteins (14
), we constructed targeted AdV that provide stringent target molecule specificity. Neither exposure of cells deficient in the target molecule nor use of AdV targeted with irrelevant bispecific scFv led to considerable transduction. The gene transfer efficiencies obtained with vectors with specificity for EGFR or EpCAM on human cancer cell lines were at or well above native AdV infectivity. This information could not be gathered before, because native AdV targeted with bispecific scFv always retained a native transduction component that precluded measurement of the true efficiency of targeted gene transfer. On most cell lines, EGFR-mediated transduction was more efficient than EpCAM-mediated gene transfer. This difference may be a reflection of differences in receptor densities on the cell surface or, alternatively, may be caused by the lower concentration of the batch of EpCAM-specific targeting molecules. The kinetics of the targeted gene delivery suggested that the internalization rate of the target molecule was a less important factor determining transduction efficiency. This implies that many cell surface molecules may serve as efficient targets for AdV-mediated gene transfer, provided that high-affinity binding to the viral capsid is established. Our bispecific scFv expression cassette serves as a versatile adapter for this purpose, since it allows simple exchange of scFv with different specificities.
Our study also provided more insight into the targeted AdV transduction process. Native AdV cell entry is promoted by αv
integrin interaction and requires PI3K activation (26
). Recently, however, it was observed that inhibition of the penton base-integrin interaction with neutralizing antibodies enhanced targeted AdV transduction (9
). This suggested that while αv
integrin interaction augments native adenovirus infection, it might not be involved in targeted AdV uptake. Integrin-independent targeted AdV transduction has so far been reported only with targeting moieties comprising active mitogenic ligands for tyrosine kinase growth factor receptors, including basic fibroblast growth factor, insulin-like growth factor 1, EGF, and tumor necrosis factor alpha (9
). Targeting toward the bombesin/gastrin-releasing peptide receptor, on the other hand, appeared to still depend on penton base-activated endocytosis (17
). Inhibitors of tyrosine kinase receptor- and PI3K-mediated signal transduction had contradictory effects on the targeted AdV transduction in different studies (16
), leaving the role of signal transduction in the targeted AdV uptake pathway unresolved. All of these previous studies used neutralizing peptides, antibodies, or Ad protein or pharmacologic signal transduction inhibitors to interfere with penton base-initiated signaling. Their conflicting results may have been caused by incomplete inhibition on the one hand or pleiotropic effects of the inhibitors on the other hand. We now for the first time targeted AdV toward alternative cell surface receptors by using vectors that inherently lack αv
integrin interaction due to absence of the penton base RGD motif. This allowed a more direct study of integrin-independent AdV entry, without any further addition of inhibitory molecules. EGFR- or EpCAM-targeted doubly ablated AdV had an efficiency similar to that of targeted native AdV and were more efficient than targeted AdV with CAR binding abolished on most cancer cell lines. The bispecific scFv that targets toward EGFR comprises the anti-EGFR scFv 425, which is an EGFR antagonist and thus should not trigger the EGFR-mediated signaling cascade (33
). Moreover, EpCAM, which is not involved in PI3K activation at all, served as an efficient target molecule for AdV transduction as well. These findings suggested, therefore, that penton base-triggered PI3K activation is not required for efficient targeted AdV entry and does not need to be complemented through tyrosine kinase activation. In fact, the interaction of targeted AdV with integrins in many cases even antagonized gene delivery. Deletion of the penton base RGD motif strongly enhanced the targeted gene transfer efficiency (three- to sevenfold) on most tested cancer cell lines. This suggested that disengaging targeted AdV transduction completely from known native entry pathways yields the most efficient targeting strategy. This was at least true for EGFR- and EpCAM-mediated transduction but may apply to other target molecules on cancer cells as well. The lack of a need for triggering cell signaling pathways could be an advantage in the context of cancer gene therapy, where cell activation is preferably avoided. In addition, it may be beneficial for in vivo gene therapy in general, because triggering of signaling events may potentiate early immune responses to the vector (3
). Thus, our findings support further exploration of AdV with penton base RGD deleted for targeted gene delivery.
The improved specificity, as indicated by the targeting index, of EGFR-targeted doubly ablated AdV on cancer cell lines was confirmed with a panel of low-passage primary glioma cell cultures. EGFR-mediated gene delivery into primary cancer cells was 2 orders of magnitude more efficient than untargeted transduction. To investigate the performance of EGFR-targeted doubly ablated AdV in a clinically relevant microenvironment for brain tumors, we employed OMS. These structures retain the complex tissue architecture present in the tumor, including (heterogeneous) tumor cells, connective tissue, immune cells, and capillaries, with intact cell-cell and cell-matrix contacts (30
). They thus represent the most clinically relevant in vitro model for human tumors. Native AdV transduced tumor OMS and brain explants with similar efficiencies, demonstrating the need for targeting strategies in gene therapy for brain tumors. EGFR-targeted doubly ablated AdV exhibited a clear preference for transduction of EGFR-expressing brain tumor OMS compared to EGFR-deficient normal brain explants. On average, the tumor-to-normal brain transduction ratio was improved 16-fold (range, 5- to 38-fold) compared to native AdV. In fact, this ratio probably underestimates the true vector specificity, because in the normal brain samples of at least two of the four patients, infiltrated tumor cells or activated EGFR-positive microglial cells were detected, and this was not ruled out in the other two cases. These observations were not unexpected, since the normal brain explants were established from tissue adjacent to the tumor and proliferating reactive microglial and astroglial cells expressing EGFR can be found in brain lesions and local inflammations (31
). Thus, targeted transduction of these EGFR-positive cells may account for most of the gene delivery measured in normal brain explants.
Taken together, our results support further consideration of doubly ablated AdV targeted with bispecific scFv as vectors for gene therapy. To this end, their efficacy and specificity will need to be confirmed under in vivo conditions. Depending on the route of administration, these conditions may be quite different from those in tissue culture. After intratumor injection, a high concentration of cells will be exposed to a high vector concentration for extended periods, while vascular vector administration may result in a very short virus-cell interaction at low concentration. In this respect, it is very promising that we observed a rather constant targeting index over a range of AdV concentrations (MOI of 10 to 10,000) and exposure times (2 min to 24 h). Moreover, selective transduction was also seen in spheroids, where the tumor and normal tissue architecture and composition were retained. Hence, the present study has encouraging implications for the development of human clinical gene therapy approaches.