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Intravesical oncolytic viral therapy using attenuated, replication-competent herpes simplex viruses G207 and Nv1020 is effective in the treatment of bladder cancer in an orthotopic syngeneic model¹

PAUL J. COZZI^*,, SANDEEP MALHOTRA, PRISCILLA MCAULIFFE, DAVID A. KOOBY, HOWARD J. FEDEROFF, BOB HURYK^*, PAUL JOHNSON^¶, PETER T. SCARDINO, WARREN D. W. HESTON^* and YUMAN FONG²

^* The George M O’Brien Urology Research Center,
Departments of Urology and
Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA,
Departments of Neurology and Medicine, University of Rochester Medical Center, Rochester, New York, USA; and
^¶ NeuroVir Therapeutics Inc., Vancouver, BC, Canada

²Correspondence: Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA. E-mail: fongy{at}mskcc.org

SPECIFIC AIMS

Attenuated, replication-competent herpes simplex virus mutants have recently been shown to replicate within and kill tumor cells while remaining at low pathogenicity to normal tissues. In this study, we investigated the effects of two promising candidates, G207 and NV1020, for their ability to infect and lyse human and murine transitional cell carcinoma (MBT-2) cells in vitro and for in vivo efficacy in a well-established orthotopic, immunocompetent animal model of bladder cancer.

PRINCIPAL FINDINGS

1. Both G207 and N1020 are capable of effective in vitro tumor cell lysis, with NV1020 significantly more effective at lower doses of virus
Both viruses are attenuated, replication-competent herpes simplex viruses that have shown particular promise as anticancer agents. G207 is a genetically engineered oncolytic virus based on wild-type herpes simplex type 1. The key features of G207 include 1) the deletion of both copies of ₁34.5 leading to highly attenuated neurovirulence and 2) insertion of a lacZ gene to interrupt the U_L39 gene, which encodes for the large subunit of ribonucleotide reductase, an enzyme required for viral DNA synthesis in nondividing cells.

NV1020 is a clonal derivative of R7020 originally constructed for vaccine studies against HSV-1 and HSV-2. NV1020 has a 700 bp deletion in the thymidine kinase locus and a 15 kb deletion across the joint region of the long (L) and short (S) components of the HSV-1 genome. The L/S junction of the NV1020 contains a 5.2 kb fragment of HSV-2 DNA inserted for previous vaccine studies and an exogenous copy of the thymidine kinase gene under the control of the HSV-1 4 promoter.

Both viruses were examined in vitro for their ability to lyse human and murine bladder cancer cell lines. At higher multiplicity of infection (MOI=1), both G207 and NV1020 demonstrated highly effective cell lysis, with greater than 70% cell kill at 4 days after treatment. At MOI = 0.1 (1 virus particle per 10 tumor cells), NV1020 was more efficacious than G207 in the poorly differentiated human transitional cell carcinoma cell lines J-82 (P<0.004), SKUB (P=0.02), and T-24 (P<0.001) (Fig. 1 ). No difference between the viability of control cultures or cells treated with the heat inactivated viruses was demonstrated throughout these experiments.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of G207 (filled boxes) and NV1020 (open boxes) in direct comparison in the human cell lines, MOI 0.1. *P < 0.05.

2. Infection efficiency correlates with oncolytic effect
G207 infected all tumor cell lines tested with high efficiency as measured by ß-galactosidase expression (% blue cells). 100% infection efficiency was seen with G207 at 24 h in MBT-2, SKUB, T-24, and RT-4 cells (MOI=1) and at 48 h in J-82 cells (MOI=2). At lower MOI, the percentage of blue cells increased from 24 h to 72 h for all cell lines, resulting in impressive X-gal staining even at the lowest MOI of 0.01 (1 virus particle per 100 tumor cells). A correlation was found between infection efficiency and cytotoxicity for all cell lines (R=0.9).

3. NV1020 is capable of more efficient viral replication than G207
Viral replication was best supported by the most susceptible cell line (RT-4) and not well supported by the least susceptible (J-82) for both G207 and NV1020. Other cell lines demonstrated support of varying levels of viral replication in between those of RT-4 and J-82. When compared directly, NV1020 replicated to higher titers than G207. Peak viral titer in the J-82 cell line was significantly greater for NV1020 at day 6 after treatment (23.4 vs. 0.05 PFU x 10⁶, P<0.001).

4. A single intravesical instillation of virus provides a high level of tumor specific infection
Efficient infection of orthotopic bladder tumors was achieved after a single intravesical instillation of G207. ß-Galactosidase expression was noted in tumor samples for 48 h after treatment at doses of 6 x 10⁵, 6 x 10⁶, or 1 x 10⁸ PFU. 1 x 10⁷ PFU was chosen as an intermediate dose to continue experiments and was administered to animals at day 8 (n=4), day 12 (n=4), and day 16 (n=5) after tumor instillation and to animals without tumor (n=4). ß-Galactosidase expression was identified within 60–75% of bladder tumors at each time point. In tumors with staining, the percentage of tumor cells stained varied between 25 and 50% and no staining of normal urothelium was observed. In multiple experiments with G207 and NV1020, no ß-galactosidase expression or signs of inflammation (hematoxylin and eosin histology) were observed in liver, brain, kidney, spleen, lung, ovary, and heart harvested from these animals.

5. A single intravesical instillation of G207 is effective at achieving cures and reducing tumor burden
Animals treated with a single instillation of G207 (1x10⁷ PFU in 50 µl PBS) at day 3 after tumor inoculation (n=15, in each group) showed a significant reduction in tumor weight as compared to controls (P=0.02, Mann-Whitney U test). All 15 animals in the control group developed tumors with a mean weight (±SD) of 310 (±130) mg compared with 9 animals in the G207 group with a mean tumor weight (±SD) of 150 (±110) mg.

6. Multiple instillation of G207 or NV1020 is effective at achieving cures and reducing tumor burden and provides comparable efficacy to intravesical bacillus Calmette-Geurin (BCG) without adverse effects
Both G207 and NV1020 were highly effective when administered by intravesical instillation weekly for 3 wk. Treatment was well tolerated, with no adverse events and no treatment-related deaths. Animals were terminated at day 21 due to signs of excessive tumor burden in the control group. Ten of 11 animals in the control (PBS only) group demonstrated bladder tumors at necropsy. Six of 12 animals in the BCG group demonstrated bladder tumors (P=0.06). A significant difference was seen in the groups treated with virus when compared to control with 5 of 13 animals in the G207 group (P=0.02) and only 2 of 12 animals in the NV1020 group with bladder tumors (P=0.001). Tumor weight was also significantly reduced between groups treated with virus when compared to control (P=0.001 for NV1020 and P=0.008 for G207). There was no statistically significant difference between viruses; similarly, there was no difference when comparing NV1020 and BCG or G207 and BCG.

In these experiments, treated animals continued to demonstrate normal activity with normal feeding and grooming habits. Control animals exhibited signs of excessive tumor burden including weight loss, hematuria, and a palpable mass. Animals tolerated the treatment well with no adverse events and no deaths.

CONCLUSIONS AND SIGNIFICANCE

Treatments that are more effective are required for patients with high-risk superficial bladder cancer. Intravesical BCG is so far the most effective treatment and has been shown to be effective at reducing disease recurrence. However, excessive morbidity and even mortality from intravesical BCG together with recent reports demonstrating no effect on the long-term risk of disease progression suggest that more effective, well-tolerated treatments for patients with high-risk superficial bladder cancer are needed.

The current studies demonstrate that these viruses are promising agents in the treatment of bladder cancer. G207 and NV1020 are able to infect, replicate and lyse preferentially in rapidly dividing cells. Our experiments have shown that G207 efficiently infects, replicates within, and subsequently kills human and murine transitional carcinoma cell lines. All five cell lines proved to be remarkably susceptible to lysis after infection by G207 and NV1020, even at low MOI. A correlation between infection efficiency and subsequent lysis was clearly demonstrated. NV1020 clearly demonstrated superior cytolytic effect at lower MOI (0.1) in all three poorly differentiated human transitional cell carcinomas (J-82, SKUB, and T-24). The clinical implications of these findings are clear. In highly susceptible tumors, such as the RT-4 cell line, either viral preparation will likely prove efficacious. Patients with tumors that are more resistant or with tumors of heterogeneous sensitivity will likely benefit from treatment with NV1020.

Bladder cancer is an ideal target for novel therapies due to the ease of intravesical delivery, allowing exposure of the tumor to large titers of vector. In addition, replication competent oncolytic viruses, which selectively infect and replicate within rapidly dividing cells, are potentially useful for bladder cancer because the umbrella cell layer (i.e., the luminal surface of the urothelium) of the bladder is not rapidly dividing and should therefore be resistant to infection and lysis.

Data from the current experiments in a well-accepted, orthotopic syngeneic model confirm safety, efficacy, and ease of delivery of oncolytic viral therapy for treatment of experimental bladder cancer. X-gal staining demonstrated that G207 was capable of infecting tumor cells while sparing normal urothelial cells and normal tissues including liver, kidney, and brain. Although both G207 and NV1020 are highly attenuated and replication restricted, therapeutic use of replicating viruses still raises concerns of viral proliferation and dissemination. Both mutants retain thymidine kinase (tk) expression, allowing anti-viral drug therapy (e.g., ganciclovir) to remain effective. Both G207 and NV1020 have recently been found to be effective in treating various experimental cancers while maintaining an excellent safety profile. This is confirmed in our current model. Treated animals continued to groom and behave normally. No adverse events were noted and there were no treatment-related deaths. Cured animals continued to gain weight whereas uncured animals lost weight toward the end of experiments due to tumor burden. Standard histology and histochemical analysis of multiple organs revealed no evidence of infection, inflammation, or other abnormality in animals treated with either virus. We and others have shown the safety of G207 and NV1020 in animal models with direct tumor injection, intravenous administration, and hepatic intra-arterial administration. The risk of intravasation of virus is low after intravesical instillation; however, should it occur, our data from previous experiments indicate that it would be rapidly cleared from the bloodstream. Furthermore, studies of NV1020 in owl monkeys, the nonhuman primate most sensitive to wild-type HSV infection, have shown markedly attenuated pathogenicity and no transmission of virus to noninfected cage mates.

We have demonstrated selective and tumor-specific cytolytic effect in this orthotopic animal model of bladder cancer after intravesical administration of G207 and NV1020. These viruses appear to be safe in the best available immunocompetent animal model of human bladder cancer currently available. Further studies are required to elicit the exact mechanism of cell kill and the immune events surrounding treatment in this model. This report encourages further evaluation of intravesical oncolytic viral therapies for bladder cancer in clinical trials.SCHEME

View larger version (27K): [in this window] [in a new window]   Scheme 1. No caption available.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0533fje ; to cite this article, use FASEB J. (March 5, 2001) 10.1096/fj.00-0533fje

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