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.

Efficient infection of tumor endothelial cells by a capsid-modified adenovirus.

Targeted antiangiogenic gene therapy is an attractive approach to treat metastatic cancer. However, the relative paucity of the receptors of the commonly used adenovirus serotype 5 in endothelial cells as compared with liver cells undermines the use of this vector for targeting the endothelial cells in tumors. To overcome this problem, we analyzed the ability of a hybrid Ad5/35 virus, where the serotype 5 fiber has been replaced with the fiber from serotype 35, to target tumor vasculature. Infection of human umbilical vein endothelial cells (HUVECs) with Ad5/35 at MOI 120 infected 100% of cells. In contrast, infection with Ad5 at the same MOI infected only 10% HUVECs. Ad5/35 was even more effective in transducing human aortic endothelial cells (HAECs), as infection with Ad5/35 at MOI 3.6 was sufficient to transduce 95% of cells. Gene expression analyses demonstrated that infection of HUVECs and HAECs with Ad5/35 resulted in between 1 and 3 orders of magnitude higher gene expression than infection with Ad5. Furthermore, various liver-derived cells were less infectable with Ad5/35 than Ad5, indicating a favorable toxicity profile for this virus. In a rat colon carcinoma tumor model, Ad5 was located mainly in the liver parenchyma after hepatic artery administration. In contrast, Ad5/35 was found only in the angiogenesis-rich border region of the tumor. Double immunostaining revealed that Ad5/35 colocalized with CD31 and Flk-1 positive endothelial cells. These results indicate that Ad5/35 may be useful in anticancer strategies targeting tumor endothelial cells.

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.

Telomerase-dependent oncolytic adenovirus for cancer treatment.

Conditionally replicative adenovirus (CRAD) is an attractive anticancer agent as it can selectively replicate in tumor cells. Expression of telomerase reverse transcriptase (TERT) is a unique tumor cell characteristic, being absent in normal postmitotic cells. Thus, we constructed a TERT promoter regulated CRAD for tumor-specific oncolysis by replacing the endogenous adenovirus E1A promoter with that of human TERT (Adv-TERTp-E1A). We showed that its replication was severely attenuated in TERT-negative cells, but that it replicated almost as efficiently as wild-type adenovirus in TERT-positive cells. Accordingly, Adv-TERTp-E1A conferred cytopathicity to TERT-positive, but not TERT-negative, cells. In vivo replication of Adv-TERTp-E1A after local administration into a xenograft model of human hepatocellular carcinoma in nude mice was demonstrated by an increase in adenovirus titers in tumor extracts by several orders of magnitude between 6 h and 3 days postvector injection. Furthermore, significant inhibition of tumor growth with substantial necrotic tumor areas staining positively for adenovirus was observed with Adv-TERTp-E1A, but not with a control replication-deficient adenovirus. There was also the absence of hepatotoxicity in tumor-bearing animals after intratumoral delivery of the CRAD. The results indicate that the TERT promoter-driven CRAD is capable of tumor-selective replication and oncolysis in vitro and in vivo, and can be utilized as an adjuvant treatment agent for cancer.

Transcriptional targeting of conditionally replicating adenovirus to dividing endothelial cells.

Conditionally replicating adenoviruses (CRADs) are a novel strategy in cancer treatment and clinical trials using CRADs targeted to tumor cells have been reported recently. We hypothesized that it would be possible to construct CRADs targeted to dividing endothelial cells, which are present in the tumor endothelium. We utilized the regulatory elements of Flk-1 and endoglin genes, which have been shown to be highly overexpressed in angiogenic endothelial cells, to construct two CRADs: Ad.Flk-1, which has adenoviral E1A gene under the control of the Flk-1 enhancer/promoter, and Ad.Flk-Endo, which harbors the same Flk-1 enhancer/promoter as Ad.Flk-1, plus it has the adenoviral E1B gene under control of the endoglin promoter. Viral titer measurements by plaque assay showed that in human umbilical vein endothelial cells (HUVECs), both CRADs replicated at levels comparable to that of wild-type adenovirus. In Flk-1 and endoglin negative Hep3B and A549 cells, however, the replication of Ad.Flk-1 and Ad.Flk-Endo was reduced by 30-fold and 600-fold, respectively. Cytotoxicity assays demonstrated that both CRADs killed HUVECs as effectively as wild-type adenovirus and their cytotoxicity in Hep3B and A549 cells was comparable to nonreplicating control adenovirus. Furthermore, there was a striking inhibition (83-91%) of capillary network formation in an in vitro angiogenesis assay when HUVECs were infected with Ad.Flk-1 or Ad.Flk-Endo as compared with the nonreplicating control virus. These results demonstrate that CRADs can be transcriptionally targeted to dividing endothelial cells with high specificity, and that the combined use of Flk-1 and endoglin regulatory elements has a synergistic effect on targeting specificity. This principle may be incorporated into novel therapeutic agents to develop anti-angiogenic treatment for cancer.

Tumor-specific transcriptional targeting of suicide gene therapy.

Transcriptional targeting of gene expression has been plagued by the weakness of tissue-specific promoters. Thus, to increase promoter strength while maintaining tissue specificity, we constructed a recombinant adenovirus containing a binary promoter system with a tumor-specific promoter (CEA; carcinoembryonic antigen) driving a transcription transactivator, which then activates a minimal promoter to express a suicide gene (HSV-tk; herpes simplex virus thymidine kinase). This ADV/binary-tk induced equal or greater cell killing in a CEA-specific manner in vitro compared with the CEA-independent killing of a vector with a constitutive viral promoter driving HSV-tk (ADV/RSV-tk). To monitor adenovirus-mediated HSV-tk gene expression in vivo, we employed noninvasive nuclear imaging using a radioiodinated nucleoside analog ([((1)31)I]-FIAU) serving as a substrate for HSV-tk. [((1)31)I]-FIAU-derived radioactivity accumulated after intratumoral injection of ADV/binary-tk only in the area of CEA-positive tumors with significantly less spread to the adjacent liver tissue than after administration of the universally expressed ADV/RSV-tk. Both viruses exhibited similar antitumor efficacy upon injection of liver metastases. Importantly, in vivo dose escalation studies demonstrated significantly reduced toxicity after intravenous administration of ADV/binary-tk versus ADV/RSV-tk. In summary, the increased therapeutic index of this novel, amplified CEA-driven suicide gene therapy vector is a proof of principle for the powerful enhancement of a weak tissue-specific promoter for effective tumor restricted gene expression.