Herpes Simplex Virus Thymidine Kinase Gene Ganciclovir

One approach to the development of more effective therapies for prostate cancer is to initiate a cascade of molecular cellular events locally within the primary tumor that generate a localized and systemic antitumor immune response through the transfer of specific immunomodulatory genes. It has been considered that it might be possible to use specific genes to generate localized antitumor cytotoxicity as well as to initiate a systemic antitumor immune response. This strategy has evolved from purely cytotoxic-based gene therapies to more immunomodulatory gene therapies and various combinations to ultimately achieve the objective. Our initial gene therapy trials used the selected herpes simplex virus thymidine kinase (HSV-t¿) gene delivered with a replication deficient adenoviral vector. HSV-t¿+ ganciclovir (GCV) gene therapy has been shown to elicit widespread cytotoxic activities through direct and well-defined bystander activities and to elicit nonspecific and specific antitumor immunity in a variety of cancers (20).

For prostate cancer, there are only limited reports of preclinical studies describing the use of nonviral vectors to deliver transgenes (e.g., liposomes) (13-15). The results of some preclinical studies have demonstrated that herpes vectors (16-18) or canarypox vectors 19) can be therapeutically effective for a variety of malignancies including prostate cancer. The use of retroviral vectors for in situ approaches has been more limited, however they are potentially useful for ex vivo infection schemes such as those used with cell mediated therapies that will be discussed below. Thus far adenoviral vector systems have emerged as the predominant form of gene delivery for prostate cancer.

Preclinical studies were designed to assess both the efficacy and toxicity of adeno-viral vector-mediated HSV-rt + GCV therapy using both in vivo and in vitro prostate cancer models. Mouse prostate cancer cell lines were generated from both primary and metastatic tumors initiated using the mouse prostate reconstitution model system. This model system involves the initiation of prostate cancer with the ras and myc oncogenes in wild-type C57Bl/6 mice (21) or mice with a targeted inactivation of one or both alleles of the p53 gene (22,23).

The mouse models used are relevant to human prostate cancer because the mouse prostate cancer cell lines are inoculated orthotopically thus incorporating features unique to the prostate milieu and also incorporating the presence of the host immune system. Furthermore, the mouse cell lines used for these models resemble human prostate cancers in their expression of relevant oncogenic pathways and specific molecular markers (24); possession of widespread metastatic activities (27); and their low intrinsic immunogenicity (28).

In our initial studies of adenoviral vector-based HSV-rt + GCV gene therapy, we documented cytotoxic activities in human prostate cancer cell lines in vitro and extensive cytotoxic activity through the induction of necrosis and apoptosis in subcutaneous tumors with the C57Bl/6 derived mouse prostate cancer cell line RM-1 (29). A significant survival advantage was documented for the treated animals. In the clinically more relevant orthotopic models cancer cells form prostate cell lines were injected into the prostate of fully immunocompetent host mice. These inoculums develop into tumors that were still relatively small and within the confines of the host prostate. In these mice HSV-t^+GCV therapy led to prolonged survival (30,31). To test the possibility that HSV-t^+GCV gene therapy could generate systemic antitumor immunity in prostate cancer, we developed a model system of pre-established lung metastasis in which mouse prostate cancer cells were simultaneously introduced into the prostate (orthotopic injection) as well as into the tail vein. Tail vein injection of these cells resulted in the establishment of lung colonies within 3 to 4 d and by the time of treatment (approx1 wk following orthotopic injection) these metastatic lesions represented pre-established metastatic targets to evaluate the systemic effects of localized in situ gene therapy. Interestingly, a single injection of HSV-rfr+GCV vectors not only suppressed the growth of local ortho-topic tumor through necrotic and apoptotic cell death, but also dramatically reduced the number and size of pre-established lung metastatic foci (20). In nontumor bearing mice local inflammation after the administration of the HSV-t^+GCV vector were minimal and vector spreading outside the prostate gland was very limited and well within the limits that were perceived as being safe for human use (32), thus, allowing to conduct a phase I trial in patients whose prostate cancer had recurred locally following initial radiation therapy but without any evidence of metastatic disease. The urologic gene therapy program Baylor College of Medicine thus conducted the first in situ gene therapy phase I clinical trial for human prostate cancer and demonstrated the safety of in situ HSV-t^+GCV gene therapy. In this clinical trial men with biochemical recurrence of localized prostate cancer following radiation therapy received a single injection of the adenoviral vector (33). Although one instance of toxicity was observed at the highest dose (1 x 1011 IU), the complications ultimately resolved completely. Minimal toxicity was observed in most patients and decrease of serum PSA levels by 50% or more was noted in 3 of 18 patients (33). An additional 18 patients received doses of 1-2 x 1010 IU, which proved to be a safe dose even when administered at multiple sites or when repeated for up to three times (34). Analysis of the data from this group of patients indicated that in situ HSV-tk+GCV gene therapy led to an increased PSA doubling time, a significant PSA reduction, and a significantly increased mean time to return to initial PSA following vector injection, both after the initial and the repeated injections. A potential immune modulatory component in the response to HSV-tk gene +GCV gene therapy was demonstrated by increased levels of activated (HLA DR+) CD8+ T-cells in the peripheral blood following treatment. Interestingly, the density of CD8+ T-cells in post-treatment biopsies was increased, which correlated with an increased number of apoptotic cells (7).

After the safety and potential efficacy of HSV- tk + GCV gene therapy had been shown in men with recurrent disease, the gene therapy was tested in a neo-adjuvant setting. Men with newly diagnosed prostate cancer and clinical markers that suggested high grade disease who elected to undergo a radical prostatectomy 4 to 6 wk after vector injection entered the trial. The radical prostatectomy specimens clearly demonstrated that in situ gene therapy induced local inflammation within prostate cancer foci accompanied by increased infiltration of CD4 and CD8 T-cells (35). In addition, necrosis within prostate cancer lesions in preference to adjacent normal prostatic tissues was noted. Additional studies confirmed that HSV-tk+GCV gene therapy (in this patient population) led to increased numbers of HLA DR+ CD8+ T-cells in the peripheral blood, again suggesting a systemic immune response (36).

An additional phase I-II trial involved 59 patients that received two to three doses of HSV-tk+GCV combined with standard of care radiotherapy. Intravenous GCV was replaced with the oral bioequivalent drug valacyclovir. Men in this trial were stratified to 3 groups, 29 men with low-stage disease, 26 men with high-stage disease, and 4 men with stage D1 (regional lymph node metastases). The latter two groups also received concurrent hormonal therapy. Mild hematologic and hepatic abnormalities found in the patients were attributed to the gene therapy whereas genitourinary and gastrointestinal side effects were typical radiation related side effects. However, there was no added toxicity attributable to the combination therapeutic approach (37). The combined radiogene therapy approach appeared to provide good local control based on biopsy data, but it was not adequate for men with prostate cancer that had already spread to the pelvic lymph nodes (38). Men with low-stage disease had evidence of activation of circulating CD4 and CD8 T-cells (36). This translational research involving adenoviral vector mediated in situ gene therapy for prostate cancer set the stage for the further development of immunomodulatory active vaccines.

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