[CANCERRESEARCH 57.466-471.February1, 1997] ABSTRACT MOdified, nonneurovirulent herpes simplex viruses (HSVs) have shown promise in the treatment of brain tumors. However, HSV-1 can infect and lyse a wide range of cell types. In this report, we show that HSV-1716, a mutant lacking both copies of the gene coding ICP-34.5, can effectively treat a localized Lp malignancy. Human malignant mesothelloma cells supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. injection of HSV-1716 into animals with established tumor nodules re duced tumor burden and significantly prolonged survival in an animal model ofnon-central nervous system-localized human malignancy without dissemination or persistence after i.p. injection Into SC@ mice bearing human tumors. These findings suggest that this virus may be efficacious and safe for use in localized human malignancies of noaneuronal origin such as malignant mesothelioma. INTRODUCTION The recent emergence of viral-based “gene therapy―as an approach to treat cancer has generated a great deal of enthusiasm and interest (1). For example, our group and others have explored the use of retrovirus-producing cells or adenoviruses to deliver the HSV& gene into localized malignancies, such as brain tumors and malignant mesothelioma (2—6). Although a “bystander― effect amplifies the efficacy of the HSVtk/ganciclovir system (7), it is likely that a major obstacle in treating actual patients with localized malignancies will be the ability to transduce a sufficient number of cells within a large tumor mass. One strategy that holds promise is the use of replicating viral vectors (8). The idea of using replicating viruses to treat tumors was suggested as early as 1904 by George Dock (9); in fact, a number of clinical trials were conducted in the l950s and 1960s with some success (10, 11), although interest in the use of viral-based cancer therapy waned thereafter. Advances in virology and molecular biology now allow the “engineering―of viruses with specific properties, suggesting that the idea of viral-based cancer therapy should be revisited. One promising virus in this regard is HSV-l. A number of HSV-l mutants have recently been identified that appear to replicate preferentially in rapidly dividing transformed cells (12, 13). Because of the natural tropism of wild-type herpes virus for neuronal tissue, the published uses of modified, replicating HSV to treat cancer have centered on tumors of CNS origin (14—16).One of these mutants is HSV-1716, a virus that contains a 759-bp deletion in the genes coding for ICP 34.5 Received 7/29/96; accepted 12/4/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I The first two authors contributed equally to this work. 2 To whom requests for reprints should be addressed, at Division of Thoracic Surgery, University of Pennsylvania Medical Center, 4 Silverstein, 3400 Spruce Street, Philadel phia, PA 19104. Phone: (215) 662-7538. 3 The abbreviations used are: HSVtk, herpes simplex thymidine kinase; HSV-l, herpes simplex-l virus; CNS, central nervous system; ICP, infected cell protein; MO!, multi plicity of infection; PFU, plaque-forming unit; MT1', 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide. (12). This mutation, through mechanisms that are still unclear, se verely attenuates the ability of HSV-l716 to replicate in normal tissues (12, 17, 18), but does not appear to affect the virus' ability to replicate in rapidly dividing malignant cells. We and others (18, 19) have shown that HSV-1716 is extremely nonneurovirulent in animal models and can be used to treat intracranial tumors in normal and immunodeficient mouse models (15, 16, 20). Neurovirulent, wild-type HSV-l is able to infect and lyse a wide variety of cell types. Therefore, we reasoned that a mutant HSV virus, such as HSV-17l6, might be efficacious in the treatment of localized, non-CNS malignancies. In addition, the use of this virus in a location distant from the CNS might offer significant safety advantages. Ac cordingly, in this paper we report on the successful in vitro and in vivo use of HSV-l716 to treat a localized, non-CNS malignancy. We have chosen human malignant mesothelioma as a model tumor system for HSV-l716 because of the localized nature of this malignancy (allow ing for direct virus administration) and because of the current lack of effective treatment (21). MATERIALS AND METHODS HSV-1716. HSV-l716 was originally isolated in the laboratory of S. M. Brown(Glasgow, Scotland)andpassagedfor use in this studyby N. W. Fraser (Philadelphia, PA). The genome of this virus contains a 759-bp deletion located within each copy of the BamHI fragment of the long repeat region of the genome (12). These deletions remove most of the gene encoding ICP 34.5, and the mutant fails to make the protein (12, 22) In Vitro Studies of HSV-1716 on a Human Malignant Mesothelioma Cell Line. A humanmalignantmesotheliomacell line call RENwas isolated, characterized, and passaged as described previously by our laboratory (23). Human lung cancer cell lines A549, H322, and H358 were obtained from the American Type Culture Collection (Rockville, MD). The human osteosarcoma cell line SAOS was obtained from T. Halazonetis (The Wistar Institute). The human melanoma cell line WM451 Lu was obtained from M. Herlyn (The Wistar Institute) and was isolated, characterized, and passaged as described previously (24). To construct single-step viral growth curves, cells were plated on six-well plates at a density of 5 X iO@cells/well and infected 24 h later with HSV-l7l6 at a MO! of 0.01 (5 X l0@PFU/well). One well was harvested at 0, 6, 12, and 24 h by cell scraping and collection of the media. The samples were freeze/thawed and titered by plaque assay on baby hamster kidney cell monolayers. A cell viability assay was performed by plating cells in 96-well plates at a density of 5 X l0@cells per well. Twenty-four h later, the cells were infected with HSV-l716 at MOIs of 0, 0.001, 0.01, and 0.1. Six wells were infected at each MO!. A sufficient number of plates were used to allow for viability assay at 24, 48, 72, and 96 h after infection. Viable cell number was assessed by a colorimetric assay (CellTiter 96 Aqueous Nonradioactive MTT Cell Proliferation Assay; Promega, Madison WI) that measures viable cell dehydrogenase activity by absorbance. The percentage of control growth is defined as the ratio of the mean absorbance of six treatment wells at 490 nm to the mean absorbance of six untreated matched controls. In Vivo Studies. A previously described model of human malignant me sothelioma growing in the peritoneal cavity of SCID mice was used for all in vivoexperimentation (23). Animal protocols were approved by the Animal Use committees of the Wistar Institute and the University of Pennsylvania in compliance with the Guide for the Care and Use of Laboratory Animals (NIH 466 Use of a “Replication-Restricted― Herpes Virus to Treat Experimental Human Malignant Mesothelioma John C. Kucharczuk,' Bruce Randazzo,' Michael Y. Chang, Kunjiata M. Amin, Ashraf A. Elshami, Daniel H. Sterman, Nabil P. Rizk, Katherine L. Molnar-Kimber, S. Moira Brown, Alasdair R. MacLean, Leslie A. Litzky, Nigel W. Fraser, Steven M. Albelda, and Larry R. Kaiser@ Thoracic Oncology Research Laboratory Ii. C. K.. M. Y. C., K. M. A., A. A. E., D. H. S.. N. P. R.. K. L M-K.. L A. L. S. M. A., L R. K.] and Department of Dermatology [B. R.]. University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104; Wistar Institute, Philadelphia, Pennsylvania 19104 (B. R., N. W. F., S. M. A.]: and Glasgow University, Neurovirology Research Laboratories, Glasgow, Scotland 651 4TF (S. M. B., A. R. M.]