Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

Enhanced oncolytic potency of vesicular stomatitis virus through vector-mediated inhibition of NK and NKT cells.

Recombinant oncolytic viruses represent a promising alternative option for the treatment of malignant cancers. We have reported earlier the safety and efficacy of recombinant vesicular stomatitis virus (VSV) vectors in a rat model of hepatocellular carcinoma (HCC). However, the full potential of VSV therapy is limited by a sudden decline in intratumoral virus replication observed early after viral administration, a phenomenon that coincides with an accumulation of inflammatory cells within infected lesions. To overcome the antiviral function of these cells, we present a recombinant virus, rVSV-UL141, which expresses a protein from human cytomegalovirus known to downregulate the natural killer (NK) cell-activating ligand CD155. The modified vector resulted in an inhibition of NK cell recruitment in vitro, as well as decreased intratumoral accumulations of NK and NKT cells in vivo. Administration of rVSV-UL141 through hepatic artery infusion in immune-competent Buffalo rats harboring orthotopic, multi-focal HCC lesions resulted in a one-log elevation of intratumoral virus replication over a control rVSV vector, which translated to enhance tumor necrosis and substantial prolongation of survival. Moreover, these results were achieved in the absence of apparent toxicities. The present study suggests the applicability of this strategy for the development of effective and safe oncolytic agents to treat multi-focal HCC, and potentially a multitude of other cancers, in the future.

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma.

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.

Auto-regulated hepatic insulin gene expression in type 1 diabetic rats.

Paradigms of insulin gene therapy for type 1 diabetes should incorporate vigorous control for insulin gene expression to be effective in correcting postprandial hyperglycemia and to be safe in preventing fasting hypoglycemia. We hypothesize that hepatic insulin gene expression auto-regulated positively by glucose and negatively by insulin might be both effective and safe in the treatment of type 1 diabetes. Expression of the glucose 6-phosphatase (G6Pase) gene in the liver is both stimulated by glucose and suppressed by insulin. The G6Pase promoter incorporated with intronic enhancers of the aldolase B gene was used to direct insulin gene expression in the liver of streptozotocin-induced diabetic nude rats. In the treated animals, blood insulin levels were elevated after feeding, and nonfasting hyperglycemia was significantly reduced. Glucose tolerance testing also illustrated that the treated animals exhibited accelerated glucose utilization rates. Upon fasting, blood glucose was reduced to normoglycemic range within 4 h and maintained at that level during the prolonged fasting of 16 h. No hypoglycemia was observed in any treated animals at any time throughout the fasting period, as blood insulin gradually declined to the normal range. These results suggest that auto-regulated hepatic insulin expression can potentially be developed as an effective and safe treatment modality for type 1 diabetes.

Hepatic insulin expression improves glycemic control in type 1 diabetic rats.

Low levels of hepatic insulin production have been shown to prevent lethal ketoacidosis associated with type 1 diabetes. To assess the beneficial effects of sustained hepatic production of insulin on glycemic control in type 1 diabetes, we have employed the adenovirus-mediated gene delivery system to transfer an engineered rat preproinsulin gene to the livers of streptozotocin-induced diabetic nude rats. Hepatic insulin production resulted in the reduction of blood glucose in treated diabetic rats, the degree of blood glucose reduction correlated with both the vector dose and the level of hepatic insulin expression. At moderate vector doses, 0.3-0.7 ng/ml of plasma insulin was produced in treated diabetic animals, resulting in significant reduction of nonfasting hyperglycemia and improvement in glucose tolerance. Furthermore, these animals maintained euglycemia after 12-h fast. At higher vector doses, greater than 1 ng/ml of plasma insulin was produced, completely reversing nonfasting hyperglycemia in treated rats. However, all of the treated animals developed severe hypoglycemia upon fasting. This study has defined the maximal tolerable level of hepatic insulin production that is sufficient to reduce the degree and ameliorate the adverse effects of nonfasting hyperglycemia without risk of fasting hypoglycemia in type 1 diabetic rats.

Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells.

The liver is an attractive target organ for insulin gene expression in type 1 diabetes as it contains appropriate cellular mechanisms of regulated gene expression in response to blood glucose and insulin. We hypothesize that insulin production regulated by both glucose and insulin may be achieved using the promoter of the glucose 6-phosphatase gene (G6Pase), the expression of which in the liver is induced by glucose and suppressed by insulin. Recombinant adenoviral vectors expressing the reporter gene CAT or insulin under transcriptional direction of the G6Pase promoter were constructed. Glucose-stimulated as well as self-limiting insulin production was achieved in vector-transduced hepatoma cells in which expression of the insulin gene was controlled by the G6Pase promoter. While insulin strongly inhibited the G6Pase promoter activity under low glucose conditions, its inhibitory capacity was attenuated when glucose levels were elevated. At the physiologic glucose level of 5.5 mM glucose, vector-transduced hepatoma cells produced a self-limited level of insulin at approximately 0.2-0.3 ng/ml, which is within the range of fasting levels of insulin in normal animals. These results indicate that the G6Pase promoter possesses desirable features and may be developed for regulated hepatic insulin gene expression in type 1 diabetes.