Nicola K. Green

Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies

Adenovirus is a widely used vector for cancer gene therapy because of its high infection efficiency and capacity for transgene expression in both dividing and nondividing cells. However, neutralisation of adenovirus by pre-existing antibodies can lead to inefficient delivery, and the wide tissue distribution of the coxsackie and adenovirus receptor (CAR, the primary receptor for adenovirus type 5) precludes target selectivity. These limitations have largely restricted therapeutic use of adenovirus to local or direct administration. A successful viral gene therapy vector would be protected from neutralising antibodies and exhibit a preferential tropism for target cells. We report here the development of a covalent coating and retargeting strategy using a multivalent hydrophilic polymer based on poly-[N-(2-hydroxypropyl)metha- crylamide] (pHPMA). Incorporation of targeting ligands such as basic fibroblast growth factor and vascular endothelial growth factor on to the polymer-coated virus produces ligand-mediated, CAR-independent binding and uptake into cells bearing appropriate receptors. Retargeted virus is resistant to antibody neutralisation and can infect receptor-positive target cells selectively in mixed culture, and also in xenografts in vivo. Multivalent polymeric modification of adenovirus is an effective way of changing its tropism and interaction with the immune system. As a non-genetic one-step process, the technology is simple, versatile and should yield vectors with an improved safety profile.

Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus

Systemic delivery of adenoviral vectors is a major goal in cancer gene therapy, but is currently prohibited by rapid hepatic uptake of virus following intravenous injection with levels of viable virus in the murine plasma typically falling to less than 0.1% after 30 min. We have used a surface-masking technique based on multivalent copolymers of poly(N-(2-hydroxypropyl)methacrylamide) to ablate all pathways of receptor-mediated infection, combined with dose modulation to achieve partial saturation of nonspecific uptake pathways. Polymer coating gave at least 100-fold decreased hepatic transgene expression at all doses and even high doses of coated virus (pc-virus) showed no weight loss or stimulation of serum transaminases. Low doses of virus and pc-virus (109 viral particles (vp)/mouse) were mainly captured by the liver (assessed by quantitative PCR), although higher doses led to greater fractional persistence in the plasma (measured after 30 min). Coated virus at a dose of 6 × 1011 vp/mouse showed nearly 50% plasma circulation, representing a 3.5-fold greater area under the concentration–time curve (0–30 min) compared to unmodified virus. Such an increase in the bioavailability of adenovirus, coupled with substantial decreases in toxicity and unwanted transgene expression is an important step towards producing systemically available tumour-targeted viruses.

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

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.

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.

Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954

Expression of the E. coli enzyme nitroreductase (NTR) in tumour cells enables them to activate the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide), leading to inter- strand DNA crosslinking and cell death. Using transfected or retrovirally transduced SKOV3 ovarian carcinoma cell clones, we show a strong correlation between sensitivity to CB1954 and level of NTR enzyme activity. Importantly for clinical application in ovarian cancer, a cisplatin-resistant ovarian tumour cell line remains as susceptible to the NTR-dependent cytotoxicity of CB1954 as parental cells. In mixed populations of NTR-expressing and non-expressing cells, we observe a marked ‘bystander killing’ effect with this system. The use of NTR-encoding retroviruses from clonal producer cell lines at titres of 5 × 105 c.f.u./ml to transduce either established or low passage primary ovarian carcinoma lines only achieves an average 10-fold sensitisation of the cultures at gene transfer efficiencies of 15–25%. Concentration of the retrovirus to 3 × 107 c.f.u./ml elevates gene transfer to 80–90% in a single exposure to target cells, resulting in up to 500-fold sensitisation of the entire, unselected SKOV3 population to CB1954. In an initial investigation of NTR/CB1954 for the treatment of tumours in vivo, we observe regression of tumours expressing NTR following administration of CB1954, resulting in significantly increased median survival.

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5‐(aziridin‐1‐yl)‐2,4‐dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a “suicide enzyme” for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication‐defective adenovirus vector (Ad‐CMV‐ntr), which allowed efficient gene transfer to SK‐OV‐3 or IGROV‐1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2,000‐fold sensitisation) with 25–50 p.f.u. per cell. Similar Ad‐CMV‐ntr‐dependent sensitisation to CB1954 was seen in 3 of 6 low‐passage primary ovarian tumour lines. Cells grown at low‐serum concentration to inhibit proliferation remained equally susceptible to the Ad‐CMV‐ntr‐dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme‐prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin‐resistant cells were sensitised towards CB1954 by Ad‐CMV‐ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin‐resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad‐CMV‐ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001). Int. J. Cancer 86:848–854, 2000.

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.

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via |[alpha]|6-integrins

Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via α6β1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an α6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-α6 integrin and anti-β1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased α6β1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.

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

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.

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.