Elena V. Shashkova

Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded

Virotherapy is currently undergoing a renaissance, based on our improved understanding of virus biology and genetics and our better knowledge of many different types of cancer. Viruses can be reprogrammed into oncolytic vectors by combining three types of modification: targeting, arming and shielding. Targeting introduces multiple layers of cancer specificity and improves safety and efficacy; arming occurs through the expression of prodrug convertases and cytokines; and coating with polymers and the sequential usage of different envelopes or capsids provides shielding from the host immune response. Virus-based therapeutics are beginning to find their place in cancer clinical practice, in combination with chemotherapy and radiation.

Preparation and Titration of CsCl-Banded Adenovirus Stocks

Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus (either replication-competent or replication-defective) in spinner cultures (e.g., KB, HeLa, or 293 cells). Protocols for harvesting cells and isolation of adenovirus by CsCl banding are presented. Methods for titering adenovirus by plaque assay are presented along with a discussion of how plaque assays can be used to determine the kinetics of cell killing and cytolysis by adenoviruses.

Modification of adenoviral vectors with polyethylene glycol modulates in vivo tissue tropism and gene expression

Polyethylene glycol (PEG) is a hydrophilic polymer that has been used to coat adenoviral (Ad) vectors to improve their pharmacology. To analyze the effects of PEG on Ad5 tropism, Ad5 was covalently modified with different sizes of PEG and in vitro and in vivo transduction was analyzed. All tested PEGs ablated in vitro transduction. When protein C (PC) and factors VII, IX, and X were added, only factors IX and X increased transduction by the PEGylated vectors with the largest effect by X. Inactivation of these factors with warfarin drastically reduced liver transduction in mice by the PEGylated vectors after intravenous (i.v.) injection. Ad5 conjugated with 5 kd PEG maintained normal liver transduction while conjugation with larger 20 and 35 kd PEGs significantly reduced liver transduction. When intraperitoneal (i.p.) injection was tested, Ad transduced the peritoneum efficiently with only low level liver transduction. When Ad5 was modified with 5 kd PEG, peritoneal transduction was reduced and the virus preferentially transduced the liver. These data demonstrate the effects of different sizes of PEG on in vivo Ad tropism and suggest that this approach may be useful in retargeting and detargeting Ad in vivo.

Chemical Modification with High Molecular Weight Polyethylene Glycol Reduces Transduction of Hepatocytes and Increases Efficacy of Intravenously Delivered Oncolytic Adenovirus

Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites.

Targeting Interferon-α Increases Antitumor Efficacy and Reduces Hepatotoxicity of E1A-mutated Spread-enhanced Oncolytic Adenovirus

Novel approaches are needed to improve the antitumor potency and to increase the cancer specificity of oncolytic adenoviruses (Ad). We hypothesized that the combination of interferon-alpha (IFN-α) expression with a specific mutation in the e1a gene of Ad could target vector replication to genetic defects in the IFN-α pathway resulting in both improved antitumor efficacy and reduced toxicity. The conditionally replicative Ad vector KD3-IFN carries the dl1101/1107 mutation in the e1a gene that eliminates binding of E1A proteins to p300/CBP and pRb. KD3-IFN expresses human IFN-α in concurrence with vector replication and overexpresses the adenovirus death protein (ADP; E3-11.6K). The antitumor activity of KD3-IFN was significantly higher than that of a control vector in established human hepatocellular carcinoma tumors in immunodeficient mice and in hamster kidney cancer tumors in immunocompetent Syrian hamsters. The dl1101/1107 mutation rendered Ad replication sensitive to the antiviral effect of IFN-α in normal as opposed to cancer cells. These results translated to reduced vector toxicity upon systemic administration to C57BL/6 mice. The combination of Ad oncolysis, ADP overexpression, and IFN-α-mediated immunotherapy represents a three-pronged approach for increasing the anticancer efficacy of replicative Ads. Exploiting the dl1101/1107 mutation provides a mechanism for additional selectivity of IFN-α-expressing replication-competent Ads.

INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents

Human adenovirus type 5 (Ad5) has been the most popular platform for the development of oncolytic Ads. Alternative Ad serotypes with low seroprevalence might allow for improved anticancer efficacy in Ad5-immune patients. We studied the safety and efficacy of rare serotypes Ad6, Ad11 and Ad35. In vitro cytotoxicity of the Ads correlated with expression of CAR and CD46 in most but not all cell lines. Among CAR-binding viruses, Ad5 was often more active than Ad6, among CD46-binding viruses Ad35 was generally more cytotoxic than Ad11 in cell culture studies. Ad5, Ad6, and Ad11 demonstrated similar anticancer activity in vivo, whereas Ad35 was not efficacious. Hepatotoxicity developed only in Ad5-injected mice. Predosing with Ad11 and Ad35 did not increase infection of hepatocytes with Ad5-based vector demonstrating different interaction of these Ads with Kupffer cells. Data obtained in this study suggest developing Ad6 and Ad11 as alternative Ads for anticancer treatment.

Anticancer activity of oncolytic adenovirus vector armed with IFN-α and ADP is enhanced by pharmacologically controlled expression of TRAIL

We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3–interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-α. We hypothesized that the anticancer efficacy of the KD3–IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON–SEAP vector was obtained. Coinfection with TetON–TRAIL augmented oncolytic activity of KD3 and KD3–IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON–TRAIL and KD3–IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-α-mediated immunotherapy, and pharmacologically controlled TRAIL activity.

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 × 1010 viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

Expanded Anticancer Therapeutic Window of Hexon-modified Oncolytic Adenovirus

One of the significant hurdles toward safe and efficacious systemic treatment of cancer with oncolytic adenoviruses (Ads) is dose-limiting hepatotoxicity that prevents the increase of a therapeutic dose. In this study, we expanded the therapeutic window of oncolytic serotype 5 Ad (Ad5) by a genetic modification of hypervariable loop 5 (HVR5) in the capsid protein hexon that prevented infection of hepatocytes due to ablation of binding to blood factors. This oncolytic virus, Ad-GL-HB, had significantly reduced levels of hepatocyte transduction in immunocompetent and immunodeficient mice as compared to parental virus Ad-GL. The hepatocyte detargeting decreased liver damage and increased the maximum tolerated dose of Ad-GL-HB tenfold relative to that of Ad-GL. Intravenous (i.v.) injection of Ad-GL or Ad-GL-HB into tumor-bearing mice produced equally increased survival rates demonstrating that while Ad-GL-HB detargeted hepatocytes, it sustained tumor cell infection after systemic administration. The significantly improved safety of the virus allowed it to be used at increased doses for improved systemic antitumor efficacy. Our results suggest that hexon modifications provide valuable strategies for systemic oncolytic Ad therapy.