Title:  Herpes simplex virus expressing foreign genes and method for treating cancers therewith

United States Patent:  6,764,675

Issued:  July 20, 2004

Inventors:  Whitley; Richard J. (Birmingham, AL); Markert; James MacDowell (Birmingham, AL); Gillespie; George Yancey (Birmingham, AL); Parker; Jacqueline Ness (Brimingham, AL)

Assignee:  The UAB Research Foundation (Birmingham, AL)

Appl. No.:  009972

Filed:  February 14, 2002

PCT Filed:  June 8, 2000

PCT NO:  PCT/US00/40165

PCT PUB.NO.:  WO00/75292

PCT PUB. Date:  December 14, 2000

Abstract

An anti-cancer pharmaceutical composition includes a herpes simplex virus (HSV) vector into which a nucleic acid sequence encoding an anti-cancer agent selected from interleukin-12, GM-CSF, and CD has been inserted. A method of treatment of a patient suffering from cancer includes administering to the patient the anti-tumor pharmaceutical composition including an HSV vector having a nucleic acid sequence encoding an anti-cancer agent selected from interleukin-12, GM-CSF, and CD inserted therein.

SUMMARY OF THE INVENTION

A method for treating a subject suffering from cancer includes administering a therapeutically effective amount of a herpes simplex virus (HSV) vector expressing a cytokine or other anti-cancer agent encoding nucleic acid sequence, such as coding for interleukin-12, into a subject inducing an anti-tumor response in the subject.

An anti-tumor pharmaceutical composition comprising a herpes simplex virus (HSV) vector comprising a nucleic acid sequence encoding for a compound selected from IL-12, GM-CSF, and CD operatively linked to a promoter, and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found according to the present invention that malignant cancer cells can be treated with a genetically manipulated and/or modified herpes simplex virus, preferably type 1, (HSV-1) expressing a foreign gene, preferably an interleukin 12 (IL-12) gene in order to produce IL-12 constitutively with a target cell and can be used as an effective anti-tumor treatment. The engineered HSV-1 expressing IL-12 can also be used for the treatment of primary and metastatic central nervous system (CNS) tumors including, but not limited to meningiomas, pituitary adenomas, and acoustic neuromas, and specifically brain tumors including glioblastoma, malignant glioma, and low-grade glioma, either with or without cognate therapies such as chemotherapy and/or radiation therapy. Additionally, the engineered HSV-1 expressing IL-12 can be utilized in the treatment of non-CNS tumors including malignant melanoma, hepatocellular carcinoma, head and neck cancers, etc., both with and without cognate therapy. The engineered HSV-1 expressing IL-12 can also be utilized as a vaccine or vaccine adjuvant and also for the treatment of infectious diseases by stimulating the immune system.

The engineered HSV-1 vectors express a foreign gene including cytokines such as IL-12, granulocyte macrophage colony stimulating factor (GM-CSF), IL-16, IL-10, IL-4, or cytosine deaminase (CD) and are constructed by inserting the foreign gene, such as a gene encoding for cytokine or other anti-tumor/anti-cancer gene product, into the viral genome of HSV-1. The foreign gene is inserted into a region under the control of promoter-regulatory regions of the viral genome. Thus, the viral genome becomes a vector for the expression of the foreign gene in target cells (e.g., tumor cells). In the present invention, a nucleic acid sequence encoding for IL-12 (see FIG. 1) or some other anti-tumor/anti-cancer agent is placed under the transcriptional control of the murine early-growth response-1 promoter (Egr-1). It should be noted that while description is given herein for use of HSV-1 which is readily available to those of ordinary skill in the art, other herpes simplex viruses including HSV-2 can also be utilized in the present invention. See FIG. 6 for an HSV-1 vector expressing GM-CSF and see FIG. 7 for an HSV-1 vector expressing cytosine deaminase.

The HSV of the present invention is preferably a neuroattenuated, replication-competent HSV. The HSV-1 of the present invention includes a deletion in the .gamma.₁ 34.5 gene rendering the virus aneurovirulent.

As described below in the Experimental Section, biologically active murine IL-12 consists of heterodimer of the p40 and p35 subunits. The nucleic acid sequences encoding for the expression of the p35 and p40 subunits are separated by an internal ribosome entry site (IRES) and are inserted as a single expression cassette into HSV to form a recombinant HSV of the present invention. The HSV expresses the murine IL-12 via bicistronic expression of the p35 and p40 subunits separated by the IRES sequence. The IL-12 produced thereby is a self-assembling, heterodimeric, functionally active molecule in HSV-1. The cytokine can then exit the HSV and contact bystander cells and/or elicit and/or enhance the patient's or subject's immune response.

For the engineered HSV vector expressing GM-CSF it was shown by ELISA that significant production of GM-CSF was achieved in Vero cells and in the Neuro2A neuroblastoma cell line of A/J mouse origin. Neurotoxicity studies performed in highly sensitive A/J strain mice revealed that the GM-CSF virus was somewhat toxic at high doses, with an LD₅₀ of approximately 5x10⁶ pfu. Intracranial studies demonstrated increased host survival in an intracranial syngeneic neuroblastoma murine model over mock-treated mice, although treatment with the GM-CSF virus at highest doses demonstrated toxicity.

For the engineered HSV vector expressing cytosine deaminase, cytosine deaminase activity was demonstrated in vitro by conversion of tritiated 5-fluorocytosine (5FC) to 5-fluorouracil (5FU). CD-expressing virus has been injected into U87MG human glioma cells intracranially xenografted into scid mice and 5FC was administered. The local expression of cytosine deaminase led to very localized tissue metabolism of drugs such as 5-fluorocytosine providing a local anti-tumor effect.

The present invention provides an anti-cancer or anti-neoplasm pharmaceutical agent or composition which constitutively produces IL-12, GM-CSF or CD and, optionally, includes a pharmaceutically acceptable carrier or diluent. The anti-tumor agent according to the present invention can be administered by any number of means and routes known in the art. For example, administration may be by subcutaneous, intravenous, intrathecal, intraventricular, intra-arterial, intramuscular, or intraperitoneal injection, by infusion, or preferably, by direct intratumoral injection. The dosage administered will be dependent upon the condition of the patient and the severity of the disease. Anti-tumor compositions comprising 10⁴ to 10⁹ virus, preferably 10⁷ to 10⁸ virus, at a dose, are administered to a patient according to the invention. The treatment can comprise several doses at spaced apart intervals, according to the necessity.

The recombinant HSV expressing IL-12 or other anti-cancer/anti-tumor agent according to the invention will be used in a method for the treatment of a patient or subject suffering from cancer, e.g., a malignant solid tumor, lymphoma or leukemia. The method of treatment includes the step of administering a therapeutically effective amount of an HSV vector expressing an IL-12 or other anti-cancer product encoding nucleic acid sequence into a patient or subject such that an anti-tumor response is induced in the subject.

The terms "patient" and/or "subject" as used herein mean all animals including humans. Examples of patients and/or subjects include humans, rodents, and monkeys.

A "therapeutically effective amount" is an amount of a HSV vector expressing IL-12 or other anti-tumor/cancer agent, that when administered to a patient or subject, inhibits tumor growth, causes tumor regression, prevents metastasis or spread of the tumor, prolongs the survival of the subject or patient, and combinations thereof.

The anti-tumor agents of the present invention can be administered to a patient or subject either alone or as part of a pharmaceutical composition of the agents admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

A preferred route of administration is direct, intratumoral injection. Compositions suitable for injection may comprise physiological acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifing, and dispensing agents. Prevention of the action of microorganisms can be controlled by addition of any of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

The invention will now be illustrated by the following examples without limiting thereto. In the examples the following Experimental Methods were employed:

Experimental

Materials and Methods

Cells. Vero cells (American Type Culture Collection [ATTC], Rockville, Md.) were grown and maintained in Minimal Essential Medium (Cellgro, Mediatech) containing 7% fetal bovine serum. The human 143 thymidine kinase minus cells (143tk-, ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) (Cellgro) supplemented with 10% fetal bovine serum. Rabbit skin cells (originally acquired from Dr. J. McClaren, University of New Mexico, Albuquerque, N.Mex., USA) were maintained in DMEM supplemented with 5% fetal bovine serum. The human malignant glioma cell lines U251MG and D54MG were obtained from D. D. Bigner (Duke University, Durham, N.C., USA) while the murine neuroblastoma cell line Neuro-2A (derived from stain A/J mice) was purchased from the ATCC (CCL 131, passage 171). These latter three cell lines were maintained in a 50:50 mixture of DMEM and Ham's Nutrient Mixture F-12 (DMEM/F12) supplemented to 2.6 mM L-glutamine and 7% FBS.

Plasmids and viruses. HSV-1 (F) strain is a low passage clinical isolate used as the prototype HSV-1 strain in our series (Post et al. (1981) Cell 25, 227-232; Jenkins et al. (1986) J. Virol. 59, 494-9). Viruses R3616 and R4009, which contain a 1 kb deletion and a stop codon, respectively, within both copies of the .gamma.₁ 34.5 gene, have been described previously (Chou et al. (1990) Science 250, 1262-1266). Construction of M002, which expresses murine interleukin 12 (mIL-12) under the transcriptional control of the murine early-growth response-1 promoter (Egr-1), is described below. This strategy is identical to that used to construct the cytokine-expressing viruses R8306 (mIL-4) and R8308 (mIL-10) (Andreansky et al. (1998) Gene Ther. 5, 121-130). The plasmids containing the p40 and p35 subunits of mIL-12 in pBluescript-SK+ (Stratagene) (Schoenhaut et al. (1992) J. Immunol. 148, 3433-3440), were kindly provided by Dr. Ueli Gubler (Hoffman-LaRoche, Inc., Nutley, N.J., USA). The p40 subunit was removed by digestion with HindIII (5' end) and BamHI (3' end) and the p35 subunit was removed by digestion with NcoI (5' end) and EcoRI (3' end). The internal ribosome entry site, or IRES, sequence was amplified from vector pCITE-4a+ (Novagen, Madison, Wis.) using polymerase chain reaction (PCR) and primers 5'-CITE (5'-CGCGGATCCTTATTTTCCACCATATTGCC-3') (SEQ ID No: 1), which has a BamHI site, and 3'-CITE (5'-GGAGCCATGGATTATCATCGTGTTTTTC-3') (SEQ ID No: 2), which has an NcoI site that retains the translational start sequence. Plasmid pBS-IL12 was constructed by three-way ligation of the murine p40, murine p35 and IRES sequences into HindIII and EcoRI sites of pBS-SK+ such that the IRES sequence separates the p40 and p35 coding sequences. This effectively duplicates a strategy previously reported for expression of the mIL-12 subunits (Tahara et al. (1995) J. Immunol. 154, 6466-6474). The IL-12 genes were entirely sequenced by the University of Alabama at Birmingham Cancer Center DNA Sequencing Facility.

The HSV shuttle plasmid pRB4878 has been previously described (Andreansky et al. (1998) Gene Ther. 5, 121-130). Plasmid 4878-IL12 was constructed as follows: pBS-mIL-12 was digested with XhoI and SpeI to remove a 2.2 kb fragment containing the entire IL-12 subunit coding regions, including the IRES, ends filled in using the Klenow fragment, and ligated into a blunted KpnI site located between the Egr-1 promoter (a mammalian promoter) and hepatitis B virus polyA sequences within pRB4878. M001 (tk-) and M002 (tk repaired at native locus) were constructed via homologous recombination as described previously (Andreansky et al. (1998) Gene Ther. 5, 121-130). Two tk-repaired viruses M002.29 and M002.211, were confirmed by Southern blot hybridization of restriction enzyme-digested viral DNAs which were electrophoretically separated on a 1% agarose, 1xTPE gel and transferred to a Zeta-Probe membrane (Bio-Rad). The blot was hybridized with the appropriate DNA probe labeled with alkaline phosphatase using the Gene Images AlkPhos Direct DNA labeling system (Amersham-Pharmacia Biotech, Piscataway, N.J.). IL-12 production was demonstrated by enzyme-linked immunosorbent assay (ELISA).

ELISA. Production of murine IL-12 by M002 was confirmed and quantified using a murine p70 ELISA kit (R&D Systems, Minneapolis, Minn.). Briefly, six well plates were seeded at a confluency of 4x10⁵ cells/well one day prior to infection with M002 or control virus at a multiplicity of infection (M.O.I.)=1 in a total volume of 0.5 ml. After two hours, the inoculum was removed, 1 ml of growth medium was overlaid onto infected wells and plates incubated 24 hr at 37^oC. The supernatant was removed, transferred to microcentrifuge tubes, and spun down briefly to remove cellular debris. Either undiluted or 10-fold dilutions of supernatants were analyzed by ELISA, according to the manufacturer's protocol. Experiments were performed at least three separate times to determine average level of cytokine production.

In vitro characterization of M001/M002. In vitro replication of M001 in subconfluent cultures of the human malignant glioma cell lines U251MG and D54MG was determined, as previously described (Andreansky et al. (1998) Gene Ther. 5, 121-130), at 12, 24, 48 and 72 hours post-infection hpi). For cytotoxicity assays, monolayers U251MG and D54MG cells, as well as the murine neuroblastoma cell line Neuro-2A were infected with M002.29 and M002.211 at an M.O.I.=1. The TD₅₀ was determined by alamarBlue.TM. assay, as described (Andreansky et al. (1997) Cancer Res. 57, 1502-1509). Dye conversion values were obtained by reading plates on a Bio-Tek EL310 plate reader (Winooski, Vt.) with the O.D. value at 590 nm subtracted from the O.D. at 562 nm. The decrease in O.D. relative to uninfected cells was plotted against number of virus plaque forming units (pfu)/ml to determine the number of pfu needed to produce a 50% reduction in O.D.

Animals. Specific pathogen-free female A/J strain mice were obtained from Charles River Laboratories and used at approximately eight weeks of age. All animal studies were conducted in accordance with guidelines for animal use and care established by The University of Alabama at Birmingham Animal Resource Program and the Institutional Animal Care and Use Committee (IACUC protocol 97K03985).

In vivo characterization of M002. For determination of M002 neurovirulence in A/J strain mice, graded numbers of virus pfu were prepared in sterile milk and 5 .mu.l of each dilution were inoculated into the right cerebral hemisphere of 3-10 mice as described (Chambers et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1411-1415). For survival studies, A/J strain mice were stereotactically inoculated with 10⁵ Neuro-2A cells in the right cerebral hemisphere. Five days later, mice were randomly divided into three cohorts and 5x10⁶ pfu of M002, R3659 or vehicle were stereotactically inoculated into each tumor. Mice were assessed daily; moribund mice were sacrificed and the date of death recorded as described (Chambers et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1411-1415).

Histopathology. Three sets of three mice each were injected with Neuro-2A, then treated with M002, R3659 or vehicle as described in the survival experiment. At days three and seven, one mouse from each group was sacrificed and its brain harvested and frozen in Tissue-Tek OCT compound. Sections 10-12 microns thick were cut through the injection site in each brain and mounted on TEPSA-coated slides, fixed in 95% ethanol and blocked in PBS-2% BSA. Sections were stained with standard hemotoxylin and eosin to determine degree of residual tumor, presence of neurotoxicity and extent of any inflammatory response. To characterize the nature of the inflammatory infiltrate, serial sections were reacted with rat monoclonal antibodies specific for mouse CD4, CD8, and macrophage markers and the antibody binding detected using biotinylated rabbit anti-rat Ig followed successively with an avidin-biotin-horseradish peroxidase complex and 1% diaminobenzidine (Andreansky et al. (1998) Gene Ther. 5, 121-130).

Results

Construction of a recombinant HSV-1 expressing murine IL-12. Previously, work by Applicants demonstrated that recombinant HSV which express murine interleukin-4 (IL-4) could significantly improve survival when injected into tumors implanted in brains of immunocompetent mice in a syngeneic murine model (Andreansky et al. (1998) Gene Ther. 5, 121-130). To extend these initial studies, Applicants evaluated recombinant HSV that would express the well-described anti-tumor cytokine IL-12.

Biologically active mIL-12 consists of a heterodimer of the p40 and p35 subunits. Therefore, recombinant HSV M001(tk-) and M002 (tk+) were constructed to express both mIL-12 subunits within a single expression cassette, separated by the internal ribosome entry site (IRES) from the 5' untranslated region of equine encephalomyocarditis virus (FIG. 1). Recombinant virus M001 was obtained by co-transfection of plasmid DNAs with R3659 viral DNA and selection of tk(-) viruses on 143tk- cells overlaid with medium containing 100 .mu.g/ml bromodeoxyuridine. The recombinant tk(-) mIL-12-expressing virus M001 was confirmed by Southern blot hybridization (data not shown). Recombinant virus M002 was obtained by cotransfection of M001 viral DNA with pRB4867, a plasmid used to repair the 501 bp deletion within the tk gene in its native locus (U_L 23), and subsequent selection in HAT medium. These recombinant viruses contain two copies of the IL-12 construct replacing both copies of the .gamma.₁ 34.5 gene. To confirm the presence of the mIL-12 insert in M002, viral DNAs were isolated, digested with NcoI, and evaluated by Southern blot hybridization as described in Materials and Methods and shown in FIG. 2. Repair of the tk gene was also verified by hybridization to a probe specific for the tk gene insert (data not shown).

Expression of mIL-12 by M002. To determine if M002 expressed physiologically relevant levels of murine IL-12, culture supernates from M002- or R3659-infected Vero and Neuro2A cells were quantified using a commercially available ELISA kit specific for mIL-12 p70 heterodimers. Applicants evaluated IL-12 production from two genetically identical subclones of M002, clone 29 and clone 211. The averaged values are indicated in Table 1. The highest production of mIL-12 by M002 was seen in Vero and Neuro-2A cells, which produced 3-4 ng/5x10⁵ cells/24 hours after infection at an MOI=1. Production was slightly lower in the D54MG and U251MG cell lines, at 1.8 and 0.8 ng/5x10⁵ cells/24 hours. Such levels are physiologically relevant and have been shown to produce anti-tumor responses in other models (Toda et al. (1998) J. Immunol. 160, 4457-4464; Zitvogel et al. (1994) Hum. Gene Ther. 5, 1493-1506).

Growth of wild-type and recombinant viruses in tumor cell lines. Before repairing the tk gene to create M002, Applicants first established the replication competence of our tk(-) IL-12 expressing HSV (M001) as compared with wild-type "F" or the backbone virus R3659, in the human glioma cell lines D54MG and U251MG. As indicated in FIG. 3, M001 replicated as well as R3659 in both glioma cell lines, and as well as the wild-type "F" strain in D54MG. This confirmed that replication competence of the IL-12 expressing virus remained intact and would be suitable for comparisons with other cytokine-expressing or parent viruses.

Viruses containing mutations or deletions within the .gamma.₁ 34.5 locus have previously been shown to have a direct cytolytic effect on D54MG and U251MG (Andreansky et al. (1997) Cancer Res. 57, 1502-1509). Applicants quantitatively measured the cytolytic activity of M002 on Neuro2A cells, as well as D54MG and U251MG, by alamarBlue.TM. assay and compared the results with cytolytic activity of the backbone virus, R3659. As shown in Table 2, the cytolytic activity of M002 was slightly higher than R3659 in all cell lines tested. Thus, this virus is at least as cytotoxic in both human glioma cells and in murine Neuro2A cells as its parent virus, and may even have a slight growth advantage.

A syngeneic model for neuroblastoma. The GL-261 cell line is a murine glioma line derived from C57BL/6 mice, and are relatively resistant to infection by HSV-1 (Lopez (1975) Nature 258, 152-153). Thus, this syngeneic model is not the ideal system for evaluating the therapeutic potential of Applicants' recombinant cytokine-expressing HSV, which replicate much more efficiently in human cells than in GL-261 cells. Therefore, Applicants tested their cytokine-expressing viruses in a syngeneic model using a murine strain that would be more susceptible to HSV infection. Strain A/J mice were utilized due to their known sensitivity to HSV-1 (Lopez (1975) Nature 258, 152-153). There are currently no syngeneic glioma models in A/J mice. However, Neuro2A cells are a neuroblastoma cell line originally derived from A/J mice. Neuro2A tumors were established in brains of A/J mice to be evaluated as a syngeneic brain tumor model system in a more sensitive murine strain. To determine optimal tumor cell dose for evaluating M002 in these tumors in vivo, 1x10³, 10⁴ or 10⁵ cells were stereotactically introduced into A/J strain mice as described in Materials and Methods, and followed to determine median survival rates for each dose. A dose-response effect was defined for survival, which ranged from 14 to 25 days. Based on this study, Applicants elected to inoculate between 5x10⁴ and 1x10⁵ cells to produce a median survival of three weeks from tumor induction in order to facilitate a rapid and stringent evaluation of the survival effects of the therapeutic viruses.

Neurovirulence. Previous studies with G207, a genetically-engineered HSV-1 currently in human trials for the treatment of malignant glioma, have shown no neurovirulence in this assay at doses of 10⁷ pfu. Thus, the maximum tolerated dose was determined (pfu/LD₅₀) for both clones of Applicants' IL-12 EL expressing virus, M002.29 and M002.211. For clone 211, up to 2x10⁷ pfu of virus could be directly injected without adverse effects, whereas the maximum tolerated dose of clone 29 was 5x10⁶ pfu (data not shown). Since clone 211 appeared to be the safer of the two, this virus was tested for experimental therapy of Neuro2A tumors, and is referred to herein as "M002."

Survival of A/J strain mice with intracerebral Neuro-2A neuroblastomas treated wit M002. To evaluate the sensitivity of Neuro2A tumors to HSV infection in A/J strain mice, 1x10⁴ tumor cells were injected intracranially into A/J female mice followed five days later by intratumoral injection of 1x10⁷ pfu (in 5 .mu.l) of either R3659 or M002 (mIL-12). As a control, 5 .mu.l of the diluent were also injected. Data shown in FIG. 4 represent a composite of three experiments, the median survival post-tumor induction in mice injected with diluent only was 19.8 days, and all animals were dead by day 34. In contrast, mice with M002-injected tumors had a significant increase on median survival of 50.5 days (p=0.00023), calculated using the log-rank test. Mice that received an intratumoral injection of R3659 had a median survival (19.5 days) that was not significantly different (p=0.556) from vehicle-treated mice. All survivors were sacrificed at 59 days and their brains examined histologically but there was no evidence of tumor.

Immunohistologic identification of inflammatory cell infiltrates. Intratumoral injection of the parent .gamma.₁ 34.5^- HSV, R3659, induced a mild but discernible immune-related inflammatory response characterized principally by macrophages and CD4⁺ T cells with a few CD8⁺ T cells. These inflammatory cells were scattered throughout the tumor mass with occasional foci predominated by macrophages or CD4⁺ T cells. In contrast, injection of M002 elicited a pronounced influx of macrophages and CD4⁺ T cells with a significant increase in CD8⁺ T cells as well. Inflammatory responses were maximal around days 5-6 and bad begun to regress by day 7 after viral injection.

Genetically engineered, neuroattenuated herpes simplex viruses (HSV) expressing various cytokines can improve survival in the treatment of experimental brain tumors. These attenuated viruses have both copies of .gamma.₁ 34.5 deleted. Recently, Applicants demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, a potent mediator of T_H -2 type responses, as compared to treatment with the parent construct (.gamma.₁ 34.5^-) alone or a virus expressing IL-10 (Gene Therapy 5: 121, 1998). The construction of a conditionally replication competent mutant expressing both subunits of murine IL-12 (M002), and its evaluation in a syngeneic neuroblastoma murine model is described. IL-12 induces a T_H -1 type response, which may induce more durable anti-tumor effects. In vitro studies demonstrated that, when infected with M002, both Vero cells and Neuro2A neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by pfu/LD₅₀, performed in HSV-1 sensitive A/J strain mice revealed that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than animals treated with R3659, the parent .gamma.₁ 34.5^- mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors revealed a pronounced influx of CD4⁺ T cells and macrophages, as well as CD8⁺ cells when compared with R3659-treated tumors. M002 produced a survival benefit via oncolytic effects combined with T_H -I mediated immunologic effects.

In view of the teaching presented herein, other modifications and variations of the present invention will readily be apparent to those of skill in the art. The discussion and description are illustrative of some embodiments of the present invention, but are not meant to be limitations on the practice thereof. It is the following claims, including all equivalents, which define the scope of the invention.

Any patents or publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
 

Claim 1 of 5 Claims

What is claimed is:

1. An anti-tumor pharmaceutical composition comprising a replication competent herpes simplex virus vector comprising a nucleic acid sequence encoding cytosine deaminase operatively linked to a promoter, and a pharmaceutically acceptable carrier.

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