Substitution of adenovirus serotype 3 hexon onto a serotype 5 oncolytic adenovirus reduces factor X binding, decreases liver tropism, and improves antitumor efficacy.

Following intravascular delivery, an important route of administration for many clinical applications, the liver is the predominant site of adenovirus serotype 5 (Ad5) sequestration, thereby posing a risk of toxicity. In this regard, it has recently been shown that the Ad5 capsid binds to the blood coagulation factor X (FX) via the Ad5 hexon protein. This interaction mediates the majority of Ad5 liver transduction. Patient FX levels can be diminished by the administration of warfarin, a vitamin K inhibitor in the liver that decreases FX production; however, warfarin is a potent anticoagulant and can have a number of undesired side effects. Therefore, genetic modification of the virus to ablate FX binding is the preferred approach. Modifications of the hexon protein, specifically within the hypervariable 5 (HVR5) and 7 (HVR7) regions, have produced Ad5 vectors that show minimal liver sequestration. Our laboratory has pioneered adenovirus hexon modifications, including insertion of peptide ligands into the hypervariable regions and substitution of the adenovirus hexon with hexon proteins from alternate serotypes. Substitution of the adenovirus serotype 3 (Ad3) hexon protein onto the Ad5 capsid has been further characterized with regard to its interaction with FX and incorporated into an infectivity-enhanced conditionally replicative adenovirus (CRAd). In vitro evaluation of these hexon-modified vectors showed decreased binding to FX and decreased cell transduction via FX-mediated pathways. Furthermore, in vivo biodistribution studies in mice exhibited a decrease in liver sequestration. With the use of xenograft tumor models, the antitumor efficacy of the hexon-modified CRAds was enhanced over nonmodified controls.

Oncolytic adenoviruses targeted to cancer stem cells.

Cancer stem cells (CSC) represent a distinct subpopulation of cancer cells of integral importance. CSCs embody the refractory nature observed among many cancers: very competent initial tumor establishment and extremely aggressive metastatic nature. Recent discoveries indicate that CSCs embody chemo- and radioresistance and have been correlated with advanced disease and resistance to current therapies, and thus help explain the treatment resistance of many cancers. As CSCs are critical for tumor initiation, progression, persistence, and the development of metastasis, the success or failure of treatment approaches may be influenced greatly by the presence and treatment sensitivity of these cells. There also seems to be a direct link between epithelial-mesenchymal transition phenomena and CSCs. Cancer cure is predicated upon effectively targeting and eradicating the CSC population. Oncolytic viruses have undergone many developments and through multiple generations offer an effective way to specifically target and eradicate CSCs, while still maintaining the ability to affect the general tumor cell population. Conditionally replicative adenoviruses (CRAd) are one virotherapy that is especially promising. Multiple advanced targeting and infectivity enhancement schemes have been developed to allow the necessary specificity and transduction efficiency required for an effective therapy. Furthermore, these advanced generation CRAds can be armed with therapeutic transgenes to generate greater antitumor effects. Although ultimately, the rewards of targeting and eradicating CSCs will be evaluated in clinical trials, there are numerous methods for isolating primary CSCs based on surface marker expression and multiple established cell lines representative of CSCs for preliminary evaluation.

Members of adenovirus species B utilize CD80 and CD86 as cellular attachment receptors.

Alternate serotypes of adenovirus (Ad), including Ads of species B, are being explored to circumvent the disadvantages of Ad serotype 5 gene delivery vectors. Whereas the majority of human Ads utilize the Coxsackievirus and adenovirus receptor (CAR), none of the Ad species B use CAR. Ad species B is further divided into two subspecies, B1 and B2, and utilizes at least two classes of receptors: common Ad species B receptors and B2 specific receptors. CD46 has been implicated as a B2-specific receptor. Ad serotype 3 (Ad3), a member of B1, utilizes CD80 and CD86 as cellular attachment receptors. The receptor-interacting Ad fiber-knob domain is highly homologous among species B Ads. We hypothesized that other members of Ad species B may utilize CD80 and CD86 as cellular attachment receptors. All tested species B members showed specific binding to cells expressing CD80 and CD86, and the Ad fiber-knob domain from both B1 and B2 Ad efficiently blocked CD80- and CD86-mediated infection of Ad3 vectors. Members of both B1 and B2 demonstrated CD80- and CD86-specific infection of CHO cells expressing CD80 and CD86. Therefore, all of the members of Ad species B utilize CD80 and CD86 for infection of cells.

Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors.

Most viruses exploit a variety of host cellular proteins as primary cellular attachment receptors in the context of successful execution of infection. Furthermore, many viral agents have evolved precise mechanisms to subvert host immune recognition to achieve persistence. Herein we present data indicating that adenovirus (Ad) serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. CD80 and CD86 are co-stimulatory molecules that are present on mature dendritic cells and B lymphocytes and are involved in stimulating T-lymphocyte activation. To our knowledge, this is one of the first demonstrations of a virus utilizing immunologic accessory molecules as a primary means of cellular entry. This finding suggests a mechanism whereby viral exploitation of these proteins as receptors may achieve both goals of cellular entry and evading the immune system.

A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism.

The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion.