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The limited efficacy of adenovirus type 5 (Ad5)-based oncolytic viruses seen in the clinic thus far may be attributable in part to variable expression of its receptor on tumor cells. Replacement of the Ad5 fiber knob with the Ad35 fiber knob generated the Ad5/35 chimeric virus, which has previously been demonstrated to have significant antitumor activity in murine tumor models, presumably by virtue of its recognition of the CD46 receptor, which is abundant on many types of tumor cells. In the current study, a CD46 receptor transgenic mouse strain (hCD46Ge) that expresses the CD46 receptor in a pattern closely mirroring that in humans was used to study the in vivo properties of Ad5/Ad35 chimeric viruses. Vector distribution was evaluated after intravenous administration to hCD46Ge mice of an Ad5-based oncolytic adenovirus or an Ad5/35 chimeric oncolytic adenovirus (designated OV-5 and OV-5T35H, respectively), a wild-type Ad5 virus (Ad5wt), or an Ad5-based, E1-deleted adenovirus (Addl312) at 1.25 x 10(12) viral particles/kg. The amount of OV-5T35H vector genomes in the liver was at least two orders of magnitude lower than that of Ad5-based viruses. Moreover, animals injected with OV-5T35H virus had significantly lower elevations of serum proinflammatory cytokines and liver enzyme levels. Mice injected with Ad5wt lost more than 20% of their body weight and died or required euthanasia because of poor clinical condition within 4 days of virus administration. Mice treated with OV-5 lost as much as 15% of their body weight over 8-9 days, but recovered within 14 days. Mice that were treated with Addl312 or OV-5T35H exhibited no body weight loss during the study period. These studies suggest that the Ad5/35-based chimeric viruses may have a better safety profile after intravenous injection compared with Ad5-based viruses.

Purpose: Evaluate the codelivery of hyaluronidase enzyme with oncolytic adenoviruses to determine whether it improves the spread of the virus throughout tumors, thereby leading to a greater overall antitumor efficacy in tumor models. Experimental Design: The optimal dose of hyaluronidase that provided best transduction efficiency and spread of a green fluorescent protein (GFP)-expressing adenovirus within tumors was combined with oncolytic viruses in tumor models to determine whether the combination treatment results in an improvement of antitumor efficacy. Results: In mice injected with the adenovirus Ad5/35GFP and an optimal dose of hyaluronidase (50 U), a significant increase in the number of GFP-expressing cells was observed when compared with animals injected with virus only (P < 0.0001). When the oncolytic adenoviruses Ad5OV or Ad5/35 OV (OV-5 or OV5T35H) were codelivered with 50 U of hyaluronidase, a significant delay in tumor progression was observed, which translated into a significant increase in the mean survival time of tumor-bearing mice compared with either of the monotherapy-treated groups (P < 0.0001). Furthermore, the mice that received the combination of Ad5/35 OV and hyaluronidase showed the best antitumor efficacy. Importantly, the combination treatment did not increase the metastatic potential of the tumors. Lastly, the increase in virus potency observed in animals injected with both enzyme and virus correlated with enhanced virus spread throughout tumors. Conclusion: Antitumor activity and overall survival of mice bearing highly aggressive tumors are significantly improved by codelivery of oncolytic adenoviruses and hyaluronidase when compared with either of the monotherapy-treated groups, and it may prove to be a potent and novel approach to treating patients with cancer.

Replication-deficient lentiviral vectors (LV) have been shown to enable the stable genetic modification of multiple cell types in vivo. We demonstrate here that vascular and hepatic delivery of a third-generation HIV-derived lentiviral vector encoding human Factor IX (LV-hFIX) produced potentially therapeutic serum levels of hFIX protein with no vector-mediated local or systemic toxicity of adult mice. Portal vein administration produced the highest serum levels of hFIX and demonstrated proportionally higher levels of gene transfer to the liver with up to 4% of hepatocytes expressing hFIX. Vascular delivery of a lentiviral vector encoding GFP resulted in genetic modification of up to 12% of liver cells. Cell proliferation was not required for hepatocyte transduction with either vector. Serum hFIX levels reached 4% of normal levels following vascular LV-mediated hFIX gene transfer and remained stable for months following vector administration.

Recombinant adeno-associated virus type 2 (AAV) vectors represent a promising gene delivery system because of their nonpathogenicity, ability to stably transduce both dividing and nondividing cells including cells from lung (5), liver (21, 22), brain (13), and muscle (8, 9, 23), and genome-integrating capability which results in long-term protein expression (16, 22). AAV-mediated gene delivery can be potentially obstructed by a host's immune response to its component proteins. In the case of recombinant AAV vectors, the primary target of the immune response is the capsid of the vector particle since these vectors do not encode any viral proteins. Several groups have shown that the failure of AAV readministration to generate further transduction events correlated with the presence of virus-neutralizing antibodies generated in response to a previous exposure to the virus. Manning et al. demonstrated that transient depletion of helper T cells during the initial exposure to AAV with anti-CD4 antibodies allowed successful readministration of AAV vectors to skeletal muscle (14). Similarly, immunosuppression during the initial exposure with anti-CD40L antibodies (which block T-cell activation of B cells) or CTLA4Ig (which inhibits T-cell activation by interfering with CD28-B7 interactions) facilitated transgene expression in mouse lung (6) and also allowed readministration of adenovirus to the mouse liver (10). The liver is a potential target for gene therapy including treatment for hemophilia (21, 22). Since this treatment will likely require delivery to individuals with established preexisting immunity to AAV (1) or repeat vector delivery, and because conclusions regarding vector delivery cannot be extrapolated from tissue to tissue, we examined the effect of preexisting immunity on the delivery of AAV to the liver. In addition, we transiently immunosuppressed the mice concomitantly with readministration of the “therapeutic” AAV, a protocol which closely reflects the reality of a clinical situation in which patients already have immunity, rather than during the primary exposure as reported by others. To delineate further the specificity of the AAV neutralizing antibody response in humans, we used serum samples and a capsid peptide scan (pepscan) in blocking enzyme-linked immunosorbent assays (ELISAs) to map linear antibody epitopes on AAV. Using pools of immunogenic peptides identified in the linear scan, we then identified six peptides that block the effect of neutralizing sera and a neutralizing mouse monoclonal antibody. This information may allow genetic manipulation to circumvent the host immune response for successful AAV vector delivery to patients with preexisting immunity. The immunogenic epitopes described here also corroborate previous genetic and structural data and identify exposed capsid regions potentially involved in the binding of AAV to cellular receptors.

In this report, a Treg-depleting anti-FR4 antibody is combined with a GM-CSF-secreting tumor cell immunotherapy (GVAX) for treatment of melanoma-bearing animals. Median survival time (MST) of animals treated with GVAX was 41 days, compared to a MST of 32 days in untreated animals. Anti-FR4 monotherapy had no effect on MST. Combination of anti-FR4 and GVAX significantly prolonged MST to 55 days, suggesting that these two agents can function cooperatively. Combination therapy increased expression of IFN-γ and granzyme B by CD8 T cells. In contrast to anti-CD25-mediated Treg depletion, administration of anti-FR4 after GVAX did not reduce efficacy, suggesting that anti-FR4 does not deplete effector cells induced by GVAX. Triple combination of a blocking CTLA4 antibody with GVAX and anti-FR4 further enhanced overall survival and reduced growth of well-established melanomas. Considered together, anti-FR4 antibody and GVAX may be a promising approach for the treatment of patients with cancer.

The present study sought to determine the impact of the route of administration of an adeno-associated virus (AAV) vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX. Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice, which typically demonstrate significant immune tolerance to hFIX. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. At all dose levels tested, delivery of AAV-hFIX by an intramuscular (IM) route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals even at doses of 2 x 10(11) DNA viral particles (vp) of AAV-hFIX. This was in stark contrast to the mice that received AAV-hFIX by intraportal vein (IPV) administration. No anti-hFIX inhibitors were observed in any of these mice and therapeutic levels of hFIX were detected in the serum of all mice that received doses of 2 x 10(10) vp AAV-hFIX and higher. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. These results suggest that the threshold of inducing an immune response against a secreted transgene product, in this case the xenoprotein hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV. (Blood. 2001;97:3733-3737)

We describe here that DE1-adenovirus vectors (AV) expressing a p27-p16 fusion molecule, termed W9, induce tumor cell apoptosis when overexpressed in a wide range of tumor cell types. However, in primary human cells derived from a variety of normal tissues, AV-W9 induced minimal apoptosis. In tumor cells AV-W9 demonstrated 5- to 50-fold greater tumoricidal activity than either of the parental molecules p16 and p27. In these studies, AV-W9 elicited apoptosis independent of the p53 and Rb status of the tumor cells. In several murine tumor models AV-W9 demonstrated p53-independent antitumor activity. It completely prevented tumor formation in two ex vivo models, whereas the parental molecules resulted in partial protection. Furthermore, AV-W9 induced tumor regression or suppressed tumor growth when introduced intratumorally into preestablished tumors in mice. This effect may be mediated through tumor cell apoptosis or antiangiogenic activity of AV-W9. Thus, this novel chimeric molecule is more potent and capable of killing a broader spectrum of tumors than the parental p16 and p27 molecules independent of the tumor cell p53 and phenotype and represents a powerful new therapeutic agent for cancer gene therapy.

Cancer of the bladder is a good target for oncolytic virotherapy because the bladder has several unique features that allow for facile instillation of virus with minimal systemic exposure. CG8840 is a conditionally replication competent oncolytic adenovirus (OAV) designed to preferentially replicate and kill uroplakin II-positive bladder tumors. CG8840 replicates in and kills bladder transitional carcinoma cells (TCC) in vitro, with highly attenuated cytotoxicity in other cell types and human primary cells. In vivo, CG8840 elicits reproducible and dose-dependent anti-tumor effects in subcutaneous human xenograft tumor models in nude mice. We have developed an orthotopic bladder tumor model in nude mice to closely mimic the location and behavior of bladder tumors in humans. Employing the human bladder transitional cell carcinoma cell line SW780 we established tumors on the luminal surface of the nude mice bladder. In order to visualize the tumor growth and anti-tumor effect following virus infection, the TCC cell line SW780 was stably transduced to express the luciferase gene product. The bladder lumen was initially seeded with 1 × 106 SW780-luc tumor cells in a volume of 100 μl after which the mouth of the urethra was closed with a purse string for 1hr to allow for tumor cell attachment to the bladder wall. Following IP injection of luciferin, the orthotopic tumors were visualized in vivo by luminescence imaging. Measurement of the photon counts emitted from the tumors following luciferin injection allowed us to monitor the growth of the orthotopic tumors. Within 2 weeks tumor size was large enough to initiate efficacy studies. Immuno-histochemical analyses of the orthotopic tumor-bearing bladder with human anti-cytokeratin staining confirmed the human origin of the tumors. Histologically the SW780-Luc orthotopic tumors closely resembled superficial bladder tumors in humans. The need for pretreatment with dodecyl-β-D-maltoside to enhance infectivity of bladder epithelium with adenovirus was demonstrated earlier employing a beta galactosidase-expressing non-replicative adenovirus (Ramesh et al., 2003). For efficient infection of orthotopic tumors with adenovirus similar pretreatment was found to be essential. Following intravesical instillations of the uroplakin-II positive bladder tumor-specific OAV CG8840 we studied the anti-tumor efficacy of the virus. Three doses of CG8840 (1 × 1010 vp/treatment weekly for 3 consecutive weeks) were efficacious in reducing the tumor burden. Similar antitumor activity was not observed following intravesical instillation of a non-bladder tumor specific OAV. Six weeks after the initiation of OAV treatment, immuno-histochemical examination of the bladder showed complete absence of tumor cells in CG8840-treated mice. This simplified technique consistently produced a high incidence of orthotopic tumors in mice. This will be a useful model to conduct intravesical anti-tumor efficacy studies of OAV or other viruses both as single agents and in combination with other therapeutic agents.