Novel Therapeutic Delivery Systems In The Treatment Of Prostate Cancer

Gene therapy involves delivering recombinant genetic material, in the form of DNA or RNA to combat disease. Major strategies involve modifying gene expression to correct deficiencies or block inappropriate gene expression, inducing 'suicide' genes to promote cancer cell death and modulating the interactions of the immune system with cancer cells. This is performed either by removing tissue and genetically altering it ex vivo or delivering the genetic material in vivo. In vivo gene therapy has been limited largely by inefficient delivery systems and improvements in gene vectors, and delivery will allow practical use of clinical gene therapy for a variety of conditions. The ideal delivery system would be non-toxic to normal cells, deliver the genetic information efficiently with specificity to the targeted system and be inexpensive yet easily administered.

Tumor cell vaccines are a typical ex vivo gene therapy strategy for cancer and rely on the ability of cancer-specific antigens to elicit an immune response. An approach is to harvest tumor cells from the patient, genetically modify the cells (usually with retroviral transfection) so that they can stimulate the immune system, irradiate the cells so that they are non-tumorigenic, and reinject the cells into the patient. The first tumor vaccine trial for prostate cancer began in 1994 when granulocyte-macrophage colony-stimulating factor (GM-CSF) was transfected using retrovirus into a patient's prostate cancer cells in vitro. The cells were then injected subcutaneously and found to be safe in phase I trials.178 Data from this trial indicate vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. These data are the first to suggest that both T-cell and B-cell immune responses to prostate cancer can be generated by treatment with irradiated, GM-CSF gene-transduced prostate cancer vaccines. A limitation in the trial by Simons etal. was generating vaccine cells in culture. This group has embarked on a new trial to estimate the efficacy of using the ex vivo, GM-CSF-transduced, prostate cancer cell lines PC-3 and LNCaP as a vaccine. They reported results on 21 patients treated and one had a partial PSA response of greater than 7 months in duration, 14 had stable disease and six patients underwent progression. By 3 months PSA velocity or slope decreased in 71% of cases. Additionally, numerous new postvaccination IgG antibodies were identified in these patients, indicating that immune tolerance to prostate cancer-associated antigens may be broken.179

The first report to demonstrate anticancer activity of gene therapy in human prostate cancer, by Herman and colleagues, used intratumoral injection of replication-deficient adenovirus carrying the thymidine kinase gene from the herpes simplex virus, followed by parenteral administration of the prodrug gancyclovir. They report promising results with some patients with local recurrence after radiotherapy obtaining a greater than 50% decrease in PSA lasting 6 weeks to 1 year.180 These data support the usefulness of cytoreductive gene therapy, by 'suicide' genes, or by increasing tumor cell sensitivity to chemotherapeutics. Replication-deficient adenovirus containing the herpes simplex virus thymidine kinase (HSV-tk) gene ('suicide' gene therapy) has also been used in men with localized prostate cancer. Multiple and/or repeat intraprostatic injections was followed by intravenous ganciclovir or oral valaciclovir 14 days after injection. A total of 52 patients were treated, with a total of 76 gene therapy cycles; toxic events were recorded in 16 of 29 patients (55.2%) who were given multiple viral injections into the prostate, seven of 20 (35%) who received two cycles of 'suicide' gene therapy and three of four (75%) who received a third course of gene therapy. All toxic events were mild (grades 1 to 2) and resolved completely once the therapy course was terminated. Mean follow-up was 12.8 months and preliminary results for 28 patients indicated a mean decrease of 44% in serum PSA in 43% of patients.181

A current study is evaluating the effect of p53 replacement therapy utilizing intraprostatic Ad5CMVp53 injection in men with localized prostate cancer prior to radical prostatectomy. Patients are followed for tumor volume response by transrectal ultrasound and magnetic resonance imaging (MRI) and injections are continued every 2 weeks until tumor shrinkage abates, after which time they undergo radical prostatectomy. Results are not available at this time as this trial is still open for enrollment.182 Logothetis et al. reported initial results using a similar gene replacement strategy of adenovirus containing wild-type p53 (AdCMVp53) driven by the CMV promoter before radical prostatectomy in 11 patients with locally advanced prostate cancer. AdCMVp53 was administered via a transperineal route under ultrasound guidance. In this phase I—II study, three patients who completed a repeat course of therapy had a greater than 25% decrease in tumor size, as measured by endorectal coil MRI after the initial treatment course and there was no grade 3 or 4 toxicity in the 14 evaluable patients. Further results on efficacy due to the apparent radiological responses, correlations with gene expression, pathological findings at prostatectomy and surgical outcomes are pending.183

Investigators have recently completed a phase I trial in 24 patients with locally advanced prostate cancer using gene-based immunotherapy. A functional DNA-lipid complex encoding the IL-2 gene was administered intraprostatically, under transrectal ultrasound guidance, into the hypoechoic tumor lesion. Patients were stratified into two groups: group 1 included patients who underwent radical prostatectomy after the completion of the treatment regimen; and group 2 consisted of patients who had failed a prior therapy. IL-2 gene therapy was well tolerated, with no grade 3 or 4 toxic reactions occurring. Post-treatment prostate specimens were attained and compared with the transrectal biopsies performed prior to therapy. Evidence of systemic immune activation after IL-2 gene therapy included an increase in the intensity of T-cell infiltration seen on immunohistochemistry of tissue samples from the injected tumor sites, and increased proliferation rates of peripheral blood lymphocytes that were cocultured with patient serum collected after treatment. Transient decreases in serum PSA were seen in 16 of 24 patients (61%) on day 1. Fourteen of the patients persisted in this decrease to day 8 (58%). However, in eight patients the PSA level rose after therapy. More patients (9-10) in group 2 responded to the IL-2 gene injections and six of the nine also had lower than baseline PSA levels at week 10 after treatment.184 This group has also reported on novel chimeric PSA enhancers that exhibit increased activity in prostate cancer gene therapy vectors. They addressed the problem of low transcriptionally active native PSA enhancer and promoter, which otherwise confers prostate-specific expression when inserted into adenovirus vectors. The investigators exploited the mechanism by which androgen receptor molecules bind cooperatively to androgen response elements in the PSA enhancer core, and act synergis-tically with AR bound to the proximal PSA promoter to regulate transcriptional output. They generated chimeric enhancer constructs by inserting four tandem copies of the proximal AREI element, duplicating the enhancer core, or by removing intervening sequences between the enhancer and promoter. They obtained chimeric PSA enhancer constructs, which were highly androgen inducible and retained a high degree of tissue specificity even in an adenoviral vector (Ad-PSE-BC-luc). They also demonstrated that augmented activity of the chimeric constructs in vivo correlated with their ability to recruit AR and critical coactivators in vitro. Furthermore, systemic administration of Ad-PSE-BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts showed that this prostate-specific vector retained tissue discriminatory capability compared with a comparable cytomegalovirus (CMV) promoter driven vector.185

Kwon and colleagues have utilized regulation of T-lymphocyte proliferation by the stimulatory HLA-B1 family with T-cell surface inhibitory receptor CTLA4 in a transgenic mouse model of metastatic prostate cancer growth after tumor resection. They modified their tumor vaccine with the addition of anti-CTLA4 antibodies and showed metastatic relapse dropped from 91.4% in controls to 44% in animals treated immediately after tumor resection.186 Additionally, anti-CTLA4 antibody treatment can delay tumor growth in a transgenic mouse model.181

In vivo gene therapies for prostate cancer utilize vectors to deliver genetic information to tissues. Viruses are commonly used and types include adenovirus, retrovirus, adeno-associated virus, herpes virus and poxvirus. Non-viral vectors include liposomes, DNA-coated gold particles, plasmid DNA and polymer DNA complexes. Overcoming the obstacle for gene therapy vector inefficiency is being approached with development of less immunogenic viral vectors and 'stealth liposomes,' which evade the first-pass destruction by the reticular endothelial system. Furthermore, developing more specific therapies by targeting specific cell receptors or using prostate-specific gene promoters (e.g. prostate-specific membrane antigen and human kallekrein II) may allow for lower effective doses and perhaps decreasing the side effects commonly seen with viral vectors.

Correction of genetic alterations of tumor suppressor genes and cancer-promoting oncogenes can target a multitude of genes in the pathways to cancer development or progression. Restoration of one of the tumor-suppressor genes (Rb, p53, p21, and p16) have been shown to alter malignant phenotype, but would have to be delivered to every cancer cell to eradicate the cancer. The retinoblastoma gene has been shown to be mutated in human prostate cancer specimens, and gene therapy has shown some effectiveness in model systems of neuroendocrine and lung tumors.188'189 Mutations in p53 are reported at a wide range of rates; however, p53 gene therapy suppressed tumorigenesis and induced apoptosis in vitro.190 Furthermore, in vivo studies have shown some benefit of p53 gene therapy in prostate tumor models.191 Similarly, p21 gene therapy has been shown to suppress growth in prostate tumor cells in vitro and in vivo.192,193

A concept that is being expanded is restoration of genes that confer increased chemosensitivity or radiosensitivity to prostate cancer tumors. One study has looked at proliferation of tumor cells after incubation with various combinations of p53 adenovirus (p53 Ad) and chemotherapeutic drugs. The authors found p53 Ad combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate or etoposide inhibited cell proliferation more effectively than chemotherapy alone in multiple human tumor cell lines, including DU-145 prostate cells. Human tumor xenografts in SCID mice dosed with intraperi-toneal or intratumoral p53 Ad with or without chemotherapeutic drugs showed greater anticancer efficacy with combination therapy in four human tumor xenograft models in vivo.194 Another study evaluated gene therapy enhancement of prodrugs and radiation therapy. Prostate tumor cells (PC-3) were transduced with adenovirus containing a fusion gene encoding the E. coli cytosine deaminase and herpes simplex virus type-1 thymidine kinase under the control of a cytomegalovirus promoter. PC-3 cells expressing this protein were sensitized to killing by the normally innocuous prodrugs 5-fluorocytosine and ganciclovir. In addition, radiation-induced killing was enhanced in virally infected cells in the presence of the pro-drugs.195 Additionally, some evidence exists that gene therapy may be cooperative with androgen deprivation. Castration was combined with HSV-tk plus GCV using an androgen-sensitive mouse prostate cancer cell line, which led to markedly enhanced tumor growth suppression in both subcutaneous and orthotopic models compared with either treatment alone. An enhanced survival was also observed, in which combination-treated animals lived twice as long as controls in the subcutaneous model and over 50% longer than controls in the orthotopic model.196 A very interesting study utilized a constructed recombinant adenovirus containing E. coli cytosine deaminase and herpes simplex virus type 1 thymidine kinase fusion gene under the control of a human inducible heat shock protein 70 promotional sequence. Heating at 41 °C for 1 hour induced strong expression of the fusion gene product. Heat-induced expression of the CD-TK protein significantly reduced the survival of PC-3 cells in the presence of both 5-fluorocytosine and ganciclovir prodrugs. These data represent a novel form of gene therapy involving the transduction and regulation of a double suicide gene in tumor cells and may provide a unique application for hyperthermia in cancer therapy.197 In the future, perhaps hsp promoters could be used in combination with thermal energy modalities.

Gene transfer trials sponsored by the National Institutes of Health are listed on the World Wide Web at http:// and currently list 40 trials.

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