The goal of this study was to explore the possibility of genetically altering the natural tropism of Ad5 by using artificially designed proteins, the affibodies. This was attempted in order to resolve a fundamental problem that has hampered progress in the development of targeted Ad vectors for in vivo gene delivery—the shortage of appropriate targeting ligands. Since these ligands are to be incorporated into the Ad capsid genetically, they have to satisfy a set of stringent structural, functional, and biosynthetic criteria, which in aggregate effectively eliminate from the list of potential ligand candidates nearly all naturally existing proteins. Therefore, instead of continuing an unpromising search for such molecules in nature, we took an alternative route of employing artificial proteins that have been rationally designed to fit the profile of a targeting ligand for Ad.
In this study, affibody proteins derived from a compact, simply structured, and stable scaffold of Staphylococcus protein A dZ were used to target knob-deleted Ad fiber proteins to Her2. The trimeric structure of these chimeras was facilitated by the inclusion of a trimerization domain of the phage T4 fibritin protein. These modifications yielded fully functional tripartite protein chimeras that proved to be an adequate replacement for the wt Ad5 fiber. Namely, they formed stable fiber-like trimers and consequently incorporated into Ad particles with efficacy of encapsidation equal to that of the wt fiber. The affibody ligands within these targeting proteins retained their Her2 binding specificity and directed the virus to the target receptor. Furthermore, this engineered tropism to Her2 allowed two of the targeted vectors to deliver the transgene to Her2-positive human breast cancer cells with efficiency equal to or greater than that of the virus with unaltered receptor specificity. Thus, this tropism modification improved both the vector's specificity for the target cells and the efficiency of gene delivery.
The following considerations suggest that for Ads, genetic modification of virus tropism with affibodies could be the targeting strategy of choice. First, both of the affibody species we tried (Zher2:7
) resulted in functional fibers and virions. These findings, taken together with the high level of structural homology among affibodies, suggest that the overall success rate with other affibodies should be high, too. Second, the efficacy of Ad vector targeting and gene delivery may be improved further by using affibody multimers as ligands. Of note, it has been shown that compared to affibody monomers, affibody dimers have higher affinities for Her2 and better tumor localization properties in vivo (26
). Alternatively, affibodies with higher affinities for their targets could be used. While the dissociation equilibrium constants (KD
) for the Zher2:4
affibodies used in this work are 50 nM and 140 nM (33
), respectively, recent affinity maturation work resulted in much better Her2 binders, with KD
s in the lower picomolar range (24
). Third, in addition to their high affinities, the affibodies show excellent target selectivity both in vitro and in vivo as they are able to discriminate between closely related members of the same receptor families (9
). Fourth, the repertoire of affibodies with specificities for disease-related targets is growing, and significant commercial interest in this technology is expected to ensure its further growth. Fifth, the fact that some affibodies trigger internalization upon binding to the target (9
) provides an opportunity to design targeted Ad vectors that would not require integrin-mediated internalization. For such vectors, integrin expression by the target tissues would not be a limiting factor for efficient gene delivery. Sixth, affibodies may be developed that are specific for molecular markers of diseases for which natural ligands do not exist, allowing Ad targeting of these molecules.
It should be noted that although the use of affibody ligands is an attractive strategy, it does not by itself guarantee that Ad vector tropism will be successfully altered. In this regard, recent attempts to target Ad5 by using an affibody-modified, knob-deleted fiber fused with the neck region peptide of lung surfactant protein D have been unsuccessful (19
). In the same study, incorporation of the affibody within the HI loop of the fiber knob resulted in a poor encapsidation of the resultant proteins in Ad virions, which was only 25% of that seen with the wt fiber. Similarly, our affibody-modified chimeras of the FF type were only marginally useful in directing the virus. It was thus the particular combination of the affibody with the improved ligand-presenting molecule, the 11F chimera, that made our strategy a success. It is possible, however, that alternative fiber-derived chimeric backbones, such as the recently developed fusions between the Ad5 fiber and reovirus protein σ1 (22
), might be successful in accommodating affibodies as targeting ligands for Ad. Regardless of which ligand-presenting protein is used to replace Ad fiber, targeting Ads to new receptors may affect virus uptake, intracellular trafficking, and nuclear import of viral genomes, and, thus, these potential problems must be considered for each new ligand/receptor pairing. Also, despite our success with the 11F chimera, it is possible that some affibodies may fail to function as ligands when fused to this scaffold.
What makes the combination of the fiber replacement approach with the use of affibodies particularly attractive is that this strategy should be equally efficient when applied to Ad serotypes other than human Ad5. Several of those serotypes are currently being explored as alternative vector prototypes with the goal of overcoming the preexisting anti-Ad5 immunity in humans (10
). The high degree of structural similarity between various Ad fibers (7
) suggests that replacement of the fiber knob in these alternative vectors with fibritin-affibody fusions is likely to be a successful approach. Targeting of those other Ad fibers by replacing their knobs with the fibritin-affibody fusions will be independent of both the identity of their natural receptors and the identification of their receptor-binding sites; thus, this strategy would be predicted to be rather straightforward.
From the standpoint of basic science research, efficient remodeling of the Ad's cell binding mechanism with affibodies makes the Ad-cell interaction manipulatable and thus allows the study of important biological questions that could not be answered using viruses with native tropism. For instance, affibody-based modification of Ad vector tropism could be used to study the dependence of the overall efficacy and temporal dynamics of the infection process on the nature of the target receptor, the localization of the virus-binding site within the receptor molecule, or its position relative to the cell surface. It is thus envisioned that the work reported here, by establishing that affibodies can be used to successfully manipulate Ad vector tropism, will lead to many new research opportunities in both the basic and applied fields of science.