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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.

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.