Adenovirus 5 recovery using nanofiber ion-exchange adsorbents.

Viral vectors such as adenovirus have successful applications in vaccines and gene therapy but the manufacture of the high-quality virus remains a challenge. It is desirable to use the adsorption-based chromatographic separations that so effectively underpin the therapeutic protein manufacture. However fundamental differences in the size and stability of this class of product mean it is necessary to revisit the design of sorbent's morphology and surface chemistry. In this study, the behaviour of a cellulose nanofiber ion-exchange sorbent derivatised with quaternary amine ligands at defined densities is characterised to address this. This material was selected as it has a large accessible surface area for viral particles and rapid process times. Initially, the impact of surface chemistry on infective product recovery using low (440 µmol/g), medium (750 µmol/g), and high (1029 µmol/g) ligand densities is studied. At higher densities product stability is reduced, this effect increased with prolonged adsorption durations of 24 min with just ~10% loss at low ligand density versus ~50% at high. This could be mitigated by using a high flow rate to reduce the cycle time to ~1 min. Next, the impact of ligand density on the separation's resolution was evaluated. Key to understanding virus quality is the virus particle: infectious virus particle ratio. It was found this parameter could be manipulated using ligand density and elution strategy. Together this provides a basis for viral vector separations that allows for their typically low titres and labile nature by using high liquid velocity to minimise both load and on-column times while separating key product and process-related impurities.

Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than serotype 5: implications for ovarian cancer therapy.

In a phase II/III clinical trial intraperitoneal (i.p.) administration of a group C adenovirus vector (Ad5) caused bowel adhesion formation, perforation and obstruction. However, we had found that i.p. group B, in contrast to group C adenoviruses, did not cause adhesions in nude BALB/c ovarian cancer models, prompting further investigation. Ex vivo, group B Ad11 caused lower inflammatory responses than Ad5 on BALB/c peritoneal macrophages. In vivo, i.p. Ad11 triggered short-term cytokine and cellular responses equal to Ad5 in both human CD46-positive and -negative mice. In contrast, in a long-term study of repeated i.p. administration, Ad11 caused no/mild, whereas Ad5 induced moderate/severe adhesions and substantial liver toxicity accompanied by elevated levels of IFNγ and VEGF and loss of i.p. macrophages, regardless of CD46 expression. It appears that, although i.p. Ad11 evokes immediate inflammation similar to Ad5, repeated administration of Ad11 is better tolerated and long-term fibrotic tissue remodelling is reduced.

Tropism ablation and stealthing of oncolytic adenovirus enhances systemic delivery to tumors and improves virotherapy of cancer.

Intravenous delivery of therapeutic virus particles remains a major goal for virotherapy of metastatic cancer. Avoiding phagocytic capture and unwanted infection of nontarget cells is essential for extended plasma particle kinetics, and simply ablating one or the other does not give extended plasma circulation. Here we show that polymer coating of adenovirus type 5 (Ad5) can combine with predosing strategies or Kupffer cell ablation to achieve systemic kinetics with a half-life >60 min, allowing ready access to peripheral tumors. Accumulation of virus particles within tumor nodules is proportional to the area under the plasma concentration/time curve. Polymer coating wild-type Ad5 in this way is known to decrease hepatic toxicity, increasing the dose of virus particles that can be safely administered. Using polymer-coating technology to deliver a replicating Ad5 systemically, virus replication and transgene expression was almost totally confined to tumor tissues, giving a much improved therapeutic index compared with uncoated virus, and complete control of human HepG2 tumor xenografts.

Cetuximab retargeting of adenovirus via the epidermal growth factor receptor for treatment of intraperitoneal ovarian cancer.

Gene and virotherapy of ovarian cancer, using type 5 adenovirus (Ad5), has demonstrated good activity in preclinical animal studies, particularly after intraperitoneal administration of virus; however, success in clinical trials has been limited by poor infectivity of ovarian cancer cells and inflammatory responses to Ad5. We previously demonstrated that covalent modification of Ad5 with reactive copolymers on the basis of poly(hydroxypropylmethacrylamide) can shield the virus, offering protection from neutralizing antibodies and enabling retargeting to cancer-upregulated receptors with peptide ligands (basic fibroblast growth factor [bFGF] and murine epidermal growth factor [EGF]). These ligands may be less than ideal for clinical use, however, because they are potential mitogens. Accordingly, in this study we investigated the use of an anti-EGF receptor (EGFR) antibody, cetuximab, to retarget adenoviral transduction of EGFR-positives in vitro and in vivo. Cetuximab retargeting altered the physicochemical characteristics of Ad5, although it did not cause particle aggregation. Although cetuximab stimulated internalization of EGFR, similarly to EGF, it inhibited EGFR phosphorylation. Adenoviral transduction was inhibited after polymer coating, but was rescued in EGFR-positive cells (and not in EGFR-negative cells) by cetuximab retargeting. Cetuximab retargeting of wild-type adenovirus serotype 5 (Ad5WT) prolonged survival in an animal model of human ovarian cancer, similar to unmodified Ad5WT, but polymer coating ameliorated stimulation of adhesion formation. We conclude that polymer coating and covalent attachment of cetuximab successfully retargeted adenovirus to EGFR-positive cells, retained in vivo efficacy of an oncolytic adenovirus, and ameliorated side effects caused by unmodified adenovirus.

Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1.

Type 5 adenovirus (Ad5) is a human pathogen that has been widely developed for therapeutic uses, with only limited success to date. We report here the novel finding that human erythrocytes present Coxsackie virus-adenovirus receptor (CAR) providing an Ad5 sequestration mechanism that protects against systemic infection. Interestingly, erythrocytes from neither mice nor rhesus macaques present CAR. Excess Ad5 fiber protein or anti-CAR antibody inhibits the binding of Ad5 to human erythrocytes and cryo-electron microscopy shows attachment via the fiber protein of Ad5, leading to close juxtaposition with the erythrocyte membrane. Human, but not murine, erythrocytes also present complement receptor (CR1), which binds Ad5 in the presence of antibodies and complement. Transplantation of human erythrocytes into nonobese diabetic/severe combined immunodeficiency mice extends blood circulation of intravenous Ad5 but decreases its extravasation into human xenograft tumors. Ad5 also shows extended circulation in transgenic mice presenting CAR on their erythrocytes, although it clears rapidly in transgenic mice presenting erythrocyte CR1. Hepatic infection is inhibited in both transgenic models. Erythrocytes may therefore restrict Ad5 infection (natural and therapeutic) in humans, independent of antibody status, presenting a formidable challenge to Ad5 therapeutics. "Stealthing" of Ad5 using hydrophilic polymers may enable circumvention of these natural virus traps.

Retargeting polymer-coated adenovirus to the FGF receptor allows productive infection and mediates efficacy in a peritoneal model of human ovarian cancer.

BACKGROUND: Transductional targeting of adenovirus following systemic or regional delivery remains one of the most difficult challenges for cancer gene medicine. The numerical excess and anatomical advantage of normal (non-cancer) cells in vivo demand far greater detargeting than is necessary for studies using single cell populations in vitro, and this must be coupled with efficient retargeting to cancer cells. METHODS: Adenovirus (Ad5) particles were coated with reactive poly[N-(2-hydroxypropyl)methacrylamide] copolymers, to achieve detargeting, and retargeting ligands were attached to the coating. Receptor-mediated infection was characterised in vitro and anticancer efficacy was studied in vivo. RESULTS: Polymer coating prevented the virus binding any cellular receptors and mediated complete detargeting in vitro and in vivo. These fully detargeted vectors were efficiently retargeted with the model ligand FGF2 to infect FGFR-positive cells. Specific transduction activity was the same as parental virus, and intracellular routing appeared unaffected. Levels of transduction were up to 100-fold greater than parental virus on CAR negative cells. This level of specificity permitted good efficacy in intraperitoneal cancer virotherapy, simultaneously decreasing peritoneal adhesions seen with parental virus. Following intravenous delivery FGF2 mediated unexpected binding to erythrocytes, improving circulation kinetics, but preventing the targeted virus from leaving the blood stream. CONCLUSIONS: Polymer cloaking enables complete adenovirus detargeting, providing a versatile platform for receptor-specific retargeting. This approach can efficiently retarget cancer virotherapy in vivo. Ligands should be selected carefully, as non-specific interactions with non-target cells (e.g. blood cells) can deplete the pool of therapeutic virus available for targeting disseminated disease.

Passive tumour targeting of polymer-coated adenovirus for cancer gene therapy.

Adenovirus provides many opportunities as a vector for delivery of cytotoxic genes to tumours. Polymer coating of adenovirus is known to increase its plasma circulation kinetics, affording the possibility of active and passive targeting to tumours. Here we show that polymer-coating adenovirus (pc-virus) abrogates its normal infectivity in vitro and also in liver following intravenous injection. The coated virus accumulates within solid subcutaneous AB22 mesothelioma tumours 40-times more than unmodified virus, and mediates higher levels of transgene expression within tumours. This is the first demonstration of passive tumour targeting of polymer-coated adenoviruses administered by intravenous injection, and also the first time pc-virus has been shown to be infectious following passive targeting to tumours in vivo. This technology provides an interesting option for delivery of therapeutic viruses to disseminated tumour masses by intravenous injection.

Vectors based on reducible polycations facilitate intracellular release of nucleic acids.

BACKGROUND: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. METHODS: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys(10)-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. RESULTS: A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 +/- 7.9%), LNCaP (55.2 +/- 6.7%) and PC-3 (66.1 +/- 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC(50) values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). CONCLUSIONS: These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity.

Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector.

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence.

Adenoviral vectors: systemic delivery and tumor targeting.

The development of a targeted adenoviral vector, which can be delivered systemically, is one of the major challenges facing cancer gene therapy. The virus is readily cleared from the bloodstream, can be neutralised by pre-existing antibodies, and has a permissive cellular tropism. Clinical studies using the ONYX virus have shown limited efficacy, but there are several hurdles to overcome to achieve an effective tumor-specific systemic therapy. In this review, we have summarized the various strategies used to overcome the limitations of adenoviral-mediated gene delivery.