WO2003073919A2

WO2003073919A2 - Treatment of metastatic institute for cancer

Info

Publication number: WO2003073919A2
Application number: PCT/US2003/006659
Authority: WO
Inventors: Yuman Fong; Michael I. Ebright
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 2002-03-01
Filing date: 2003-03-03
Publication date: 2003-09-12
Also published as: AU2003217925A1; WO2003073919A3

Abstract

The invention provides methods of treating metastatic or primary cancer of the pleura, involving the use of replication-competent, attenuated herpes viruses.

Description

TREATMENT OF METASTATIC OR PRIMARY PLEURAL CANCER

Field of the Invention

This invention relates to methods of treating cancer of the pleura.

Background of the Invention

Metastatic disease of the pleura, which is the thin, transparent membrane that covers the lungs and lines the thoracic cavity, is a common and significant management problem for the thoracic oncologist. This disease is associated with a grim prognosis, with most patients surviving only a few months after diagnosis (Sahn et al., Ann. Intern. Med. 108:345-349, 1988). It occurs most frequently in patients with locally advanced or disseminated cancer, but occasionally occurs as a disease of unknown primary. The pleural tumor itself usually manifests as a malignant effusion, but it can also present as a pleural-based mass. The most common etiologies of pleural tumors are lung, breast, ovarian, lymphoid, uterine, and gastrointestinal cancers (Johnston, Cancer 56:905-909, 1985; DiBonito et al., Acta Cytol. 36:329-332, 1992; Chernow et al., Am. J. Med., 63:695-702, 1977), with the majority of cases being adenocarcinoma by histotype. Systemic chemotherapy has a poor response rate in treating pleural malignancies. Several chemotherapeutic agents have been used to treat pleural disease by intrapleural administration, with no more than modest success (Figlin et al., Chest 106:(6

Suppl):363S-366S, 1994; Tohda et al. Chemotherapy 45:197-204, 1999). As a result, new therapeutic modalities are needed to treat this condition.

Summary of the Invention The invention provides methods of treating metastatic or primary cancer of the pleura in patients, such as humans. The methods involve administration of attenuated, replication-competent herpes viruses to patients. The viruses can be administered systemically (e.g., intravenously) or, preferably, intrapleurally. Examples of cancers that metastasize to the pleura, and thus which can be treated using the methods of the invention when so metastasized, include lung, breast, ovarian, and lymphatic cancers. An example of a primary pleural cancer that can be treated using the methods of the invention is pleural mesothelioma.

Herpes viruses that can be used in the methods of the invention include herpes simplex virus-1 (HSV-l)-derived viruses. These viruses can include mutations, such as, for example, an inactivating mutation in a γ34.5 gene and or an inactivating mutation in a U_L 4 gene. A specific example of a herpes virus including both of these mutations, which can be used in the invention, is NV1020. The viruses can also include an inactivating mutation in the ribonucleotide reductase gene of the virus, optionally in combination with the mutations noted above. A specific example of a virus including inactivating mutations in a γ34.5 gene, a U_L24 gene, and a ribonucleotide reductase gene is G207.

Optionally, the herpes virus administered according to the methods of the invention includes a heterologous nucleic acid molecule encoding a therapeutic product, which can be, for example, a cytotoxin, an immunomodulatory protein, a tumor antigen, an antisense nucleic acid molecule, or a ribozyme.

The herpes virus treatment of the invention can also be carried out in conjunction with a second (or more) anticancer treatment. For example, it can be carried out in conjunction with chemotherapy, biological therapy, radiation therapy, gene therapy, and surgery. Examples of types of chemotherapeutic agents include alkylating agents, antmeoplastic antibiotics, antimetabolites, and natural source derivatives, and specific examples of each of these types of chemotherapeutic agents are as follows: alkylating agents: busulfan, carboplatm, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa; antmeoplastic antibiotics: bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, pentostatin, and plicamycin; antimetabolites: fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fludarabine, flurouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine; and natural source derivatives: docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol, prednisone, and tamoxifen.

An example of a type of biological therapeutic agent that can be used in the methods of the invention is immunomodulatory molecules, such as cytokines, chemokines, complement components, complement component receptors, immune system accessory molecules, adhesion molecules, and adhesion molecule receptors.

Specific examples of cytokines include, for example, interleukins, interferons, tumor necrosis factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, and granulocyte colony stimulating factor. Also included in the invention is the use of the viruses mentioned above and elsewhere herein in the preparation of medicaments for use in the prevention or treatment of metastatic or primary pleural cancer.

The invention provides several advantages. For example, as is discussed further below, mutant herpes viruses that can be used in the invention replicate in, and thus destroy, dividing cells, such as cancer cells, while not affecting other, quiescent cells in the body. These herpes viruses can also be multiply mutated, thus eliminating the possibility of reversion to wild type. Moreover, if necessary, the replication of herpes viruses can be controlled through the action of antiviral drugs, such as acyclovir, which block viral replication, thus providing another important safeguard. The theoretical advantage of using a replication-competent virus is that only a fraction of tumor cells need to be infected initially before the virus propagates in permissive cancerous tissue.

The invention thus provides safe and effective methods for treating pleural cancer. Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.

Brief Description of the Drawings Fig. 1 is a graph showing the in vitro cytotoxicity of NV 1020 against A549 cells expressed as a fraction of control. Cell counts were measured daily for 7 days. Assays were performed in triplicate, and error bars indicate SEM. Fig. 2 is a graph showing NV1020 replication in A549 cells after in vitro infection with an MOI of 1.0 (5x10⁴ PFU). Viral titers were evaluated by standard plaque assay. Assays were performed in triplicate, and error bars indicate SEM.

Fig. 3 is a graph showing mean intrapleural tumor weight in rats treated 3 hours after tumor inoculation. Rats were treated with intrapleural PBS, intravenous NV1020 (lxlO⁷ PFU), or intrapleural NV1020 (lxlO⁷ PFU). Error bars indicate SEM. The p- value for the intravenous treated group is 0.29, and for the intrapleural group is 0.001. Fig. 4 shows representative sections of tumor implants evident 3 days after intrapleural tumor inoculation. Shown are samples (20x) from mediastinal fat (A), chest wall (B), and diaphragm (C).

Fig. 5 is a graph showing mean intrapleural tumor weight in rats treated 3 days after tumor inoculation. Rats were treated with intrapleural PBS or intrapleural NV1020 (lxlO⁷ PFU). Error bars indicate SEM. The p-value using a non-parametric Wilcoxon test is 0.046.

Fig. 6 is a representative section of pleural tumor from rats treated with intrapleural virus 72 hours earlier. Sections were analyzed by immunohistochemistry using a polyclonal anti-HSV primary antibody. Brown cells are positive for viral antigen.

Fig. 7A is a graph showing the cytotoxic effect of NV1066 on A549 cells. NV1066 effectively kills A549 cancer cells in vitro at multiplicities of infection (MOI) of 0.1 or 1.0. Results are expressed as cell survival compared to untreated control cells grown under identical conditions. Fig. 7B is a graph showing an analysis of in vitro replication of NV1066. NV1066 replicates effectively in A549 cells, with peak viral titers demonstrating a 54-fold increase over the original infecting dose of virus. Fig. 7C is a graph showing EGFP expression in NV1066-infected cells. Following treatment with NV1066, infected A549 cells express EGFP, as determined by flow cytometry. Results are reported as percent of live cells in the population expressing EGFP. While initially dose-dependent, following viral replication, EGFP expression incrementally increased to nearly 100% with both MOIs.

Fig. 8 are graphs showing the antitumor effect of NV 1066 in vivo. Fig. 8 A shows organ weights (heart, lungs, and mediastinal tissue). Intrapleural injection of NV1066 decreased tumor burden, as compared to PBS treated mice at 3 weeks.

Extensive tumor deposits led to organ weights that were 53% greater than those seen in NV1066 treated mice (p < .001, t-test). Fig. 8B shows chest wall nodules. Similarly, intrapleural injection of NV1066 decreased chest wall tumor nodules, compared to treatment with PBS alone (p < .001, t-test). Seven of 9 mice treated with NV1066 had no evidence of chest wall nodules at time of sacrifice, while all 5 mice treated with PBS alone had developed macroscopic tumor nodules. Fig. 9 shows in vivo EGFP expression. EGFP expression was easily visualized by fluorescent microscopy. Figures 9A and 9C represent global views, while 9B and 9D represent close-up views of tumor deposits in the thoracic cavity. Tumors were visualized in brightfield (9A and 9B) and subsequently in green fluorescent mode (9C and 9D) using an Olympus stereotactic microscope with appropriate filters. EGFP expression localized to tumor deposits, sparing normal tissues, and could be used to localize foci of tumor less than even 2 mm in diameter.

Fig. 10 shows thoracoscopic identification of EGFP expression. Figs. 10A-D demonstrate images obtained using the fluorescent thoracoscopic system in animals with A549 pleural carcinomatosis. Tumor deposits were visualized with conventional (10A and 10B) and fluorescent (IOC and 10D) thoracoscopy. Non-tumor bearing organs did not fluoresce when examined through the EGFP filter and were easily distinguished from tumor.

Fig. 11 shows viral specificity for tumor. Tissue specimens were selected by EGFP expression under stereomicroscopy in intact animals. Serial sectioning was performed and specimens were examined under fluorescent microscopy (11 A), then H & E stained (1 IB) for identification of tumor cells. Representative sections of a nodule taken from the surface of the pleura are shown. Following intrapleural administration of NV1066, strong EGFP expression was noted in the tumor nodule. All sections that expressed EGFP had tumor cell infiltrates.

Detailed Description The invention provides methods of treating cancer of the pleura. These methods, involve the administration of attenuated, replication-competent herpes viruses, which, as is discussed furtlier below, we have shown are effective at selectively destroying cancer cells that are present in the pleura. Use of these viruses in the methods of the invention, as well as experimental results showing the efficacy of these methods, are described further below.

Pleural cancers that can be treated using the methods of the invention can be metastatic or primary. The most common primary malignant neoplasms that metastasize to the pleura are lung cancer (36%), breast cancer (25%), lymphoma (10%), ovarian cancer (5%), and gastric cancer (2%), with the most common cell type metastasizing to the pleura being the adenocarcinoma. Additional types of cancers that can metastasize to the pleura include uterine cancer, gastrointestinal tract cancer, thymoma, leukemia, myeloma, and renal cell carcinoma. An example of a primary cancer of the pleura is mesothelioma, which is caused by exposure to asbestos fibers. The methods of the invention can be used in the treatment of any of these and other types of cancers that are present in the pleura, whether in a malignant effusion or a tumor mass.

Viruses that can be used in the methods of the invention can be derived from any of the members of the family Herpesviridae. For example, herpes simplex virus- 1 (HSV-l)-derived viruses can be used. Additional examples of herpes family viruses from which viruses that are used in the invention can be derived are herpes simplex virus-2 (HSV-2), vesicular stomatitis virus (VSV), cytomegalovirus (CMV), Epstein- Barr virus (EBV), human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7), and human herpes virus-8 (HHV-8). A central feature of the viruses that can be used in the methods of the invention is that they are replication-competent, and thus are able to infect, replicate in, and lyse malignant cells, while at the same time they are sufficiently attenuated to not adversely affect normal cells.

One specific example of an HSV- 1 -derived virus that can be used in the methods of the invention is NV1020. The U_L24 gene, as well as one of the two copies of the HSV major neurovirulence gene, γ34.5, are deleted in this virus. NV1020 is described in further detail below. An additional specific example of an HSV- 1 -derived virus that can be used in the methods of the invention is G207 (Yazaki et al., Cancer Res. 55(21):4752-4756, 1995). This virus has deletions in both copies of the γ34.5 gene, as well as an inactivating insertion in UL39, which is the gene that encodes infected-cell protein 6 (ICP6), the large subunit of HSV ribonucleotide reductase. Still a further specific example of a herpes virus that can be used in the invention is G47Δ (Todo et al., Proc. Natl. Acad. Sci. U.S.A. 98(11):6396-6401, 2001), which is a multimutated, replication-competent HSV-1 vector that was derived from G207 by a 312 basepair deletion within the non-essential a47 gene (Mavromara-Nazos et al., J. Virol. 60:807-812, 1986). Because of the overlapping transcripts encoding ICP47 and US11 in HSV, the deletion in a.47 places the late US11 gene under control of the immediate-early a.47 promoter, which enhances the growth properties of γ34.5' mutants. Additional examples of attenuated HSV viruses that can be used in the methods of the invention include hrR3, which is ribonucleotide reductase-defective (Spear et al., Cancer Gene Ther. 7(7):1051-1059. 2000), NV1023 (Wong et al., Hum. Gene Ther. 12:253-265, 2001), HF (ATCC VR-260), Maclntyre (ATCC VR-539), MP (ATCC VR- 735), HSV-2 strains G (ATCC VR-724) and MS (ATCC VR-540), as well as any viruses having mutations (e.g., inactivating mutations, deletions, or insertions) in any one or more of the following genes: the immediate early genes ICPO, ICP22, and ICP47 (U.S. Patent No. 5,658,724); the γ34.5 gene; the ribonucleotide reductase gene; and the VP16 gene (i.e., Vmw65, WO 91/02788, WO 96/04395, and WO 96/04394). The vectors described in U.S. Patent Nos. 6,106,826 and 6,139,834, as well as other replication-competent, attenuated herpes viruses, can also be used in the methods of the invention.

The effects of the viruses used in the methods of the invention can be augmented, if desired, by including heterologous nucleic acid sequences encoding one or more therapeutic products in the viruses. For example, nucleic acid sequences encoding cytotoxins, immunomodulatory proteins (i.e., proteins that enhance or suppress patient immune responses to antigens), tumor antigens, antisense RNA molecules, or ribozymes can be included in the invention. Examples of immunomodulatory proteins that can be encoded by the heterologous nucleic acid sequences include, e.g., cytokines (e.g., interleukins, for example, any of interleukins 1-15, α, β, or γ-interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines (e.g., neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, and macrophage inflammatory peptides MlP-la and MIP-lb)), complement components and their receptors, immune system accessory molecules (e.g., B7.1 and B7.2), adhesion molecules (e.g., ICAM-1, 2, and 3), and adhesion receptor molecules. Appropriate heterologous nucleic acid sequences for use in the methods of the invention can be readily selected by those of skill in this art. The heterologous nucleic acid sequences can be inserted into the viruses for use in the methods of the invention in a location that renders them under the control of regulatory sequences of the viruses. Alternatively, the heterologous nucleic acid sequences can be inserted as part of an expression cassette that includes regulatory elements, such as promoters or enhancers. Appropriate regulatory elements can be selected by those of skill in the art based on, for example, the desired tissue-specificity and level of expression. For example, a cell-type specific or tumor-specific promoter can be used to limit expression of a gene product to a specific cell type. This is particularly useful, for example, when a cytotoxic, immunomodulatory, or tumor antigenic gene product is being produced in a tumor cell in order to facilitate its destruction, and provides a further safeguard of specificity. In addition to using tissue- specific promoters, local (e.g., intrapleural) administration of the viruses of the invention can result in localized expression and effect. Tumor specific promoters can also be selected for use in the invention, based on the etiology of the cancer. Examples of promoters that function specifically in tumor cells include the stromelysin 3 promoter, which is specific for breast cancer cells (Basset et al., Nature 348:699, 1990); the surfactant protein A promoter, which is specific for non-small cell lung cancer cells (Smith et al., Hum. Gene Ther. 5:29-35, 1994); the secretory leukoprotease inhibitor (SLPI) promoter, which is specific for SLPI-expressing carcinomas (Garver et al., Gene Ther. 1 :46-50, 1994); the tyrosinase promoter, which is specific for melanoma cells (Vile et al., Gene Therapy 1 :307, 1994; WO 94/16557; WO 93/GB1730); the epidermal growth factor receptor promoter, which is specific for squamous cell carcinoma, glioma, and breast tumor cells (Ishii et al., Proc. Natl. Acad. Sci. U.S.A. 90:282, 1993); the mucin-like glycoprotein (DF3, MUCl) promoter, which is specific for breast carcinoma cells (Abe et al., Proc. Natl. Acad. Sci. U.S.A. 90:282, 1993); the mtsl promoter, which is specific for metastatic tumors (Tulchinsky et al., Proc. Natl. Acad. Sci. U.S.A. 89:9146, 1992); the NSE promoter, which is specific for small-cell lung cancer cells (Forss-Petter et al., Neuron 5:187, 1990); the somatostatin receptor promoter, which is specific for small cell lung cancer cells (Bombardieri et al., Eur. J. Cancer 31A:184, 1995; Koh et al., Int. J. Cancer 60:843, 1995); the c-erbB-2 promoter, which is specific for pancreatic, breast, gastric, ovarian, and non-small cell lung cells (Harris et al., Gene Ther. 1:170, 1994); the c-erbB-3 promoter, which is specific for breast cancer cells (Quin et al, Histopathology 25:247, 1994); and the c- erbB4 promoter, which is specific for breast and gastric cancer cells (Rajkumar et al., Breast Cancer Res. Trends 29:3, 1994). Examples of non-tissue specific promoters that can be used in the invention include the early Cytomegalovirus (CMV) promoter (U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al., Mol. Cell Biol. 5:281, 1985). Also, HSV promoters, such as HSV-1 IE and IE 4/5 promoters, can be used.

Any of a number of well-known formulations for introducing viruses into cells in patients can be used in the invention. (See, e.g., Remington 's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, PA.) However, the viruses can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without an adjuvant or carrier. The amount of virus to be administered can readily be determined by those of skill in this art, and depends on factors such as, for example, the condition of the patient intended for administration (e.g., the weight, age, and general health of the patient), the mode of administration, and the type of formulation. In general, an effective dose of, e.g., from about 10¹ to 10¹⁰ plaque forming units (pfu), for example, from about 5xl0⁴ to lxlO⁶ pfu, e.g., from about lxlO⁵ to about 4xl0⁵ pfu, is administered, although the most effective ranges may vary from patient to patient, as can readily be determined by those of skill in this art. Also, the administration can be achieved in a single dose or repeated at intervals, also as determined to be appropriate by those of skill in this art.

The viruses can be administered to patients using any conventional route used in medicine. For example, the viruses can be administered systemically by, e.g., intravenous infusion. Alternatively, the viruses can be administered directly into the pleura, using methods that are known to those of skill in the art, for example, by use of a pleural trocar or a thoracostomy tube.

The methods of the invention can employ replication competent, attenuated herpes viruses as sole therapeutic agents or, alternatively, these agents can be used in combination with other anticancer treatments. Examples of additional therapies that can be used include chemotherapy, biological therapy, gene therapy, radiation therapy, antisense therapy, and therapy involving the use of angiogenesis inhibitors (e.g., angiostatin, endostatin, and icon). Selection of any of these types of therapies for use with replication-competent, attenuated herpes in the methods of the invention can readily be carried out by those of skill in the art. Specific examples of chemotherapeutic agents that can be used in the methods of the invention are provided as follows. These compounds fall into several different categories, including, for example, alkylating agents, antmeoplastic antibiotics, antimetabolites, and natural source derivatives. Examples of alkylating agents that can be used in the methods of the invention include busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (i.e., cytoxan), dacarbazine, ifosfamide, lomustine, mecholaretliamine, melphalan, procarbazine, streptozocin, and thiotepa; examples of antmeoplastic antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, and plicamycin; examples of antimetabolites include fluorodeoxyuridine, cladribine, cytarabine, floxuridme, fludarabine, flurouracil (e.g., 5-fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine; and examples of natural source derivatives include docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol, prednisone, tamoxifen, asparaginase, and mitotane.

The biological therapy that can be used in the methods of the invention can involve administration of an immunomodulatory molecule, such as a molecule selected from the group consisting of tumor antigens, antibodies, cytokines (e.g., interleukins, interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines, complement components, complement component receptors, immune system accessory molecules, adhesion molecules, and adhesion molecule receptors. The methods of the invention, as described herein, are based, in part, on the experimental results that are described as follows.

Experimental Results

Summary NV1020 is a multi-mutated, replication-restricted herpes-simplex virus (HSV) that is under investigation for its ability to selectively kill tumors by direct cell lysis. We examined NV1020 in a rat model of pleural-based lung cancer. Briefly, cytotoxicity and viral proliferation were evaluated in vitro by exposure of the human non-small cell lung cancer cell line A549 to virus. NV1020 was also tested in an in vivo pleural-based cancer model established by injecting lxl 0⁷ A549 cells into the tlioracic cavity of nude rats. Intrapleural treatments (1x10 viral particles) were given 3 hours or 3 days after tumor injection to model treatment of microscopic or macroscopic disease (n=8- 9/group). Tumor burden was assessed at 5 weeks. NV1020 infection and dissemination within the thoracic cavity was determined by immunohistochemistry. We found that, in vitro, at multiplicities of infection (viral particles/tumor cell) of 0.01, 0.1, and 1.0, cell kill of A549 by NV1020 was 66%, 90%, and 97%, respectively, at 7 days post-infection. Viral burst occurred by day two. Intrapleural treatment was effective in both the microscopic (p<0.001) and macroscopic (p<0.05) in vivo tumor models. Virus was detectable by immunohistochemistry in tumor, but not in adjacent, normal mtrathoracic tissues. Based on these studies, we conclude that NV1020 is not only highly cytotoxic to the human lung cancer line A549 in vitro, but can be delivered in a clinically relevant fashion to safely and effectively treat pleural-based tumor in vivo in a rat model. Details of these experimental results are provided below.

Ln vitro cytotoxicity

The oncolytic efficacy of NV1020 against A549 cells in vitro was assessed. Viable cells remaining over time (as a fraction of control) are demonstrated in Fig. 1. Significant cell lysis begins after 48 hours post-infection. By day 7, NV1020 killed 66%, 90%, and 97% of tumor cells at MOI levels of 0.01, 0.1, and 1, respectively. A clear dose-response relationship exists. Similar results were seen with the human lung squamous cell HI 57.

Ln vitro viral proliferation

To assess the ability of A549 to permit viral replication, cells were infected at an MOI of 1 (5xl0⁴ PFU). Virions were recovered from both supernatant and cells and titered daily over 5 days (Fig. 2). By 48 hours, a viral burst occurred, producing a 17- fold increase in viral titer (8.8x10⁵ PFU). This burst activity corresponds to the sharp decline in viable cells observed at day three in the cytotoxicity curve.

Treatment of rat model of microscopic pleural tumor

To evaluate the efficacy of NV1020 in the treatment of free-floating microscopic disease, animals were treated 3 hours after injection of tumor cells. By five weeks, control animals developed bulky disease with the largest nodules (1-5 mm) on the pleura and pericardium. In several cases, the diaphragm was partially replaced with tumor. The internal chest wall also exhibited large tumor nodules. Smaller nodules were occasionally seen on the surface of the heart and lungs (<1 mm). A non-bloody pleural effusion was present in 3/9 animals in the control group. Disease was limited to the thoracic cavity. At sacrifice, extrathoracic organs (brain, liver, spleen, kidney, retroperitoneum, and spine) showed no evidence of macroscopic disease upon sectioning. Direct injection of NV 1020 into the pleural cavity significantly suppressed tumor growth, and in 5/9 animals completely prevented tumor growth (Fig. 3). Mean tumor weight ±SEM in the control group was 2.07±0.51 g, as compared to 0.02±0.01 g in the intrapleural treatment group (p<0.001). Intravenous therapy, however, had no significant effect of reducing tumor burden (mean tumor weight, 1.30±0.48 g). Animals in all tliree groups gained weight throughout the experimental period, without exhibiting tachypnea or other outward signs of distress.

Treatment of a rat model of macroscopic pleural tumor

To document the presence of tumor implantation and macroscopic pleural disease at 3 days post-inoculation, two animals were sacrificed 3 days after A549 cells were injected into the pleural space. Multiple small (0.5-1.0 mm) nodules were grossly visible on the pleura, chest wall, and diaphragm, as well as within the mediastinal fat. Tissues were fixed and sections were stained with hematoxylin and eosin for microscopic confirmation (Figs. 4A-4C). On microscopic evaluation, tumor was also detected in the mediastinal fatty tissue. Rats inoculated 3 days prior with intrapleural A549 were treated with either intrapleural NV1020 (lxlO⁷ PFU) or intrapleural PBS to evaluate the ability of the virus to treat implanted, macroscopic disease. The rats were sacrificed 5 weeks later and tumor burden was assessed. Animals treated with intrapleural virus had significantly less tumor burden than controls (p<0.05)(Fig. 5). Mean tumor weight ±SEM in the control group was 1.02±0.44 g, while mean tumor weight in the treated group was

0.23±0.20 g. A non-bloody pleural effusion developed in 2/8 rats in the control group. In the treated group, 4/9 rats developed no tumor.

Lmmunohistochemical analysis of thoracic tissues Three rats were inoculated with A549 cells and injected with intrapleural

NV1020 four weeks later. Three days after NV1020 injection, at the peak of viral proliferation (as determined by prior studies), rats were sacrificed and intrathoracic tissues and tumor were harvested (Bennett et al., J. Mol. Med. 78:166-174, 2000). In all 3 rats, tumor specimens displayed immunohistochemical evidence of the presence of viral antigen (Fig. 6). Positive staining for viral antigen progressed beyond the surface to involve cell layers deep in the tumor. Normal tissues tested were lung, heart, diaphragm, and internal chest wall. Viral presence was not detectable in any of these normal tissues in any of the three animals. In animals that were not administered tumor cells, viral presence was also undetectable in all of the normal tissues after viral treatment.

NV1066 based EGFP Localization and Treatment of Pleural Cancer

As is shown in detail in Figs. 7-11 and is discussed above in the Brief

Description of the Drawings, we have also used an attenuated, replication-competent derivative of HSV- 1 including coding sequences for enhanced green fluorescent protein (EGFP) (NV1066) in experiments showing the efficacy of treating pleural cancer with such viruses. In particular, as is shown in the figures, we showed that NV1066 has a cytotoxic effect in A549 cells (Fig. 7), and that NV1066 has antitumor effects in vivo when injected intrapleurally (Fig. 8). Further, we showed that EGFP expression in vivo was specifically localized to tumor cells (Figs. 9-11).

Materials and Methods

Cells

The human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection (Manassas, VA), and was maintained in Ham's F-12 medium containing 10% fetal calf serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained in a 5% CO₂ humidified incubator at 37°C. Cell viability for all experiments was >95%, as determined by trypan-blue exclusion.

Virus

NV1020 (Medigene Inc., San Diego, CA) is an attenuated, replication-competent derivative of herpes simplex virus type-1 (HSV-1) (Delman et al., Hum. Gene Ther. 11 :2465-2472, 2000). NV1020 is a non-selected clonal derivative from R7020, a candidate HSV- 1/2 vaccine strain that was obtained from Dr. B. Roizman (Meigner et al., J. Infect. Dis. 158:602-614, 1998). The structure of NV1020 is characterized by a 15 kilobase deletion encompassing the internal repeat region, leaving only one copy of the following genes, which are normally diploid in the HSV-1 genome: ICPO, ICP4, the latency associated transcripts (LATs), and the neurovirulence gene γι34.5. A fragment of HSV-2 DNA encoding several glycoprotein genes was inserted into this deleted region. In addition, a 700 basepair deletion encompasses the endogenous thymidine kinase (TK) locus, which also prevents the expression of the overlapping transcripts of the UL 4 gene. An exogenous copy of the HSV-1 TK gene was inserted under control of the α4 promotor. Virus was propagated in Vero cells and harvested by freeze thaw lysis to release virus from the cell fraction. Cell lysates were clarified by centrifugation, and viral titers were determined on Vero cells by plaque assay. All virus preparations were formulated in D-PBS-10% glycerin and stored at -80°C.

Animals

All animal work was performed with the prior approval of the Memorial Sloan- Kettering Institutional Animal Care and Use Committee. Athymic male rats 6 to 8 weeks old were purchased from the National Cancer Institute (Bethesda, MD). Animals were subjected to a 12:12 hour light: dark cycle, housed two per cage, and allowed food and water ad libitum.

Ln vitro cytotoxicity

The ability of NV1020 to infect and lyse A549 cells in vitro was assessed. Cells were plated at 5xl0⁴ cells/well in 12-well plates (Corning Inc., Corning, NY) and were infected with NVl 020 at multiplicities of infection (MOI) of 0.01 , 0.1 , and 1.0. Control wells were treated with culture medium alone. Cell viability was assessed by counting live cells via trypan blue exclusion at 24-hour intervals, carried out to 7 days. All assays were performed in triplicate.

Ln viti'o viral proliferation

To demonstrate viral replication in A549 cells, a viral growth curve was established. Cells (5xl0⁴) were plated in 6-well plates (Coming Inc., Corning, NY) and infected with NV1020 at a MOI of 1.0 (5xl0⁴ PFU). Cells and supernatants were harvested daily for 5 days post-infection. Three cycles of freeze thaw lysis were performed and viral titers were determined by standard plaque assay using Vero cells. All infections were performed in triplicate.

Establishment and treatment of pleural tumor in rats

A cellular suspension of A549 cells was implanted in the thorax of athymic rats as previously described (Kucharczuk et al., Ann. Thorac. Surg. 60:593-598, 1995). Anesthesia was induced by a single intraperitoneal injection of pentobarbital (50 mg/kg). Rats were placed in the right lateral decubitus position. The left chest was shaved and prepared with 10% povidone-iodine solution. A 2 cm skin incision was created over the 5^th or 6^th intercostal space. Sharp dissection was carried out through the muscle layers anterior to the latissimus dorsi. A number 10 scalpel was used to carefully shave away the intercostal muscles, exposing the parietal pleura without breaching it. The underlying expanding lung was thereby easily visualized through the thin membrane. Three hundred μl of A549 cellular suspension (lxlO⁷ cells) was slowly injected through the pleura with a 27 gauge needle. Puncture of the lung was easily avoided, as the needle tip was clearly observed to be superficial to the lung surface during injection through the transparent parietal pleura. After injection, the muscle layer was reapproximated with interrupted 4-0 nylon suture, and the skin was closed with surgical staples. Recovery was observed for two hours before the rats were returned to their cages.

Intrapleural treatment with virus was performed in a similar fashion either 3 hours or 3 days after tumor implantation. NV1020 (lxlO⁷ PFU) was administered in 300 μl PBS. Control animals were treated with an equal volume of PBS. For intravenous treatment, the same dose of virus was administered in 1 cc of PBS via penile vein injection. Rats were regularly assessed for weight loss and tachypnea throughout the experimental period. To quantify tumor bulk at five weeks post-implantation, rats were sacrificed and all visible tumor in the chest was excised. Total tumor weight was assessed. In the first experiment, the ability of NVl 020 to treat free-floating microscopic disease was assessed. Twenty-seven rats (n=9/group) were injected with A549 into the pleural space and chest incisions were closed. Tliree hours later, rats were treated with either intrapleural PBS, intrapleural NV1020, or intravenous NV1020. Five weeks later, the animals were sacrificed and intrapleural tumor weight was determined. In a second experiment, NVl 020 was evaluated for the treatment of macroscopic pleural disease. Two groups of rats (n=8-9/group) were implanted with A549 and treated 3 days later. Two additional rats were injected with tumor and sacrificed at 3 days to verify the presence of macroscopic disease.

Histologic analysis of the extent ofNV1020 infection and dissemination in vivo

To assess viral dissemination in tumor and in non-cancerous tissues, control animals with no pleural tumor, as well as animals with four-week old A549 tumors within the pleural cavity, were injected with virus (n=3/group). Three days later, rats were sacrificed and intrathoracic tissues were harvested. Tissues (tumor, heart, lung, chest wall, and diaphragm) were fixed in 4% paraformaldehyde and embedded in paraffin using a tissue processor (TP1050, Leica Microsystems, Deerfield, IL). Eight μm paraffin sections were stained immunohistochemically to assess the presence of heφes simplex virus type-1, using a polyclonal rabbit anti-HSV antibody (#PU084-UP, Biogenex, San Ramon, CA) in conjunction with the ABC Elite kit (Vector Labs, Burlingame, CA) detection system. Sections were counterstained with Gill's hematoxylin, dehydrated, and mounted in Permount.

What is claimed is:

Claims

1. A method of treating cancer in the pleura of a patient, said method comprising administering to the patient an attenuated, replication-competent heφes virus.

2. The method of claim 1, wherein said cancer is metastatic cancer.

3. The method of claim 2, wherein said cancer has metastasized from the lung, breast, ovary, or lymphatic system.

4. The method of claim 1, wherein said cancer is a primary pleural cancer.

5. The method of claim 4, wherein said cancer is pleural mesothelioma.

6. The method of claim 1, wherein said heφes virus is a heφes simplex- 1- derived virus.

7. The method of claim 6, wherein said heφes virus comprises an inactivating mutation in a γ34.5 gene.

8. The method of claim 6, wherein said heφes virus comprises an inactivating mutation in a Ur24 gene.

9. The method of claim 6, wherein said heφes virus comprises an inactivating mutation in a γ34.5 gene and in a U_L24 gene.

10. The method of claim 9, wherein said heφes virus is NVl 020. ,

11. The method of claim 6, wherein said heφes virus comprises an inactivating mutation in the ribonucleotide reductase gene of said virus.

12. The method of claim 6, wherein said heφes virus comprises an inactivating mutation in a γ34.5 gene, a U_L24 gene, and a ribonucleotide reductase gene.

13. The method of claim 11, wherein said heφes virus is G207.

14. The method of claim 1, wherein said heφes virus is intrapleurally administered to said patient.

15. The method of claim 1, wherein said heφes virus is systemically administered to said patient.

16. The method of claim 1, wherein said heφes virus comprises a heterologous nucleic acid molecule encoding a therapeutic product.

17. The method of claim 16, wherein said therapeutic product is selected from the group consisting of cytotoxins, immunomodulatory proteins, tumor antigens, antisense nucleic acid molecules, and ribozymes.

18. The method of claim 1, further comprising administering a second anticancer treatment to the patient.

19. The method of claim 18, wherein the second anticancer treatment therapy is selected from the group consisting of chemotherapy, biological therapy, radiation therapy, gene therapy, and surgery.

20. The method of claim 19, wherein the chemotherapy comprises administration of an alkylating agent, an antineoplastic antibiotic, an antimetabolite, or a natural source derivative.

21. The method of claim 20, wherein the alkylating agent is selected from the group consisting of busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa.

22. The method of claim 20, wherein the antineoplastic antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, pentostatin, and plicamycin.

23. The method of claim 20, wherein the antimetabolite is selected from the group consisting of fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fiudarabine, flurouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine.

24. The method of claim 20, wherein the natural source derivative is selected from the group consisting of docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol, prednisone, and tamoxifen.

25. The method of claim 19, wherein the biological therapy comprises administration of an immunomodulatory molecule.

26. The method of claim 25, wherein the immunomodulatory molecule is selected from the group consisting of cytokines, chemokines, complement components, complement component receptors, immune system accessory molecules, adhesion molecules, and adhesion molecule receptors.

27. The method of claim 26, wherein the cytokine is selected from the group consisting of interleukins, interferons, tumor necrosis factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, and granulocyte colony stimulating factor.

28. Use of an attenuated, replication-competent heφes virus in the preparation of a medicament for treating cancer in the pleura of a patient.

29. The use of claim 28, wherein said heφes virus is a heφes simplex- 1 -derived virus.

30. The use of claim 28, wherein said heφes virus comprises a heterologous nucleic acid molecule encoding a therapeutic product.