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

Abstract

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.

Figure 1

Substitution of Ad3 hexon onto Ad5 capsid ablates binding to FX. A, Slot blot assay. Viruses bound to PVDF membrane were incubated with FX or with polyclonal anti-Ad capsid antibody. B, Surface Plasmon Resonance (SPR) analysis of FX binding to hexon variants. Purified human FX was covalently coupled to a SA biosensor chip. Ad vectors were then exposed to the chip and binding interactions were analyzed. B, Overlay sensograms are presented. The sensograms presented are representative of two replicates at 37° C (additional replicates were performed at 25° C showing similar results, data not shown).

Figure 2

Hexon modifications decrease FX-mediated uptake in vitro. Ad vectors were pre-incubated with increasing concentrations of FX and then allowed to infect either A549 (high CAR expression), HepG2 (liver representative, moderate CAR expression), or SKOV3 (low CAR expression) cells. Luciferase transgene expression was measured at 36 hours and normalized for protein and divided over vectors without pre-incubation with FX. Ad5 vectors with wild-type hexon (Ad5) had an increase in transgene expression with increasing FX concentrations. * = p-value of < 0.05 compared to Ad5.

Figure 3

Substitution of Ad3 hexon onto Ad5 capsid results in improved biodistribution in mice. A, Modification of Ad5 hexon decreases liver transduction in mice. C57BL/6 mice were injected with Ad vectors and at 36 hours organs were harvested and luciferase transgene expression was assayed. * = p-value of < 0.05. B, Substitution of Ad3 hexon onto Ad5 capsid results in improved tumor to liver transgene ratio. Athymic nude mice with skov3.ip1 flank tumors underwent tail vein injections with Ad vectors. Luciferase transgene transcript copy numbers in liver and tumors were measured by quantitative PCR and normalized for genomic β–actin DNA. Furthermore, comparison was made between infectivity enhanced vectors with substitution of Ad3 fiber knob domain (right grouping) compared to vectors with Ad5 fiber knob domain (left grouping). * = p < 0.05 compared to liver control, ** = p < 0.05 compared to tumor control.

Figure 4

Incorporation of hexon modification into CRAd context ablates FX binding but does not impair cell killing. A, Surface Plasmon Resonance (SPR) analysis of purified human FX binding to CRAd vectors. Purified human FX was covalently coupled to a SA biosensor chip. Ad vectors were then exposed to the chip and binding interactions were analyzed. A, Overlay sensograms are presented. The sensograms presented are representative of two replicates performed at 37° C. B, Crystal violet assay demonstrating no impairment of cell killing by substitution of Ad3 hexon onto CRAd capsid. Cytocidal effect of vectors on skov3.ip1 cells was assayed using crystal violet dye assay at 10 days post-infection (vp/cell ratio in bottom left corner of sample). Images presented are representative of three replicates performed for each vector and dose.

Figure 5

Hexon modification results in effective systemic targeting of oncolytic Ad. Athymic nude mice with skov3.ip1 subcutaneous flank tumors were treated with the panel of infectivity enhanced CRAds. A, Tumor size was followed up to 28 days. Ad5Δ24E1F5/3 = wild-type Ad5 hexon-containing vector (dark gray/triangle), Ad5Δ24E1F5/3 + warfarin = wild-type Ad5 hexon-containing vector pre-treated with warfarin at days -3 and -1 (gray/circles), Ad5Δ24E1H3F5/3 = Ad3 hexon substituted vectors (light gray/diamonds) B, Viral replication was measured by E4 copy number in liver and tumor specimens. Closed bars = liver, open bars = tumor. * = p < 0.05 compared to placebo.