Combination Suicide Gene And Cytokine Gene Therapy

Based on the initial immune responses generated after suicide gene therapy, there has been a recent interest in combining this strategy with cytokine gene therapy. Cyotokine gene therapy has arisen because of the growing opinion that tumor-specific antigens are expressed in many if not all human tumors, but the immune system fails to generate an adequate immune response against these antigens (53). Designing or discovering a means to augment baseline immune activity or generate tumor-specific responses would greatly enhance any cancer treatment strategy. Systemic infusions of cytokines has demonstrated tumor regression in both animal tumor models and some human clinical trials; however, the severe toxicity associated with the high concentrations required to achieve antitumor efficacy significantly limits this strategy (54-56). Cytokine gene therapy may circumvent the toxicity of systemic infusions and provide both larger and sustained local concentrations of cytokine within the tumor environment. This augmentation of the immune response with cytokine production may also be synergistically enhanced by the addition of suicide gene therapy.

Upon treatment of a tumor with suicide gene, direct cyto-toxicity and tumor cell death occurs. This tumor killing releases large amounts of tumor antigens as well as other cellular proteins and debris. As has been demonstrated previously, this results in variable levels of immune activity in and of itself. The suicide gene therapy not only reduces the gross tumor burden, but also primes the local tumor immune environment for the beneficial effects of cytokine production. In addition, the actual suicide gene proteins themselves are believed to be immunogenic and may act as superantigens for both nonspecific and tumor-specific lymphocyte activation. By introducing high levels of local cytokine production using combined gene transfer techniques, this overall antitumor immune response may be greatly magnified.

Pioneering this area has been Chen and Woo et al. who investigated the effects of combination HSV-tk and IL-2 gene therapy for colorectal cancer liver metastases (57). Upon injecting the liver metastases with a combination of HSV-tk and IL-2, a significantly increased level of tumor regression was noted as compared with either HSV-tk or IL-2 alone. A long-term survival benefit was not seen. The combination treatment did generate an initial systemic immunity against a second challenge of wild-type tumor at a distant site. This response was tumor specific, but was not sustained. Immune studies revealed a predominantly CD8-positive T lymphocyte response. Interestingly, the treatment of the liver metastases with HSV-tk alone did not demonstrate a significant effect with respect to protection against a second- site tumor challenge. When applied to a head and neck orthotopic squamous cell carcinoma model, the combination of HSV-tk and IL-2 gene therapy demonstrated both a synergistic antitumor response and increased survival as compared with single gene therapy (58). The combination of HSV-tk and IL-2 in this head and neck cancer model also generated an immediate systemic immunity and protection against a second-site challenge of wild-type tumor (53). Immunohistochemical analyses local tumor environments revealed a predominance of CD8-positive T cell infiltration, but also CD4-positive T cells were identified.

Further investigations with the combination of HSV-tk, IL-2, and GM-CSF have demonstrated both enhanced tumor regression and animal survival. Also, this triple combination has shown that systemic immunity can be sustained as long-term protection against second wild-type tumor challenges could be generated (59). It is hypothesized that the addition of GM-CSF not only stimulates antigen presentation cells within the local tumor environment, but induces a long-term antitumor memory that is mediated by CD4-positive T cells (60) However, not all tumors respond to triple combination therapy, and there may inherent factors within the tumor type or even the anatomical region of the body that influences such responses to the addition of GM-CSF (60a).

Given the low in vivo gene transfer efficiency of many viral vectors, certain strategies have been studied that may circumvent these limitations. VP22 is a protein normally produced by the HSV-1 virus and has a unique capability of achieving intercellular spread (60b). The VP22 protein is actively exported from the cytoplasm of cells producing the protein, and then it is subsequently transported across neighboring cell membranes where it is delivered to the cell nucleus. VP-22 fusion proteins and genes fused with VP22 (i.e., VP22-tk, VP22-p53, CD-VP22) are able to retain these intercellular transport properties (60c,60d). Wybranietz et al. demonstrated a significant benefit of adenovirus-mediated CD-VP22 fusion gene delivery vs. adenovirus CD suicide gene therapy alone in multiple tumor cell lines. Gene therapy strategies employing VP22 fusion proteins present new horizons for improving the efficiency and overall efficacy of suicide gene therapy strategies.

Another possibility for improved suicide gene therapy is to combine two powerful suicide genes into one delivery vehicle or viral vector. Various studies have investigated the potential for adenovirus-mediated HSV-Tk and CD double suicide gene therapy in both preclinical and clinical investigations (60e). Moriuchi et al. did not identify a benefit of HSV-TK and CD double suicide gene therapy with respect to killing of glioma tumor cells (insert reference Moriuchi S). A replication-defective adenovirus vector was used in these experiments. A number of investigators, however, have demonstrated in preclinical studies the benefit of double suicide gene therapy strategies in combination with radiation for various tumors cell lines (60f,60g). The application of double suicide gene therapy in human clinical trials are discussed at the end of the chapter.

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