Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.

Development of a targeted gene vector platform based on simian adenovirus serotype 24.

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

Modification of adenovirus capsid with a designed protein ligand yields a gene vector targeted to a major molecular marker of cancer.

The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2-expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.

Treatment of ovarian cancer with a tropism modified oncolytic adenovirus.

Ad5-Delta 24RGD is an adenovirus that is selectively replication competent in cells defective in the Rb/p16 pathway, such as ovarian cancer cells. The fiber of Ad5-Delta 24RGD contains an integrin binding RGD-4C motif, allowing Coxsackie adenovirus receptor-independent infection of cancer cells. Oncolysis of cell lines was similar to that of a wild-type control, and replication in primary tumor material was shown using a novel three-dimensional spheroid model. Finally, an orthotopic murine model of peritoneally disseminated ovarian cancer was used to test i.p. administration to tumor-bearing animals. Injection of the agent resulted in eradication of i.p. disease, whereas control animals expired (P < 0.0001). These results suggest that Ad5-Delta 24RGD could be useful for treatment of ovarian cancer in humans.

Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.

Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells.

Gene delivery efficiency in clinical cancer gene therapy trials with recombinant adenoviruses (Ads) based on serotype 5 (Ad5) has been limited partly because of variable expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human primary cancer cells. As a means of circumventing CAR deficiency, Ad vectors have been retargeted by creating chimeric fibers possessing knob domains of alternate Ad serotypes. In this study, we have constructed an Ad5-based vector, Ad5/3luc1, with a chimeric fiber protein featuring a knob domain derived from Ad3. This virus is retargeted to the Ad3 receptor and, therefore, has different tissue tropism. A novel knob binding assay was used to measure expression of CAR and the Ad3 receptor. Further, to evaluate the correlation of receptor expression and infectivity by Ad, a panel of ovarian cancer cell lines and purified primary cancer cells were infected with Ad5luc1 and Ad5/3luc1 at 50, 200, and 1000 viral particles/cell. Our results confirm that Ad5/3luc1 is retargeted to the Ad3 receptor. Furthermore, the Ad3 receptor is present at higher levels than CAR on ovarian adenocarcinoma cells. Also, the amount of binding to primary receptor appears to be the major factor determining the efficiency of transgene expression. The Ad5/3 chimera displays enhanced infectivity for ovarian cancer cell lines and purified primary tumor cells, which could translate into increased efficacy in clinical trials.

Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting.

Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (K(D) 1.35 nM) and fused this protein with a DARPin specific for Her2, an established cell-surface biomarker of human cancers. The stability of the complex formed by this bispecific targeting adapter and the Ad virion resulted in insufficient gene transfer and was subsequently improved by increasing the valency of adapter-virus binding. In particular, we designed adapters that chelated the knob in a bivalent or trivalent fashion and showed that the efficacy of gene transfer by the adapter-Ad complex increased with the functional affinity of these molecules. This enabled efficient transduction at low stoichiometric adapter-to-fiber ratios. We confirmed the Her2 specificity of this transduction and its dependence on the Her2-binding DARPin component of the adapters. Even the adapter molecules with four fused DARPins could be produced and purified from Escherichia coli at very high levels. In principle, DARPins can be generated against any target and this adapter approach provides a versatile strategy for developing a broad range of disease-specific gene vectors.

A strategy for adenovirus vector targeting with a secreted single chain antibody.

BACKGROUND: Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly. METHODOLOGY/PRINCIPAL FINDINGS: We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer. CONCLUSIONS/SIGNIFICANCE: This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors.

Genetically targeted adenovirus vector directed to CD40-expressing cells.

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge.

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.